Micro- and nanoelectrochemistry for surface patterning

Micro- and Nanoelectrochemistry for Surface Patterning, Biosensing and

Electrocatalysis

Dissertation

zur Erlangung des Grades eines Doktors der Naturwissenschaften

Fakultät für Chemie und Biochemie

Ruhr-Universität Bochum

vorgelegt von

Jan Clausmeyer

- Bochum, Februar 2016 -

This work was carried out in the period from May 2012 to February 2016 in the Department of

Analytical Chemistry under the supervision of Prof. Dr. Wolfgang Schuhmann, Ruhr-Universität

Bochum, Germany.

First Examiner: Prof. Dr. Wolfgang Schuhmann

Second Examiner: Prof. Dr. Martin Muhler

Acknowledgements

I have been very fortunate to work with some very inspiring individuals and I hope my efforts

may be an inspiration to others. I am thankful to everyone who believed in me, advised me,

shared a piece of knowledge, created some opportunities or supported me otherwise. My

gratitude goes especially to these people without whom this work would not have been possible.

I thank Prof. Dr. Martin Muhler for agreeing to co-examine this work.

I am very grateful to all former and present members of Prof. Dr. Yuri Korchev’s group at the

Imperial College London who were involved with this amazing collaboration. Especially, I thank

Yuri Korchev for his contagious passion in science, the opportunity to work in his lab and to take

over some responsibilities. Dr. Paolo Actis openly shared his knowledge about nanoelectrodes,

encouraged me to go further and gave many helpful hints. I am also very thankful to Dr. Ainara

López Córdoba for welcoming me, being a good friend and sharing her knowledge about cells.

Working together with Dr. Yanjun Zhang was a pleasure because of his expertise, kindness and

his persistence that turned the collaboration into a very successful story.

I thank Dr. Nicolas Plumeré for his support at an early stage of my project as well as Dr. Justus

Masa and Dr. Edgar Ventosa for their inspirational input on electrocatalysis and battery

electrochemistry. It was my pleasure to collaborate with marvelous Dr. Lutz Stratmann and Dr.

Dominik Schäfer who both contributed tremendously. I am also grateful to Dr. Thomas Erichsen

and Dr. Kirill Sliozberg for their technical help on all kinds of issues and to Bettina Stetzka for

her unselfish support.

I thank all students I used to work with and who became esteemed colleagues or continue their

way elsewhere: Alexander Botz, Stefanie Stapf, Tuba Simsek, Youssef Slibi, João Junqueira,

Tsvetan Tarnev, Anna Tymoczko, Denis Öhl, Tobias Löffler and Patrick Wilde. I would like to

thank especially Anna Muhs and Miriam Marquitan for their tremendous efforts and

contributions.

I am grateful to all my great colleagues and friends who made life and work in the past years

amazing and unforgettable. Special thanks go to my parents and Monika for supporting me and

giving me everything to make me ready to start an endeavor like this. Most of all, I thank

Katharina for her love, support and understanding throughout the past years.

I am most thankful to Prof. Dr. Wolfgang Schuhmann for not missing a single chance to support

me. He provided countless opportunities for me and always found the right balance between

advising me and giving me the freedom to develop myself.

All experiments were conceived, analyzed and performed by the author of this thesis, if not noted

otherwise. If applicable, references to collaborations and contributions of other persons are

indicated in the beginning of each chapter. It is also indicated whether content appearing in this

thesis was published elsewhere.

Abstract

Exploiting the physiology of living organisms at the single-cell level, electrochemical

characterization of single nanoparticles, and high-throughput studies of biomolecule function all

share a common need for advanced micro- and nanotechnological methods as well as chemical

analysis with high spatial resolution. This work describes how the merits of micro- and

nanoelectrodes are applied for electrochemical surface patterning, the detection of various

analytes inside and around single cells, and electrocatalytic studies at individual nanoparticles.

For the selective electrochemical patterning, carbon surfaces are modified with organic

electrochemically addressable surface functionalities such as protected p-hydroquinone

moieties as well as nitrophenyl groups. Highly localized activation of the redox active films is

achieved using two different scanning probe techniques: The Scanning Electrochemical Droplet

Cell (SDC) as well as Scanning Electrochemical Microscopy (SECM). After selective local

modification, the activated surface groups can capture and immobilize molecules which allow

for the construction of high-density analytical biomolecule arrays.

For the analysis of cell metabolism and oxidative stress conditions, molecular oxygen and

hydrogen peroxide are detected at single cells using needle-type carbon nanoelectrodes.

Modification with Prussian Blue creates nanosensors for the selective detection of H2O2. Due to

the small size of the sensors, reactive oxygen species (ROS) are detected outside and inside the

cell. To increase the sensitivity of the amperometric sensors, an alternative scheme based on

potentiometric sensing is proposed.

Moreover, to increase the sensitivity and allow highly flexible sensor designs, field effect

transistor (FET) sensors are built on dual carbon nanoelectrodes. Polypyrrole deposited on the

tip of the dual probe acts as the transistor channel. The high-aspect ratio FET nanosensor is used

to detect the local acidification in the microenvironment of cancer cells. Modifying the transistor

channel with hexokinase allows for sensitive ATP measurements at single cells.

Finally, the merits of nanoelectrodes are exploited for the investigation of nanoparticle

electrocatalysts. Individual Ni(OH)2 nanoparticles deposited on nanoelectrodes are

characterized in non-ensemble measurements with respect to their properties for energy

storage and electrocatalytic activity for the oxygen evolution reaction (OER). Charging by

oxidation of Ni(OH)2 is limited by diffusion of protons in the particle bulk and the OER activity is

independent of particle size.

Contents

1 Introduction ............................................................................................................ 1

2 State of the Art ........................................................................................................ 2

2.1 Electrochemical Patterning toward Biomolecule Immobilization ....................................................... 2 2.1.1 Patterned electrode arrays ............................................................................................................................................. 3 2.1.2 Patterning via electrophoretic delivery of reagents ........................................................................................... 4 2.1.3 Patterning by means of Scanning Electrochemical Microscopy .................................................................... 4

2.1.3.1 Local production of reagents ................................................................................................................................ 4 2.1.3.2 Direct electrochemical reaction at the sample surface ............................................................................. 6

2.2 Nanoelectrodes – Motivation, Fabrication and Handling ........................................................................ 8 2.2.1 Nanoelectrode fabrication .............................................................................................................................................. 9

2.2.1.1 Insulated STM tips and fibers ............................................................................................................................... 9 2.2.1.2 Metals fused in capillaries................................................................................................................................... 10 2.2.1.3 Carbon-filled nanopipettes ................................................................................................................................. 11 2.2.1.4 Other nanoelectrode fabrication protocols ................................................................................................. 12

2.2.2 Special precautions and considerations for studies at nanoelectrodes .................................................. 13 2.2.2.1 Theory of electrochemistry at the nanoscale ............................................................................................. 13 2.2.2.2 Technical and instrumental aspects ............................................................................................................... 15

2.3 High-Resolution Electrochemical Imaging ................................................................................................. 16

2.4 Cell Analysis ........................................................................................................................................................... 19

2.4.1 Detection of reactive oxygen species ...................................................................................................................... 19 2.4.2 Electrochemical single-cell analysis ........................................................................................................................ 22

2.5 Field Effect Transistor Sensors ....................................................................................................................... 27

2.6 Nanoparticle Electrochemistry ....................................................................................................................... 31

3 Aim of the Work .................................................................................................. 35

4 Results and Discussion ..................................................................................... 37

4.1 Electrochemical Surface Patterning ............................................................................................................. 37 4.1.1 Global electrode modification with p-hydroquinone layers ......................................................................... 37 4.1.2 Potentiostatic patterning in the SECM ................................................................................................................... 39 4.1.3 Galvanostatic patterning in the SECM .................................................................................................................... 40

4.1.3.1 Patterning of TBDMS-protected p-hydroquinone layers ...................................................................... 40 4.1.3.2 Patterning of nitrophenyl layers ...................................................................................................................... 42

4.1.4 Patterning in the Scanning Droplet Cell ................................................................................................................. 47

4.2 Nanoelectrode Fabrication and Characterization ................................................................................... 51

4.3 Amperometric Nanosensors in Biological Applications ........................................................................ 54 4.3.1 Platinized nanoelectrodes for oxygen measurements .................................................................................... 54 4.3.2 Prussian Blue-modified nanoelectrodes for the detection of H2O2 ........................................................... 57 4.3.3 H2O2 measurements at single living cells .............................................................................................................. 65

4.4 Potentiometric Sensors on Nanoelectrodes............................................................................................... 71

4.5 Field Effect Transistor Sensors on Nanoelectrodes ................................................................................ 78

4.5.1 pH-sensitive polypyrrole FETs on dual carbon nanoelectrodes................................................................. 78 4.5.2 pH measurements at cells ............................................................................................................................................ 84 4.5.3 PPy-FET nanobiosensors for ATP measurements ............................................................................................ 86 4.5.4 ATP detection at cells ..................................................................................................................................................... 89

4.6 Single Nanoparticle Electrochemistry ......................................................................................................... 94 4.6.1 Electrocatalyst studies .................................................................................................................................................. 94 4.6.2 Study of energy storage materials ......................................................................................................................... 103

5 Conclusions and Outlook ............................................................................... 106

6 Experimental Procedures ............................................................................. 110

6.1 Syntheses ..............................................................................................................................................................110

6.2 Global Electrode Modification and Characterization ...........................................................................112 6.2.1 Surface modification with TBDMS-protected p-hydroquinone groups ................................................ 112 6.2.2 Surface modification with nitrophenyl groups ............................................................................................... 112

6.3 SECM Patterning and Imaging .......................................................................................................................113 6.3.1 Fabrication of microelectrodes............................................................................................................................... 113 6.3.2 Surface modification with TBDMS-protected p-hydroquinone groups ................................................ 113 6.3.3 Surface modification with nitrophenyl groups ............................................................................................... 113

6.4 Scanning Droplet Cell Patterning .................................................................................................................114

6.5 Atomic Force Microscopy ................................................................................................................................115

6.6 Fabrication and Handling of Carbon Nanoelectrodes ..........................................................................115

6.7 Setups for Electrochemical Measurements and Positioning of Nanoelectrodes ........................117

6.8 Protocols for the Modification and Characterization of Carbon Nanoelectrodes ......................120 6.8.1 General............................................................................................................................................................................... 120 6.8.2 SEM imaging .................................................................................................................................................................... 120 6.8.3 Platinization .................................................................................................................................................................... 121

6.8.4 Fabrication and characterization of PB-based H2O2 nanosensors .......................................................... 121 6.8.5 Fabrication and characterization of PPy FET nanosensors ....................................................................... 121 6.8.6 Immobilization of hexokinase on PPy-FET nanosensors ............................................................................ 122 6.8.7 Deposition and characterization of Ni(OH)2 nanoparticles ....................................................................... 122

6.9 Local Measurements at Cells..........................................................................................................................123 6.9.1 H2O2 detection in macrophage cells ..................................................................................................................... 123 6.9.2 pH and ATP measurements using PPy-FET nanosensors ........................................................................... 123

6.10 Cell Culture and Tissue Preparation ........................................................................................................123 6.10.1 Brain slice preparation ............................................................................................................................................ 123 6.10.2 Dorsal root ganglia (DRG) neuronal culture .................................................................................................. 124 6.10.3 Macrophage cell culture .......................................................................................................................................... 124 6.10.4 Melanoma and melanocyte culture .................................................................................................................... 124

6.10.5 Isolation of rat ventricular myocytes ................................................................................................................ 125

7 References .......................................................................................................... 126

8 Annex .................................................................................................................... 146

8.1 Abbreviation List ...............................................................................................................................................146

8.2 Publications .........................................................................................................................................................147

8.3 Conference Contributions ...............................................................................................................................148 8.3.1 Oral presentations ........................................................................................................................................................ 148 8.3.2 Poster presentations ................................................................................................................................................... 149

1 Introduction

1

1 Introduction

Parts of the introduction and state of the art are published in ref. [1]: “Clausmeyer, J.; Schuhmann,

W.; Plumeré, N.; TrAC Trends Anal. Chem. 2014, 58, 23–30” and ref. [2]: “Clausmeyer, J.;

Schuhmann, W.; TrAC Trends Anal. Chem. 2016, DOI: 10.1016/j.trac.2016.01.018”

From the metabolism and communication mechanisms in biological organisms to current

industrial technologies for energy conversion and storage, electrochemistry governs many

processes relevant to our present and future existence. Metabolic pathways and signal

transduction mechanisms in cells are largely dictated by the electrochemical properties of their

components, namely redox active molecules. At the same time, powerful analytical techniques

based on electrochemical phenomena help in investigating cells and biomolecules and thus

contribute to the understanding of life’s principles. Moreover, in order to cover our high demand

for energy in the future, electrochemistry has to provide sustainable strategies for the

conversion and storage of energy without relying on fossil fuels. Not only new catalyst and

energy storage materials with outstanding properties need to be developed but also suitable

techniques for assessing their performance are necessary to turn the gained knowledge into

more rational design of materials.

The entities of study, e.g. biological cells or nanostructured materials are very small and their

properties result from physicochemical phenomena taking place at the nano- and microscale.[3,4]

Thus, analytical methods based on nano- and microelectrodes are promising tools to obtain

analytical information from these small entities.[5–7] For instance, the physiology of single cells

can be studied or catalytic reactions occurring at single nanoparticles can be investigated. In

addition, highly resolved electrochemical imaging[8,9] yields information concerning the

heterogeneous electrochemical activity in biological systems[10,11] and energy materials.[12] These

techniques aim at obtaining information that is difficult to get using conventional analytical

methodologies. However, techniques based on nano- and microelectrodes are not only restricted

to the sole detection and analysis but electrochemical probe techniques offer manifold

possibilities for specific local manipulation of samples, localized delivery of reagents and the

generation of micro- and nanoscale structures. Electrochemical techniques are promising

alternatives to classical patterning schemes[13–16] due to their ability both for surface patterning

at the micro- and nanoscale as well as for high-resolution visualization of the patterned surface

chemistry.[17]

2 State of the Art

2.1 Electrochemical Patterning toward Biomolecule Immobilization 2

2 State of the Art

2.1 Electrochemical Patterning toward Biomolecule Immobilization

Microarrays of biomolecules patterned onto a solid support are powerful tools for high-

throughput investigation of biomolecules interactions.[13,14,18,19] DNA and protein arrays were

implemented for function determination, diagnostics and drug screening. In recent years much

effort was spent to reduce the dimensions of generated biomolecule patterns in order to

increase the density of information on a given surface area. Biomolecules need to be

immobilized on small patterns with high control over the surface chemistry. However, with

decreasing patterning dimensions it becomes increasingly challenging to maintain and

demonstrate the chemoselectivity of the immobilization procedure. Many characterization tech-

niques fail to provide information about the surface chemistry used for attachment of molecules.

In the light of these considerations, new concepts that push forward the limits of array

generation with high spatial resolution are necessary. Moreover, novel analytical methods for

localized characterization of patterned surfaces are needed. Special attention needs to be paid to

a critical assessment of the surface chemistry and chemoselectivity of immobilization

procedures.

The goal in the fabrication of bioarrays is to assemble as many different samples of biological

recognition elements as possible on a given surface area to increase throughput of the biological

assay. At the same time the amount of consumed sample for each spot is reduced with

decreasing pattern dimensions. The spatial information to create a laterally heterogeneous

surface, the patterning, may originate from various sources. Classically, different specimens to

immobilize are dispensed to discrete areas on a surface with the help of a nozzle or pin for

printing or spotting, respectively (Figure 1 a).[18,19] Recently, the spatial resolution of patterning

was significantly improved by using atomic force microscopy (AFM) integrating cantilevers with

a fluidic channel to dispense reagents.[20] These approaches imply a serial patterning process as

opposed to parallel patterning employing photolithographic techniques.[16] Parallel patterning

techniques typically use a photomask and light is irradiated to restricted areas on the sample

triggering chemical reactions to crosslink[21] or remove material such as photo-cleavable

protection groups or biomolecule repelling films from the surface.[22] The necessity for a

template limits the flexibility when designing the biomolecule array. Additionally, protocols

from classical photolithography as used in microelectronics fabrication cannot be easily adapted

to sensitive biomolecules because the reaction conditions may affect the structure and activity of

biomolecules already immobilized on the array surface.

2 State of the Art


2.1.1 Patterned electrode arrays

Photolithography is used to fabricate arrays of small electrodes rather than triggering local

attachment or detachment of biomolecules to the sample surface directly. Individual electrodes

at different locations on the sample may be contacted to trigger an electrochemical reaction for

the immobilization of biomolecules. The individual on/off switching allows for the fabrication of

arrays with multiple biological molecules on multiple electrodes. Various electrochemically

triggered immobilization strategies including electrografting of aryldiazonium salts,[23,24] various

reactions using surface-confined quinone groups,[25,26] quinone-based protecting groups,[27,28]

polymer entrapment[29–31] and unspecific adsorption[32] were exploited. The readout of binding

events at individual electrodes without relying – as classical bioarrays do – on fluorescence

microscopy can be achieved using electrode arrays. As most proteins and DNA are not redox

active by themselves, labeling with redox active reporter molecules is often required. On

commercialized electrode arrays consisting of up to 12,544 individually addressable electrodes,

each 44 µm in diameter, DNA hybridization events[33] as well as antibody recognition[32] were

successfully detected. Ultimately, instead of classical immobilization of pre-synthesized bio-

polymers on a surface, the electrode arrays were utilized for the in-situ synthesis of both

DNA[34,35] and peptide arrays[36]. Electrochemical generation of protons replaced the addition of

acids to deprotect the pH-sensitive protective group at the growing chain as in standard solid-

phase synthesis procedures. This makes the array fabrication faster than serial spotting of

reagents[37] but more flexible and less costly than array synthesis based on photochemical

cleavage of protecting groups using photomasks.[38–40]

a

b

c

d

Figure 1. Scanning probe techniques in various configurations are employed to induce local surface modifications. Patterning schemes as for spotting/DPN (a), electrophoretic delivery (b), microreagent mode SECM (c), direct mode SECM (d). Adapted from [1].

2 State of the Art


2.1.2 Patterning via electrophoretic delivery of reagents

Owing to the fact that most biomolecules are charged they may be locally delivered from micro-

and nanopipettes to the surface by applying a voltage between an electrode inside the pipette

and an external one (Figure 1 b). This configuration is also used in Scanning Ion Conductance

Microscopy (SICM)[41] to precisely control the distance between the fragile nanopipette tip and

the sample surface by measuring the ionic current through the pipette orifice (see paragraph

2.3). The advantage of this technique is that e.g. immobilized DNA[42] or proteins[43] are in

solution at all times during patterning which prevents from possible denaturation. The

requirement is a surface chemistry that is reactive enough to capture the delivered molecules

instantaneously in order to avoid dilution or broadening of the biomolecule patterns. Instead of

using the SICM-based distance control the nanopipette was also coupled to an AFM[44]. This

technique, often called nanofountain, allowed also for parallel patterning using multiple tips,[45]

however in air. Recently, a nanopipette was filled with an organic solvent containing an

aryldiazonium salt species which was driven out from the pipette and electroreduced at the

sample electrode to generate surface patterns.[46]

2.1.3 Patterning by means of Scanning Electrochemical Microscopy

Scanning Electrochemical Microscopy (SECM) employs a micro- or nanoelectrode which can be

moved with high precision along the three directions of space. Typically, an electrochemical

reaction is carried out at the microelectrode tip and the tip is brought close to the sample

surface. This results in a perturbation of the typical electrochemical behavior of the

microelectrode observed in bulk solution. For instance, kinetics of electrochemical reactions or

production of electrochemically active species at the sample can be detected, quantified and

visualized.[47] The advantage of SECM for patterning of surfaces is that interfacial reactions with

biomolecules can be locally triggered without prior loading of the tip as it is necessary in dip-pen

nanolithography (DPN) or nanofountain technology. Surface patterning using the SECM splits up

in two modes of operation, the microreagent mode (Figure 1 c) and the direct mode (Figure 1 d)

whose advantages and disadvantages will be discussed below.

2.1.3.1 Local production of reagents

Active reagents can be locally generated at a micro- or nanoelectrode by electrochemical

conversion of precursors available in bulk solution (Figure 1 c). The chemistry at the

microelectrode tip is precisely controlled by the applied potential. A follow-up chemical reaction

with specific functional groups at the sample surface may promote localized attachment,

detachment or deactivation of biomolecules. This principle is referred to as microreagent mode

or indirect mode of SECM. In the microreagent mode, changes in surface functionality may be

induced by local changes in the pH value invoked by electrochemical water splitting at the

2 State of the Art


microelectrode tip. For instance, the protective SiO2 layer on silicon was etched by anodic

generation of H+ in an electrolyte solution containing F- ions (Figure 2 b).[48] The resulting

corrosion pits were backfilled by the reduction of aryldiazonium salts. The modified areas could

be further (bio)-functionalized in subsequent steps. The localized cleavage of ester

functionalities from self-assembled monolayer (SAM) modified gold electrodes induced by

proton production at the SECM tip was demonstrated. An alkylalcohol residue was removed

from a sample surface modified with an ester-terminated SAM while carboxyl functionalities

remained at the surface which were later activated and used to covalently attach proteins.[49]

Since the electrochemical reaction takes place at the microelectrode, an advantage of patterning

in the microreagent mode of SECM is that the sample surface does not necessarily have to be

electrically conductive. For instance, glass substrates were locally functionalized through click

chemistry[50] or polystyrene slides were oxidized by the generation of reactive radicals from Ag+

or nitrate in solution.[51] The resulting functional groups on the polystyrene surface were

suitable for the unspecific attachment of proteins and cells. A cathodic pathway was

demonstrated by performing a localized Fenton’s reaction which gives rise to hydroxyl radicals

to corrode various alkylsilane layers.[52] Through unspecific adsorption or after further

bioconjugation steps, the sample surface was patterned with an enzyme. The most widespread

patterning scheme is to use the microelectrode for the electrochemical conversion of bromide

into bromine/hypobromous acid. A homogenous layer of enzyme immobilized on the surface

was locally inactivated through the local oxidation by bromine.[53] When the sample surface was

covered with protein-repelling coatings prior to patterning, the locally produced bromine

degraded the film and allowed for spatially restricted immobilization of cells at these positions

(Figure 2 a).[54,55] To increase the intrinsically low speed of serial patterning techniques

significantly, the local generation of bromine has been also used in combination with a scanning

multiple tip consisting of eight individually addressable electrodes.[56]. However, the

microreagent mode often employs rather aggressive conditions to induce the local surface

modification. Constructive patterning, i.e. a surface functionality was locally introduced rather

than locally removed, was achieved by electrochemical grafting of aryldiazonium salts. Upon

cathodic reduction of aryldiazonium salts, organic moieties are tethered to carbon or metal

surfaces.[57,58] To assure localized grafting, the aryldiazonium precursor was generated at the

microelectrode and attached to the surface by reductive grafting at a suitable potential applied

to the sample electrode.[59,60]

2 State of the Art


a

b

c

Figure 2. Patterning schemes using the SECM. Proteins and cells locally adhere to a surface after removing a repellant coating by local production of Br2 in the microreagent mode (a). Etching of a passivating SiO2 layer by local electrochemical generation of H

+ yields reactive spots that can be further

bio(functionalized) (b). The direct mode of SECM was used for local carboxylation of a graphene substrate (c) a: Reprinted from [55] with permission from John Wiley and Sons. Reprinted with permission from [48]. Copyright (2010) American Chemical Society c: Reprinted with permission from [61]. Copyright (2015) American Chemical Society.

2.1.3.2 Direct electrochemical reaction at the sample surface

Instead of producing or delivering a reagent from a scanned tip in proximity to the sample

surface, SECM allows also performing a localized electrochemical reaction at the sample surface

directly. This requires a rather peculiar configuration of the electrodes, namely the direct mode

of SECM. The sample surface to be locally modified is used as working electrode whereas the

SECM tip serves as counter electrode (Figure 1 e). If a short potential pulse is applied to the

sample, the current necessary to drive the localized electrochemical reaction is restricted to the

area directly underneath the microelectrode tip. While the potential at the sample is controlled,

the potential applied to the counter electrode is driven to high values to provide the necessary

current to charge the large substrate electrode and to carry out the faradaic reaction at it. Hence,

in most cases the electrochemical reaction taking place at the microelectrode tip is electrolysis

of the solvent. In the direct mode of SECM, reagentless pattern generation was previously

achieved by electrografting[62,63] or removal[64] of surface groups or by changing their redox state

[65–67] However, little attention is paid on the chemoselectivity of the method, that is the chemical

nature of the local surface modification remains elusive. In contrast, Torbensen et al. recently

demonstrated the activation of graphene layers using the direct mode of SECM (Figure 2 c).[61]

Dissolved CO2 was electrochemically reduced at the areas of the substrate electrode under the

SECM tip which led to the attachment of carboxyl functionalities to the graphene substrate. The

local carboxylation was proved by various methods including x-ray photoelectron spectroscopy

(XPS) mapping and Raman microscopy. For patterning with biomolecules the direct mode of

2 State of the Art


SECM was applied to locally remove a SAM and backfill the resulting holes with a differently

functionalized alkanethiol to which glucose oxidase was coupled.[68] Also exploiting SAM

formation, gold was locally deposited and redox enzymes were bound to the gold spots.[69] A

SAM terminated with nitro groups was locally reduced electrochemically to give rise to spots of

amino/hydroxylamino groups. Using classical coupling reagents, enzymes could be immobilized

exclusively to the modified areas.[66] Alternatively, the direct mode allows to locally deposit

biomolecules in one step by incorporation into electrodeposited polymers. As tested for

macroscopic electrodes and electrode arrays, incorporation of oligonucleotide-modified pyrrole

monomers into a polypyrrole backbone may be used to generate DNA arrays.[70] Similarly, the

enzyme glucose oxidase can be co-deposited physically inside a chitosan matrix by generating a

pH gradient in the gap between microelectrode and sample surface through proton reduction at

the sample and water oxidation at the counter electrode [71]. The same enzyme was also

deposited by the more specific avidin-biotin interaction with biotinylated electropolymerized

polypyrrole.[72] In general, the direct mode allows for patterning without reagents in solution. A

surface uniformly modified with a redox-active species may be locally activated to capture

biomolecules from solution. High patterning resolution was achieved by applying a voltage

between an AFM or Scanning Tunneling Microscopy (STM) tip and the sample: Nanostructured

surfaces suitable for biofunctionalization were generated through localized metal reduction[73]

or changes in organic surface functionalities.[65,74] In these cases, high voltages have to be applied

and the problem of an ill-defined surface chemistry is even more pronounced because of the

absence of supporting electrolyte and a reference electrode.

2 State of the Art

2.2 Nanoelectrodes – Motivation, Fabrication and Handling 8

2.2 Nanoelectrodes – Motivation, Fabrication and Handling

A nanoelectrode is a solid electrochemical interface whose size in at least two dimensions is

substantially below 1 µm. Recently, much progress has been made in the low-cost fabrication

and implementation of such tools into modern analytical chemistry. Needle-type nanoelectrodes

are of particular interest as their high-aspect ratio is a prerequisite for many applications, for

instance for their use in scanning probe techniques and sensors for electrochemical analysis in

small confined volumes.[75] The small size of nanoelectrodes dictates their special

electrochemical properties which substantially deviate from the electrochemical behavior

observed at macroscopic electrodes. These features of nanoelectrodes are exploited in their

electroanalytical applications. First, since the diffusion layer of a substance produced or

consumed in an electrochemical reaction scales proportionally with electrode size, highly

localized measurements can be performed at nanoelectrodes. Second, the greatly increased mass

transport rates of reactants or products to and from the nanoelectrode allow to study catalytic

reactions without mass transport limitation and assure a high signal-to-noise ratio in sensing

schemes. However, there is still a lack in understanding size-dependent effects on the

electrochemical behavior of very small nanoelectrodes. As the electrode dimension approach the

ones of molecules and atoms, classical theory to describe electrochemical processes no longer

holds true.[76].

The use of nanometric electrochemical sensors is motivated by the small dimensions of samples

that require the sensor to be of smaller or at most equal size compared to the entity of interest.

Nanoelectrodes allow the study of electrochemical processes at single nanoparticles.

Investigating individual particles rather than whole statistical ensembles will help in elucidating

the relationship between particle size and catalytic activity. In contrast to a statistical ensemble,

the properties (size, activity, composition, geometry) of a single particle are not distributed over

a wide range and hence direct connections between these measures can be made. In addition,

electrocatalytic turnover at single particles exhibits high mass transport rates which allows to

investigate reaction kinetics without mass transport limitation.

In biological systems, microelectrochemical techniques have been used to study cell metabolism,

factors leading to pathogenic conditions as well as intercellular communication via the release of

neurotransmitters.[7,11,77,78] These techniques allow to detect metabolites and messenger

molecules released from individual cells to study cell function at the single-cell level. Individual

cell fates can be monitored and often analytical information complementary to standard optical

methods is obtained. Exploiting these novel technologies will bring about a deeper

understanding of physiological processes occurring inside living cells. Rare and unstable

substances can be detected directly at their location of production inside the cell.

2 State of the Art


Nanoelectrodes were also increasingly implemented in scanning probe techniques to achieve

chemical mapping of analytes with unprecedented spatial resolution. In biological and non-

biological systems, electrochemical imaging reveals inhomogeneous reactivity of samples. As the

size of the detecting probe decreases, not only improves the spatial resolution but also the

technique becomes increasingly non-invasive.

2.2.1 Nanoelectrode fabrication

2.2.1.1 Insulated STM tips and fibers

Metal electrodes with dimensions smaller than 1 µm were originally designed for

neurophysiology measurements already in the 1960s.[79] However, it was the success of STM

that first boosted the progress in nanoelectrochemistry and in particular the fabrication of

nanometric electrodes.[80] For a useful electrochemical interpretation of experimental data,

nanoelectrodes have to be fabricated with a defined geometry that allows to model electrode

processes and mass transport. The first electrodes used in an electrochemical context were Pt/Ir

rods that were, just like STM tips, etched in acidic solution by applying an AC voltage and then

later insulated using various coating materials to leave only the apex of the electrode exposed.

The electrode is moved through hot wax[81] or molten glass[80] to cast the insulating sheath.

Excavating the very tip of the electrode can be achieved by elaborate procedures, for instance

mounting the electrode in an STM instrument, applying a voltage between the electrode and the

sample and approaching the tip towards the sample until an electric discharge between the two

electrodes ruptures the insulating cap and leaves the tip exposed.[82] Alternatively, as insulating

material, electrodeposition paints have been commonly used for carbon fiber microelectrodes in

neurophysiological studies[83] and adapted to the fabrication of nanoelectrodes.[84–86] Upon heat

curing, the insulating sheath shrinks and retracts to leave the nanometric tip protruding from

the insulator. Alternatively, “inverted deposition”, where the electrode tip just bulges out of the

deposition paint solution was proposed.[87] The electrochemically active parts of electrodes

produced according to these methods are typically sphere segments or cones. Mirkin et al.

developed analytical expressions to describe the behavior of these electrodes in Scanning

Electrochemical Microscopy,[82] however, for most applications disk geometry is desirable to

make the nanometric electrode most sensitive to electrochemical processes occurring only at

the very tip. Moreover, the nature of electrodes derived from STM tips or carbon microfibers

precludes later polishing steps which is often necessary to obtain defined electrode geometries

and to regenerate electrodes between experiments. On the other hand, the very pointy shape

allows to insert these electrodes into small volumes while maintaining high sensitivity.

Especially flame-etched carbon fibers are promising probes for measurements in small

volumes.[88,89] After etching micrometric carbon fibers to create nanotips, the fibers are inserted

into glass capillaries for handling and electrical connection. Their conical shape is characterized

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by a small diameter at the tip while maintaining a relatively large surface area which ensures

still high sensitivity.

2.2.1.2 Metals fused in capillaries

Another widespread method to produce nanoelectrodes with good control of the electrode

geometry and high reproducibility is to pull nanopipettes together with an incorporated metal

wire using a laser-assisted pipette puller (Figure 3 a).[90,91] A piece of solid metal wire with a

diameter ranging between 10 and 100 µm is inserted in a borosilicate or quartz glass capillary

and gently heated using the laser puller while applying vacuum to the two ends of the capillary.

This causes the glass capillary to collapse concentrically and thus to tightly enclose the

micrometric wire in the capillary. In a second step, more heat is applied and the two ends of the

capillary are strongly pulled apart whereas both the glass sheath and the metal wire reduce their

dimensions to yield two nearly identical nanoelectrodes. This method has been most commonly

used to produce Pt electrodes, but also Au[92] and Ag wires[93] can be processed. After removing

excess glass from the tip by either chemical etching in HF or electrode polishing the electrodes

are well-defined metal disks fused in a coplanar and concentric glass insulator. The radii of the

active electrode range between a few nanometer to a few micrometer, whereas the overall

diameter including the insulator is typically 5 to 20 times larger than the active area. Zhang’s

group used an extension of this method to obtain extremely small electrodes of down to 1 nm.[94]

In order to ensure sufficient mechanical stability for mechanical polishing, the pulled

nanoelectrode was inserted into a second capillary and pulled again so that the electrode was

surrounded by a thicker insulating sheath (Figure 3 b).

a

b

Figure 3. Fabrication protocols to produce nanoelectrodes. Fabrication by laser-assisted fusing of Pt followed by pulling and polishing yields needle-type nanoelectrodes (scale bar 5 µm) (a). Reinforcement with an additional capillary and polishing yields a Pt electrode with a size of 3 nm (b). a: Adapted from [91] with permission from John Wiley and Sons. b: Reprinted with permission from [94]. Copyright (2009) American Chemical Society.

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The method requires additional polishing protocols to ensure high control over the tip geometry

and a defined state of the electrode surface.[91,95] The long tapered shape and the shear modulus

of the glass allows to bring the nanoelectrode tips into mechanical vibration and exploit the

electrode’s resonant frequencies for positioning using shear force Scanning Electrochemical

Microscopy (SF-SECM).[96,97] However, for high-resolution imaging of high-aspect ratio sample,

and measurements in microenvironments and inside single cells the large diameter of the

insulating glass sheath limits the electrodes’ applications.[98]

2.2.1.3 Carbon-filled nanopipettes

An alternative fabrication route to high aspect ratio nanoelectrode probes with small electrode

sizes has gained attention. Takahashi et al. described the pyrolytic decomposition of carbon

precursor gases inside pulled quartz glass nanopipettes which gives rise to nanometric carbon

electrodes with small overall dimensions at the probe tip.[99,100] The method is based on earlier

work conducted in Ewing’s group.[101] A glass capillary is first pulled to a pipette exhibiting a

nanometric or micrometric orifice. Then the pipette is connected to a carbon gas source that

provides the precursor gas (methane, acetylene, propane, butane or a mixture of the latter) at

high pressure. To exclude oxygen, the pipette is inserted into a second, unpulled capillary which

is connected to an argon or nitrogen gas cylinder with a slight gas flow. By applying heat with a

jet torch or Bunsen burner the gas inside the pipette pyrolyzes to yield a conductive carbon

remainder covering the inside walls of the pipette. When using relatively large pipette openings,

the carbon-filled pipette remains open unless it is otherwise clogged with other material.[101]

When the inner diameter of the pipette at its tip is sufficiently small, the procedure yields disk-

shaped carbon electrodes surrounded by an insulating glass sheath.[102]

The pyrolysis method provides possibilities for the construction of multifunctional

electrochemical probes when using multi-barrel capillaries as the template for the electrode

fabrication.[99] Before pyrolysis one of the two barrels is blocked to exclude the carbon gas from

one barrel while conductive carbon is exclusively deposited in the open one. The resulting

probes find applications in high-resolution electrochemical and topographical imaging by means

of SECM in combination with SICM. Accordingly, also dual probes with two individually

addressable carbon nanoelectrodes[103] or even probes with quadruple functionality can be

made by using capillaries with multiple barrels and blocking the desired number of channels to

perform carbon pyrolysis in the remaining ones.[104]

Unlike the two previously discussed families of nanoelectrode fabrication procedures which are

both top-down approaches of nanofabrication, pyrolytic filling of capillaries is a combination of a

top-down and a bottom-up approach. A macroscopic capillary is manipulated to form a template

with nanometric dimensions but then atomic building blocks (gas molecules) form larger

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structures to fill the capillary with the actual electrode material. This makes the resulting

electrode material difficult to characterize concerning its composition, electronic structure and

other physicochemical properties, especially because most spectroscopic tools for the analysis of

surfaces fail at such nanometric dimensions. It is commonly known that the kinetics of

heterogeneous electron transfer at carbon electrodes strongly depend on the extent of

graphitization, orientation of the graphitic lattice and the termination with functional groups.

Raman spectroscopy is the method of choice to assess the nature of carbon surfaces as it

distinguishes between the basal plane and edge plane of graphitic carbon and allows to estimate

the content of defects in the sp2 lattice. However, a sufficiently large surface area is necessary to

acquire Raman information of the carbon material. McNally and Wong addressed this issue by

fabricating relatively large micrometric carbon cone electrodes using similar parameters for the

fabrication as would be necessary to fabricate smaller disk-shaped carbon nanoelectrodes

(pyrolysis of acetylene in nitrogen atmosphere in a Bunsen burner flame).[105] Their Raman

spectroscopic investigation lead to the conclusion that the material contains only little defects

and that mainly the basal plane of graphite is exposed, making the material somewhat similar to

highly oriented pyrolytic graphite (HOPG). However, the electrochemical reversibility of

different redox species contradicted this observation pointing rather towards structural

similarity with hydrogenated glassy carbon (GC).

A different, however conceptionally similar method to fabricate carbon probes with nanometric

tips is the deposition of carbon inside pulled capillaries by means of chemical vapor deposition

(CVD). After filling the pulled pipette with a dilute solution of CVD catalyst and allowing to dry,

the carbon is deposited in a furnace at temperatures above 800 °C while in a methane/argon

flow. The resulting electrodes are hollow carbon pipettes whose outside glass insulator can be

etched chemically to expose the outside of the carbon to the solution.[106] By varying the

deposition temperature, gas mixture and geometric properties of the pipettes, the CVD method

offers fine tuning of the final carbon pipettes including the extent of graphitization of the carbon

material as investigated by Raman spectroscopy.[107,108] The carbon pipettes have been used for

injection of chemicals[109] into living cells and for intracellular potential measurements[110]. For

highly localized electrochemical measurements they are limited to rather specific applications

due to their hollow shape.[111]

2.2.1.4 Other nanoelectrode fabrication protocols

Very small electrodes are obtained by attaching a single carbon nanotube[112,113] or metal

nanowire[114] to a macroscopic holder to contact and handle the nanometric object. These probes

have extremely small diameters which qualifies them as excellent tools for measurements in

microdroplets and living cells. Another strategy is to coat pulled nanopipettes with a conductive

film by sputter deposition of metals.[115,116] Similar metallic coatings were reported on

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nanometric optical fibers.[117–119] After insulating the sheath with non-conducting polymer films,

the electrodes are typically ring-shaped and surrounding the open orifice of the capillary or

aperture of the optical fiber, which gives the possibility to couple the electrochemical

measurements with a complimentary technique such as SICM or optical microscopy techniques.

For combined electrochemical and AFM imaging, many studies use modified AFM cantilevers.

The active electrode can serve as the AFM tip itself or is separated from the AFM tip. Cantilevers

with the nanoelectrode fused in the tip were first fabricated from a kinked Pt wire that was

electrochemically sharpened, then pressed to form the cantilever and then insulated.[120] Later,

single carbon nanotubes were attached to AFM cantilevers and insulated to act as the electrode

tip.[121,122] In a different fabrication strategy, commercial pyramidal AFM tips were modified by

metal deposition followed by insulation. Then, by means of Focused Ion Beam (FIB) milling, a

smaller tip was cut out, leaving a rectangular metal electrode at the base of the AFM tip.[123,124]

2.2.2 Special precautions and considerations for studies at nanoelectrodes

2.2.2.1 Theory of electrochemistry at the nanoscale

Nanoelectrodes commonly find applications in quantitative investigation of the kinetics of

heterogeneous electron transfer reactions. Also, the diffusion-limited currents for conversion of

soluble redox mediators are used to determine the active electrode size based on models

describing the diffusion layer. However, a number of theoretical and experimental studies show

that the classical theory developed to describe the electrochemical behavior of macroscopic

electrodes or even microelectrodes predicts false results. In laboratory practice, the size of the

electroactive area of electrodes with rotational symmetry is normally estimated using the

equation for the steady-state diffusion-limited current at a microelectrode, Iss = AnFDcr, with Iss

the current, A a factor to describe different electrode geometries, n the number of transferred

electrons, F the Faraday constant, D the diffusivity of the redox probe, c its bulk concentration

and r, the radius of the electroactive disk. By comparing the calculated electrode radii from the

limiting current for [Ru(NH3)6]3+ reduction to the geometrical radii as measured by Scanning

Electron Microscopy (SEM), Agyekum et al. observed a systematic underestimation of the

electrode size based on the limiting currents.[125] The deviation was observed for electrodes with

geometrical radii smaller than 50 nm and became more pronounced with decreasing electrode

size. A number of effects have to be considered for the quantitative analysis of electrochemical

phenomena at the nanoscale. Most of these effects are derived from the fact that the thickness of

the diffusion layer (also often termed depletion layer or concentration distribution layer)

linearly decreases with decreasing electrode size. While in classical theory the diffusion layer

and the electrochemical double layer are treated separately from each other, this separation

may no longer be justified for treatment of nanometric electrodes. As the electrode dimensions

decrease, the electrochemical double layer occupies an increasingly large fraction of the

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diffusion layer and electroactive species start to interact with the interfacial electric field. This

effect is referred to as the electrochemical double layer (EDL) effect. In the presence of an excess

of supporting electrolyte, when the Debye length is short, this effect can be neglected for

electrodes larger than 10 nm.[126] With smaller electrodes or in the absence of supporting

electrolyte however the mass transfer rates may be increased or decreased by the electric field

in the electrochemical double layer, depending on the charge of the electroactive species with

respect to the electrode surface.[127] He et al. proposed a holistic model for a dynamic

electrochemical double layer that takes into account that the concentration distribution of

species in the diffusion layer is coupled with the structure of the electrochemical double layer

and that the latter itself is dependent on the reaction rate.[76,128] For instance, for the reduction of

[Ru(NH3)6]3+ the dynamic EDL model predicts slightly larger limiting currents than expected

from the classical theory which contradicts the experimental results obtained by Agyekum et al.

who found smaller currents compared to the expectation from the geometric electrode size.

Also the size-dependence of electron transfer rates have been discussed controversially. Since

the early applications of nanoelectrodes to measure the rate constants conducted by Penner et

al. [80] who reported excessively fast electron transfer at electrodes of small dimensions, various

groups have published results contradicting these findings.[92,129] Liu et al. stated that electrode

kinetics are not adequately described by a single value for the heterogeneous electron transfer

rate for electrodes in the low nm regime. Instead, the rate constants vary radially on the small

electrode surface because of an extended range of electron tunneling.[130] According to the model

taking into account these edge effects the predicted voltammetric response for a reduction of

cations seems more reversible (i.e. faster electron transfer with respect to the mass transport

rate) than predicted by conventional voltammetric theory. It has to be kept in mind that electron

transfer reactions at the nanoscale must be considered as stochastic events and hence are

temporally varying so that the concept of a rate constant may become useless.[131,132] As a

consequence of this, García-Morales and Krischer stated that the electrode potential itself

fluctuates, which lead them to the conclusion that electron transfer rates at nanoelectrodes must

be a priori faster than the ones observed at larger electrodes.

For electrodes having the size of only a few atoms it is the experimental uncertainty regarding

the electrode properties that makes it difficult to validate or reject proposed theoretical models.

Often “apparent” or “effective” electrode sizes have to be assumed for small electrodes to

account for the uncertainties concerning their geometry.[90] However, there seems to be general

consent that predictions and interpretation of results made by classical theory lead to fairly

precise results for all but the very smallest electrodes i.e. for r > 10 nm.[126,133,134] Generally,

despite great progress has been made both in the fabrication and handling of nanoelectrodes as

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well as concerning the theoretical prediction of their electrochemical behavior there is still an

existing gap between the experimental and the theoretical approach.

2.2.2.2 Technical and instrumental aspects

Knowledge of the electrode geometry is indispensable for the correct interpretation of

experimental results. Except estimating geometrical parameters by electrochemical methods,

SEM and Transmission Electron Microscopy (TEM) are the common tools for characterization of

electrodes. SEM fails to provide good images of electrodes whose critical features are smaller

than about 20-50 nm. TEM has been shown to be a powerful tool to measure electrode radii of

down to 1 nm,[94,135,136] however has no topographical resolution. Unfortunately, small deviations

from the expected electrode geometry are detrimental for the correct interpretation of

experimental results. For instance, it is most difficult to identify slightly recessed or protruding

electrodes. When looking at the voltammetric response of nanoelectrodes, the limiting currents

are diminished if the active electrode is recessed in the insulating sheath on account of the extra

distance to the electrode surface that has to be passed by the electroactive species by slow one-

directional diffusion.[90] To identify this recession of the electrode only by means of voltammetry

is impossible and consequently, the active electrode size is underestimated. In case of a

protruding electrode, the electrode radius is overestimated. SECM gives additional possibilities

to evaluate the electrode geometry. Mirkin’s group proposed AFM as an additional

characterization tool that allows good topographical resolution and hence to identify recessed or

protruding electrodes.[137]

Even after using elaborate fabrication techniques and thorough polishing procedures,

nanoelectrodes are very likely to change size, shape and state of the surface during handling,

storage and electrochemical experiments. Amemiya’s group observed severe damages to Pt

nanoelectrodes caused by electrostatic discharges (ESD) when the experimenter touches the

electrodes.[138] The electric charges accumulated in the human body are transferred to the

electrode and lead to extreme electric fields at the nanoscale tip which cause the electrode tip to

melt when a discharge with a nearby object occurs. As a countermeasure, discharge protection

gear such as conductive wrist straps or shoes and ESD-safe lab coats, gloves and tweezers in

combination with grounded conductive mats can be used. Another issue arises from the use of

certain potentiostats which transmit a current peak to the electrode when the cell is switched

on. The rapid charging may also lead to substantial electrode disruption.[138] To avoid these

glitches voltammetric amplifiers working in the 2-electrode system or patch clamp amplifiers

are often used.

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2.3 High-Resolution Electrochemical Imaging 16

2.3 High-Resolution Electrochemical Imaging

For an electrochemical reaction at an electrode surface, the diffusion layer scales with the size of

the electrode. When a micro- or nanoelectrode is positioned in close proximity to a sample

surface so that their diffusion layers overlap, highly resolved maps of local electrochemical

activity are obtained when scanning the micrometric probe over the surface. SECM is the only

scanning probe technique that yields truly chemical information about the samples in

question.[8–10] In the most basic operational mode, the feedback mode, a redox mediator in

solution is oxidized or reduced at the microscopic SECM tip electrode under mass transport-

limited conditions. When the sample surface reaches into the diffusion layer of the SECM tip, the

current recorded at the tip is altered. For an electrochemically inactive sample, diffusional

access to the microelectrode is blocked (referred to as negative feedback) whereas an

electrochemically active sample permits recycling of the redox mediator between the SECM tip

and the sample. This leads to an increase of tip current as the two diffusion layers overlap

(positive feedback). Nanoelectrodes are necessary to achieve high-resolution electrochemical

images.

The precise control of the tip-to-sample distance still continues to be a challenge for

implementation of small nanoelectrodes into high-resolution SECM. Using the electrochemical

tip current to control or measure the tip-to-sample distance is problematic because the

information regarding sample topography and electrochemical activity of the sample are

convoluted. True distance control independent from the local electrochemical activity is

achieved by coupling other techniques such as AFM, shear force detection [96,139,140] or SICM to

SECM.[8,9] These strategies require nanoelectrodes with an additional functionality which is

exploited for the topography measurement (as discussed further below).

Carbon nanoelectrodes made by carbon deposition inside nanopipettes exhibiting radii down to

some 10 nm were used to obtain high-resolution images of various cell types including hair cells.

These cells are very difficult to image because of their high aspect ratio.[100] The key for obtaining

such highly resolved images lies in the use of high aspect ratio probes with a thin insulating

layer. Despite the difficulties in positioning electrodes in the lower nm size regime, Mirkin and

coworkers recently reported the imaging of individual catalytically active Au nanoparticles using

nanoelectrodes of down to 3 nm radius in the basic feedback mode of SECM.[141] In addition, they

used the nanoelectrode to detect H2 electrogenerated from the hydrogen evolution reaction

(HER) at the nanoparticles.

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a

c

b

Figure 4. High-resolution and topographical imaging using carbon nanoelectrodes in SECM. SECM images combined with SICM imaging for distance control using double-barrel bifunctional probes to image a Pt band electrode array and living neurons (a). Deposition of pH-sensitive iridium oxide on double-barrel carbon probes affords pH mapping of a calcite crystal in combination with SICM imaging (b). The electrocatalytic production of hydrogen peroxide during the oxygen reduction reaction at Au nanoparticles is visualized by means of SECM-SICM with very small nanoprobes (c). a: Reprinted from [99] with permission from John Wiley and Sons. b: Adapted with permission from [142]. Copyright (2013) American Chemical Society. c: Reproduced from [143] with permission of The Royal Society of Chemistry.

Recently, SICM[144] has gained attention in combination with SECM for combined topographical

and electrochemical imaging. In SICM an open nanopipette is filled with electrolyte solution and

contacted typically with a Ag/AgCl wire inserted from the back. Upon applying potential

between the pipette and an external electrode, ionic current is passed through the capillary

opening. The passage of current is obstructed when the pipette is approached to a sample

surface. The current decrease is used as the signal to record the sample topography. Bifunctional

nanoelectrodes made by pyrolytic decomposition of carbon in Θ-shaped double barrel

capillaries[99] have simplified the probe fabrication which facilitated high-resolution

electrochemical imaging. For combined SICM-SECM one of the two barrels is blocked and carbon

is deposited exclusively in the other barrel. The open capillary is used for SICM distance control

while the carbon-filled electrode serves for local electrochemical measurements. Topographical

and electrochemical imaging with high spatial resolution using SICM-SECM was demonstrated at

various substrates (Figure 4). The electrochemical properties of the carbon electrode can be

altered by deposition of small amounts of platinum to increase the sensitivity for reactions that

are kinetically hindered on carbon surfaces.[145] Deposition of iridium oxide on the carbon side

has been used to create a small pH sensor for combined topographical and pH mapping of small

structures (Figure 4 b).[142]

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While the initial studies were rather dedicated to the investigation of biological samples,

O’Connell and Wain used SICM-SECM for electrocatalyst studies.[143,146] Due to the high

resolution and precise distance control they were able to resolve individual Pt nanoparticles and

investigate their consumption of oxygen during the ORR. Both the nanoelectrode and the

substrate modified with a small concentration of nanoparticles were polarized to reduce O2 and

thus compete for the free reactant in solution.[147] They also visualized the diffusion layers of

hydrogen peroxide generated during the incomplete reduction of O2 on individual gold

nanoparticles (Figure 4 c).[143] The platinized carbon probe was polarized at a potential to collect

generated H2O2 by anodic detection. By changing the potential applied to the nanoparticle-

modified substrate, different amounts of H2O2 were detected from the single Au particles.

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2.4 Cell Analysis 19

2.4 Cell Analysis

2.4.1 Detection of reactive oxygen species

Reactive oxygen species are radicals or otherwise unstable molecules generated during cell

metabolism. Their main source is the incomplete reduction of dioxygen to form superoxide O2∙-

which then further reacts to H2O2 via decomposition catalyzed by superoxide dismutase (SOD)

(Figure 5). Also external sources such as UV irradiation or toxic chemicals can cause ROS

production. H2O2 is the precursor for the highly reactive hydroxyl radical ∙OH. In the presence of

Fe2+ hydrogen peroxide undergoes Fenton reaction to form ∙OH. The hydroxyl radical can extract

hydrogen from any organic compound. Thus, an overproduction or imbalance of ROS damages

parts of the cell and leads to irreversible chemical modifications of lipids, proteins and

DNA.[148,149] Oxidative cell stress can be the cause for cell ageing,[150] inflammation,[151]

neurodegeneration,[152] pain,[153] heart failure[154] and cancer.[155] Apart from their detrimental

effect on cell viability, ROS also play a role in signaling and defense against pathogens.[156] For

instance macrophage cells, an essential part of the immune system, kill bacteria by engulfing

them into phagosomes and attack them by excess amounts of O2- and NO which are released into

the phagosomes by activity of the NADPH-dependent oxidase (NOX) and nitric oxide synthase

(NOS), respectively.[157] The controlled release of ROS requires antioxidative protection

mechanisms. Apart from SOD these include the enzymes catalase and gluthathione peroxidase

which neutralize H2O2. Also other oxidizable substances such as ascorbic acid, glutathione itself

and other antioxidants scavenge ROS.[150] The generation of ROS is closely related to the

generation of reactive nitrogen species (RNS), the most important of which are nitric oxide

(∙NO), peroxynitrite (ONNO∙) and nitrite (NO2-) (Figure 5). Analytical information regarding the

production of reactive oxygen species from single cells can help to understand the development

of various pathogenic conditions. Hence, micro- and nanoelectrodes are of particular interest as

analytical tools in the context of oxidative cell stress.[77] The reliable extracellular and

intracellular detection and quantification of ROS is crucial to evaluate the pathogenic effect of

oxidative stress on cells and thus has been subject to extensive research.[7,77,156,158,159] The

detection of ROS is a difficult analytical task. Due to their very unstable nature many ROS have a

short lifetime in the cell and their abundance is limited to small volumes in or around the cell.

Hence, analytical tools with high sensitivity, capabilities for real-time detection and high spatial

resolution are required. In addition, to describe the effects of particular members of the ROS

family on the cells viability, selective measurements are desirable.

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Figure 5. Generation and evolution of ROS and RNS species. Scheme modified from [77] and [160].

A number of detection strategies based on various principles has been proposed. For instance,

the O2∙- production was quantified photometrically by reduction of cytochrome c which changes

its optical absorbance when being reduced. Chemiluminescence upon reduction of lucigenin by

superoxide is also commonly used to detect O2∙-. Electron spin resonance (ESR) detects radical

species by absorption of microwave energy, which occurs on transition between the energetic

states of unpaired electrons in an applied magnetic field. To increase the lifetime of the radical

species and thus the sensitivity, specific compounds are used as spin traps. The method is very

sensitive and specific to particular radicals but requires large technical efforts.[158] Methods

based on fluorescence are more common due to the lower cost of instruments and ease of use.

The two most common dyes for the detection of ROS are 2′,7′-dichlorofluorescein diacetate

(DCFH-DA) as well as 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red). DCFH-DA forms a

fluorescent product with various ROS which is detected with high sensitivity.[148,149,161,162] Only

extracellular H2O2 is detected when Amplex® Red is oxidized to form resorufin. The lack of

chemoselectivity of the classical fluorescent ROS probes has led to the development of various

novel strategies. For all fluorescent dyes for ROS detection it is important to minimize the

chemical interactions with other constituents of the complex cellular matrix. Novel fluorescent

probes based on boronate-modified fluorescent dyes[163,164] and specifically modified

nanomaterials[165–167] have been proposed. In addition, cells have been genetically engineered to

express a protein conjugate that acts as a fluorescent probe to detect H2O2 without adding any

supplementary dyes.[168] In general, methods based on the detection of fluorescent products

upon reaction of ROS with the corresponding dyes are very sensitive and offer a good spatial

resolution in the fluorescence microscope. For instance, ROS production in small organelles such

as phagosomes or mitochondria can be visualized (Figure 6 a).[164] However, the methods face

some drawbacks such as toxicity and photobleaching of the dyes. Most importantly, the reaction

between the ROS species in question and the dyes are irreversible so that the analytical signal is

measured as an accumulated ROS response over a long time. This hampers the use of these

techniques for the real-time monitoring of ROS production.

Hydrogen peroxide has a crucial biological role, not only in the context of oxidative stress. It was

shown that a low level of H2O2 is necessary to maintain cell viability and proliferation.[150,169]

Depletion of H2O2 as well as excessive levels during oxidative stress lead to a halt of cell growth

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or to cell death. Several studies show that H2O2 is involved in cellular signaling and some

oxidative biosynthesis pathways. Thus, precise quantification of H2O2 levels in cells with high

spatial and temporal resolution is of great importance to understand its physiological role.

However, its non-radical nature precludes some of the aforementioned detection schemes and

generally a lack of specificity is encountered for many analytical methods. Compared to other

ROS species H2O2 has a relatively long lifetime and can diffuse to every compartment in the cell

or out of the cell. Yet, H2O2 is the precursor of the most detrimental of ROS, the hydroxyl radical

∙OH. Amperometric electrochemical detection allows continuous monitoring of the H2O2

generation in biological samples.[7,170] Various H2O2 sensor designs based on microelectrodes

have been proposed and some have been successfully applied for in vivo measurements.[171] The

simplest amperometric detection is achieved at umodified carbon fiber microelectrodes

electrodes using fast scan voltammetry. By sweeping the potential very fast and detecting anodic

peaks corresponding to H2O2 oxidation, high temporal resolution was achieved for

measurements in living brain slices.[172] However, due to the high potentials that need to be

applied, the method lacks selectivity.

a

b

Figure 6. Detection of ROS from single cells using electrochemical and non-electrochemical techniques. Boronate-modified dyes selectively reveal the H2O2 production via fluorescence detection (a). Release of ROS from cells is detected using carbon microfiber electrodes (b). a: Adapted with permission from [164]. Copyright (2011) American Chemical Society. b: Reprinted with permission from [173]. Copyright (2008) John Wiley and Sons.

To overcome this problem, Amatore’s group demonstrated the deconvolution of current signals

from the oxidation of H2O2, ONOO∙, ∙NO and NO2- at platinized carbon fiber microelectrodes

(Figure 6 b). All four compounds are oxidized at different potentials. The current is monitored at

four different potentials, each potential being slightly above the detection potential of one of the

species, so that the contribution of each species to the overall current signal can be calculated.

This principle was applied for various single cell analyses, including the response of macrophage

cells to mechanical stress[174] and immuno-stimulation.[173,175] The sum of all oxidizable

intracellular substances was evaluated by inserting Pt nanoelectrodes into macrophage cells.[176]

The authors reported oxidative bursts upon stimulation by a second micropipette probe. Despite

their small active electrode area, the electrodes had a total diameter of nearly 1 µm and thus the

authors described the mechanical disturbance caused by the electrochemical probe itself.

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More selective detection, however at the cost of more time-consuming sensor preparation, is

achieved by immobilizing specific peroxidase enzymes on the microelectrode surface.

Cytochrome c contains a heme group and reduces the overpotential for the reduction of H2O2.

Cytochrome c was immobilized on gold microelectrodes[177] and ZnO nanosheets.[178] The latter

allowed the extracellular detection of H2O2 released from living human hepatoma (kidney

cancer) cells. Similarly, electrodes modified with horseradish peroxidase (HRP) were applied for

monitoring hydrogen peroxide release from isolated mitochondria[179] and extracellular H2O2 in

the brain.[171] Interferences by other oxidizable substances in complex biological environments

are critical to the selectivity of ROS sensors. For instance, ascorbic acid and uric acid are the

interferents that are most likely to be co-oxidized at the sensors. To exclude access of these

compounds to the electrode, selective membranes are used. Platin microelectrodes are

themselves sensitive to many oxidizable or reducable species but membranes, for instance made

from poly-(o-phenylenediamine), largely reduced interferences.[180] Such membrane-coated Pt

microsensors were used for in vivo detection in leukocyte cultures[181] and oilseed rape leaves

after pathogen infection.[180]

Instead of enzymes with peroxidase activity, the “artificial peroxidase” Prussian Blue (PB) was

used to create selective H2O2 sensors.[170,182] The inorganic hexacyanoferrate salt can catalyze the

electrochemical oxidation as well as the reduction of hydrogen peroxide, whereas cathodic

detection is more selective to H2O2. Microelectrodes were modified with PB and applied in SECM

for H2O2[183] and glucose [184] mapping. The latter study made use of glucose oxidase which

generates hydrogen peroxide in the presence of glucose and oxygen. Salazar et al. showed the

successful application of a PB-modified microelectrode for in vivo monitoring of the glucose

concentration in the prefrontal cortex of anesthetized rats.[185]

2.4.2 Electrochemical single-cell analysis

Electroanalytical techniques using nanoelectrodes allow measurements in small and confined

sample volumes where other analytical methods often fail either because of a lack of sensitivity

or because of a lack of spatial resolution. A nanometric electrochemical sensor senses the

concentration of species within its diffusion layer. The dimensions of the diffusion layer scale

with the size of the electrode, thus ensuring high spatial resolution of the measurement. If

additionally the overall dimensions of the nanometric sensors are sufficiently small, the

electrodes can be positioned for highly localized measurements in small chemical

microenvironments. Needle-type nanoelectrodes are ideal tools to bridge the gap between the

microscopic sample and the macroscopic analytical environment. They have a macroscopic

“handle” that allows manipulation and positioning by the experimenter as well as easy

interfacing to the electronic measurement apparatus and an electrode shaft that tapers to

2 State of the Art


microscopic dimensions on the opposite end. These capabilities are most called for in the

chemical analysis of biological samples, tissues and single cells. In fact, the development of

nanoelectrode fabrication protocols was to a large extent driven by the search for answers to

biological questions. Compared to the ubiquitous optical methods in the life sciences,

electrochemical detection of substances with biological relevance have a direct correlation

between the measured signal and the amount of analyte via Faraday’s law of electrolysis. In

particular, this applies to the quantification of neurotransmitters, most of which are

electroactive substances. Discrete neurotransmitter release events are detected when a

microelectrode is positioned close to a cell and the content of all incident neurotransmitter

vesicles is electrochemically converted at the electrode.[186] From the charge and shape of the

resulting current spikes size, shape and behavior of the neurotransmitter-containing membrane

vesicles are characterized.[187] This way, electrochemistry contributed tremendously to the

understanding of intercellular communication between cells. Moreover, by in vivo

voltammetry,[188,189] where microelectrodes are placed in the intact brain of living animals, the

functionality of the complex central nervous system can be correlated to the animal’s behavior

and external stimuli that act on the animal. In these applications, neurotransmitters are

normally detected outside the cell and hence, electrodes with dimensions similar to the cell’s

dimensions (i.e. a few µm) are desirable to assure a high efficiency of collection of

neurotransmitters. Similarly, ROS release from single cells was detected at microelectrodes

(Figure 6 b).

A sensor small enough (4 µm) for the intracellular detection of oxygen was already reported in

1967.[190] Much later, inspired by the great success of patch clamping techniques, where

micropipettes are used to study transport processes in biological membranes, researchers

sought for more ways to explore the intracellular space. Ewing’s group and others made

substantial progress to the field by showing the feasibility of intracellular detection of O2,[191]

neurotransmitters,[192,193] glucose[194] and nitric oxide.[195] However, the diameter of the

employed electrochemical probes was rather in the micrometer range. For the feasibility studies

often cell models exhibiting particularly large cell bodies were chosen. As electroanalytical

techniques for the analysis of cells are trying to emerge towards real biomedical problems, the

dimensions of the probes need to be reduced to avoid damages caused to the cell when sensors

are inserted. Another often neglected challenge is to minimize the influence of the

electrochemical conversion of the analyte for detection at the micro/nanosensor on the

physiological state of the cell. For amperometric sensors it needs to be taken into account that

the electrode used for detection of the species in question is a consumer or producer of

electroactive species itself. These invasive effects of the detecting probe can be minimized by

reducing the dimensions of the probe with respect to the size of the studied objects, so that the

2 State of the Art


electrochemical turnover at the probe is negligible. The influence of the tip reaction was

investigated systematically at cells secreting the redox mediator menadione due to the activity

of NAD(P)H oxidizing enzymes inside the cells.[196] When detecting the secreted mediator at a

SECM tip, only nanoelectrodes of 200 nm or smaller could visualize the diffusion layer of the

secreted mediator without disturbance by the tip reaction itself. In contrast, larger probes

actively dragged out the mediator from the cell and thus made the diffusion layer appear larger

than its actual dimensions. Thus, nanoelectrodes are ideal tools for the detection of species

released from cells or detection of intracellular substances. Thus, it is the quest to make the

detecting electrode an “innocent spectator” that motivates the development of smaller

electrochemical sensors. Electrochemical methods for the analysis of single cells where surveyed

in 2007[197] and it was stated that nanoelectrodes had found no or only little application in the

investigation of biological phenomena up to that date. Even today only a limited number of

studies involving nanoelectrodes for the analysis of cells has been published which might be due

to the previously existing difficulties in fabrication and handling of nanoelectrodes and the high

technical requirements.

a

b

Figure 7. Sensitive chemical detection by inserting nanoelectrodes into the intracellular space of living cells or the synaptic cleft. In PC 12 cells discrete catecholamine vesicles are detected inside the cell and show a higher catecholamine amount (red) than extracellular vesicles released during exocytosis (black) (a). With a nanoelectrode inserted into a synapse the signals for the oxidation of neurotransmitter vesicles shows distinct and more complex current patterns compared to detection outside of the synapse. (b) a: Adapted with permission from [88]. Copyright (2015) John Wiley and Sons. b: Reprinted with permission from [89]. Copyright (2014) John Wiley and Sons.

Nanoelectrodes have been employed for the intracellular detection of neurotransmitters. With a

flame etched carbon fiber nanoelectrode inserted into the cytosol, vesicle collisions with the

electrode were detected from inside the cell. Interestingly, the vesicles were larger and

contained significantly more catecholamine molecules than the ones detected outside of the cell

2 State of the Art


(Figure 7 a).[88] This suggests that during exocytosis, the content of vesicles is not completely

released into the exterior but that the vesicles only fuse partially with the external membrane

and then pull back into the cytosol. For the extracellular detection nanoelectrodes can be

positioned close to different parts of the cell, allowing subcellular resolution in assessing the

exocytosis rate[198]. The extremely small size and high aspect ratio of a flame etched fiber

nanoelectrode allowed even to slide the electrode into the synaptic cleft and thus reside where

the highest exocytotic activity is expected to be (Figure 7 b).[89] Indeed, in the confined space the

frequency of release events was higher as compared to the situation when the nanoelectrode

was placed over the axon. Moreover, a large fraction of the events had a more complex current

pattern that corresponds to a series of exocytotic events within short time. This suggested that a

previously unrecognized exocytosis mechanism may act in the synapse. Apart from detection of

neurotransmitters in the synapse and intracellular detection, nanoelectrodes were also used for

in vivo voltammetry in the central nervous system of Drosophila melanogaster larvae, a structure

that is itself only a few micrometers small.[199]

The method of electrode fabrication by pyrolysis of carbon inside nanopipettes offers the

possibility to build multifunctional probes. To perform electrochemical measurements in

microdroplets, these probes can be modified to contain both electrodes of a 2-electrode circuit

so that a bulky external counter/reference electrode is not necessary. One barrel of a Θ-capillary

was filled with carbon which then acted as the sensor (i.e. the working electrode) whereas the

other barrel was filled with electrolyte solution and a Ag/AgCl wire was inserted.[200] This

electrode served as the internal counter/reference electrode. The utility of this probe was

demonstrated in measurements in microscopic wells containing only a few HeLa cells. The cells

were identified and quantified by their alkaline phosphatase activity which leads to the

formation of an electroactive product that is detected at the nanoelectrode.

First demonstrations of the electrochemical detection of ROS in or around single cells using

nanoelectrodes are promising. Zheng et al. reported on a PB-modified optical fiber nanoprobe

for combined electrochemical detection of H2O2 and optical detection of the overall oxidative

response at single cells.[117] The authors used this probe for extracellular measurements at

cancer cell lines and detected differences in the behavior with respect to the generation of H2O2

and other ROS. However, inconsistencies in the characterization of the sensors leave doubts

about its actual size. The current observed for the reduction of H2O2 at a given concentration is

nearly 100 times larger than expected for the maximum possible current according to equation

(1). This suggests that the sensor was actually much larger than reported by the authors.

Moreover, inserting nanoelectrodes into cells offers the possibility to detect ROS and RNS close

to the location of their generation. Mirkin’s and Amatore’s group used Pt nanoelectrodes to

penetrate the cell membrane and detect intracellular oxidative outbursts.[176,201] The anodic

2 State of the Art


signal occurring upon cell penetration was composed of various contributions from the

oxidation of RNS/ROS. Yet, despite the radii of the active electrodes was only tens of nm, the

overall diameter of the probes approached 1 µm, leading to considerable disruption of the cell’s

natural function in the moment of and after penetration. When using smaller probes, such

disturbance is largely avoided.[108,113]

2 State of the Art

2.5 Field Effect Transistor Sensors 27

2.5 Field Effect Transistor Sensors

The weak amperometric signal generated by nanometric electrodes at small analyte

concentrations is limited by the electronic capabilities of commercial current amplifiers.

Potentiometric sensing schemes can overcome this sensitivity limit because already a small

number of molecules is sufficient to change the electric potential at a nanometric interface

significantly.[202] Field effect transistor (FET) sensors go even one step beyond. FET sensors

show superior sensitivity towards chemical detection because the change of electric potential

caused by the interaction between an analyte and the sensitive channel of the FET is intrinsically

amplified. This is achieved by measuring a current passing between two electrodes that are

connected by a sensitive transistor channel (Figure 8). The two contacts are generally referred

to as drain and source. As in a classical metal oxide semiconductor field effect transistor

(MOSFET), the electric field at the transistor is controlled via an external gate electrode. For

chemical FET sensors, the transistor channel is in contact with the gate electrode via the

electrolyte solution. By applying an external voltage VG between the gate electrode and the

semiconducting transistor channel, the passage of current through the channel can be enabled or

blocked to open or close the transistor. Additional to the external VG, the potential at the

transistor channel is determined by its chemical environment. As an analyte specifically

interacts with the semiconducting transistor channel, the resulting potential shift induces a

change of the current IDS that passes through the channel between the drain and source

electrodes. This current, driven by an applied voltage between the two electrodes VDS, serves as

the analytical signal for the FET sensor.

Figure 8. Schematic of a field effect transistor sensor. The interfacial electric potential imposed to the transistor channel by interaction with an analyte (green spheres) changes the effectively applied gate voltage.

Despite the transistor channel can have small nanometric dimensions, IDS is generally a large and

thus an easy-to-measure current signal. The interaction of the analyte with the transistor

channel is a surface phenomenon, while current is passed through the bulk of the channel. Thus,

2 State of the Art


the sensitivity of the FET sensor increases with decreasing thickness of the transistor channel.

Nanowires and nanosheets from various materials such as silicon,[203–205,205–210] metal oxides,[211]

conducting polymers,[212–217] carbon nanotubes (CNT),[218,219] graphene[220–222] or composite

materials[223] have been exploited as transistor channels to create highly sensitive FET sensors.

To provide specificity of detection to the FET sensors, the transistor channel can be modified

with recognition elements that selectively interact with one particular analyte. There are

numerous examples for specifically modified FET sensors. For instance, DNA aptamers were

immobilized on carbon nanotubes comprising the transistor channel which then recognized

certain proteins.[219] Sensitive detection of DNA binding was achieved with peptide nucleic acid

(PNA) capture probes on FET sensors.[210] Cui et al. used Si nanowires modified with pH-

sensitive groups and proteins for the selective detection of protons, protein-protein interactions

and Ca2+.[203] Similarly, olfactory receptors were attached to a graphene transistor to comprise an

artificial bioelectronic nose.[222]

FET sensors are generally assembled on chip-like structures exhibiting relatively large (several

micrometer) electrode dimensions (Figure 8). The large electrodes are in contact with the

electrolyte solution and thus need to be insulated to reduce electrochemical noise. The electrode

dimensions have limited their ability to perform highly localized measurements in small

volumes, especially in biological samples such as individual cells. The chip-like FET sensors

cannot be easily moved to target single cells in culture. Yet, there are strategies to use FET

sensors to analyze electrical communication of cells and cells’ secretion of chemicals.[224] While

most of them address a population of cells, substantial progress in the design of FET devices has

been made to use these sensors to target single cells. The first reports about the electric

communication between cells and FET devices used flat, chip-like sensors with a micrometric

transistor channel to which single cells could adhere (Figure 9 a).[225] The fast response time of

the FET allowed to measure single extracellular action potentials. To extend the scope of FET

sensors to intracellular measurements Duan et al. installed a high-aspect ratio needle-like

branch on a flat Si FET (Figure 9 b).[226] If cells settled on these FET devices, the spiky extension

punctured the cell membrane and intracellular action potentials were measured. Further

evolution of FET design lead to kinked Si nanowires.[205] Variations in the reactant pressure

during the synthesis of semiconductor structures produced nanowires with defined kinks. The

two ends of this kinked wire were attached to drain and source electrode that bent upwards

from a flat structure. Because of its sharp kink, this FET sensor was able to penetrate into cells

placed on the device. Only recently, Lieber’s group enhanced the capabilities of kinked wire

probes.[204] The kinked transistor channel was mounted on a narrow chip probe containing the

drain and source contacts. The resulting free-standing nanowire probe was freely movable in

2 State of the Art


space and thus could target arbitrary individual cells and be inserted into the cell membrane

(Figure 9 c).

a

b

c

d

Figure 9. Review of strategies to target individual cells using FET sensors. A cell is placed on a flat micrometric silicon p-n-p FET and electric potential recording are performed after electrical stimulation of the cell. (a) A SiO2 nanotube is installed on a Si nanowire FET channel to penetrate the cell membrane for intracellular potential measurements (b). Kinked Si nanowires are mounted on high-aspect ratio chips to create a freely movable FET for potential measurements in three-dimensional space and intracellular measurements (c). Gold drain and source conctacts are deposited on a nanopipette and by modifying with carbon nanotubes and receptor molecules, intracellular Ca

2+ detection is performed. a: From [225].

Reprinted with permission from AAAS. b: Adapted by permission from Macmillan Publishers Ltd: Nature Nanotechnology [226], copyright (2012). c: Reprinted by permission from Macmillan Publishers Ltd: Nature Nanotechnology [204], copyright (2014). Reprinted with permission from [218]. Copyright (2011) American Chemical Society.

The above mentioned FET sensor technologies were yet restricted to measurement of the

electric potential in or around cells. Son et al. fabricated a FET sensor that is freely movable to

target single cells and perform intracellular detection of chemicals (Figure 9 d).[218] One side of a

glass nanopipette was sputter-coated with gold, followed by a 180° rotation around its long axis

and repeated gold deposition. The resulting probe had two individually addressable gold

contacts on either side of the pipette which were separated by a thin gap where no gold was

deposited. By immersing the pipette into a suspension of CNTs, the gap was bridged by a

2 State of the Art


network of CNTs which comprised the transistor channel. The CNTs were functionalized with a

fluorescent dye that acted as a capturing agent for Ca2+ ions. Using these probes, the authors

were able to perform Ca2+ determination inside single cells.

2 State of the Art

2.6 Nanoparticle Electrochemistry 31

2.6 Nanoparticle Electrochemistry

Nanoparticles and nanostructured materials are ubiquitous in modern chemistry. Especially

they are irreplaceable in catalysis because of their enhanced turnover rate for chemical and

electrochemical conversion reactions. Intensive research was dedicated to the synthesis,[227]

characterization[228] and application of nanoparticles in homogeneous and heterogeneous

catalysis.[229] The development of advanced materials with enhanced catalytic activity requires a

deep understanding of the underlying mechanisms and principles that govern the special

performance of nanostructured materials:[230] a) Adsorption and desorption behavior of

reactants and products during catalytic reactions are altered with respect to bulk materials

because of the high number of under-coordinated surface and edge atoms in nanoparticles. b)

Additionally, nanoparticles can have special electronic properties because the energy of

electrons in the particles is affected by the tight spatial confinement in the small particle

volume.[228] c) The high surface area creates a large interface for chemical reactions and the

small dimensions ensure fast diffusional transport of the reactants and products but also may

change the selectivity of the reaction.[231] The first two effects may have an impact on the kinetic

rates of a catalytic reaction. Compton coined the term “true nano effect” to describe a kinetic

acceleration due to the size of the nanoparticle.[232] For the design of new catalyst materials, the

interplay of these effects has to be understood. However, classical analytical tools for the

analysis of nanoparticles are hindered by the polydispersity of nanoparticle ensembles. Thus,

methods addressing individual particles, rather than particle ensembles have to be developed.

Electrochemical measurements associated with individual particles are performed in the nano-

impact (or nano-collision) method.[233] A microelectrode is used to measure electrochemical

responses when nanoparticles in solution collide with the electrode surface. The first reported

example of this method was a gradual blocking of an otherwise active Au electrode surface when

non-active latex particles settled on the microelectrode.[234] The stochastic events of particle

collisions yield information about the particle size or reaction rates for electrocatalytic

reactions. For instance, the charge transmitted for anodic oxidation of silver nanoparticles[235] or

reduction of Fe3O4 particles[236] correlates to the particle volume and thus allows to determine

the size distribution of particles. A key advantage of all techniques addressing the

electrochemistry at single nanoparticles is the extremely fast mass transport of reactants and

reaction products to and away from the electrode surface which allows to study electrocatalytic

reactions with a minimum influence of mass transport limitations. Moreover, upon intermittent

or permanent contact of particles at the surface, the particles are electrically connected and

electrocatalytic reactions such as the hydrogen evolution reaction[232,237] or oxygen evolution

reaction[238] are driven. Kahk et al. investigated the effect of particle size of Ag particles for the

2 State of the Art


HER.[232] The height of current spikes when particles came in short contact with the electrode

depends on the applied potential and follows Butler-Volmer kinetics. The authors calculated the

heterogeneous electron transfer rates and found a size-dependence for Ag particles but not for

Au particles. However, the transient nature of the nano-impact method prevents the correlation

of particle size and electrochemical activity of the same particle. A single particle cannot be

studied over a long time in steady-state measurements so that either its size or its activity can be

determined but not both. This is especially problematic for particle populations with high

polydispersity.

As an alternative, high-resolution SECM or scanning electrochemical droplets were proposed for

the study of single electrocatalytically active particles.[141,143,146,239,240] These studies show the

feasibility of detecting the products of catalytical conversion and at the same time, the

electrochemical image gives an estimation of the particle size. The production of hydrogen was

detected from Au particles.[141] For the ORR, the oxygen consumption was visualized on Pt

particles[146] as well as the generation of H2O2 during incomplete oxygen reduction on Au

particles.[143] However, due to the technical difficulties, these techniques did not yet afford a

systematic correlation of structural parameters and particle activity.

Nanoelectrodes themselves are very good tools to study the size and catalytic activity without

the technical difficulties associated with positioning and distance control of small electrodes.

Nanoelectrodes can serve as a template for the synthesis of a single particle. If the electrode

dimensions are sufficiently small, controlled electrochemical deposition yields particles with

similar or slightly larger size than the electrode (Figure 10 a).[231,241,242] Alternatively, pre-

synthesized nanoparticles have also been immobilized to the electrode surface using different

chemical surface modification strategies[135,136] or simply physisorption (Figure 10 b, c).[243]

Chen and Kucernak pointed out that when investigating the ORR at individual Pt

nanoparticles[231] the mass transport rates at such small electrodes exceed by far the ones

obtainable in rotating disk electrode (RDE) experiments. Figure 10 b shows the mass transport

rates corresponding to different particle sizes. To achieve the same mass transport rate as found

at an electrode as large as 1 µm one would have to rotate an RDE with a technically impossible

rotation speed of 106 rpm. By first depositing Pt electrochemically on nanoelectrodes and

performing the ORR in acidic medium the authors describe two interesting phenomena.

Whereas macroscopic Pt electrodes are known to reduce oxygen completely via the 4-electron

pathway to water, more hydrogen peroxide via the 2-electron reduction was produced at

smaller particles. This observation is an effect of the enhanced mass transport. For a sequential 2

by 2-electron transfer, the high mass transport rate leads to a quick escape of intermittently

produced H2O2 before further reduction occurs. Hence, the electrocatalyst at too small particle

2 State of the Art


size is less efficient due to the decreased electron yield. Figure 10 b also shows the decreasing

number of transferred electrons n as a function of decreasing particle size. Moreover, the

authors observed an effective deceleration of the electron transfer rate as indicated by higher

Tafel slopes at smaller particles and they attribute this finding to the EDL effect that diminished

the driving force for the reaction within the double layer. A similar phenomenological

observation seems to occur at Au nanoparticles (NP) which were attached to Pt nanoelectrodes

by silane chemistry.[136] From voltammograms at these electrodes the authors extracted a size-

dependent shift of half wave potentials E1/2 and concluded that Au particles with 24 nm had a

higher specific electrocatalytic activity than those with 14 nm. E1/2 is a measure of the

reversibility of the electrode process, i.e. the electron transfer rate with respect to the mass

transport rate. Since also the mass transport rate changes with particle size, the observed shift in

E1/2 is not necessarily equivalent to an increased specific catalytic activity.

a

b

c

d

Figure 10. Single nanoparticles deposited on nanoelectrodes allow non-ensemble electrocatalyst studies at high mass transport rates. A single Pt nanoparticle is deposited on a nanoelectrode (a) and during the oxygen reduction reaction (ORR) at the single particles, the number of transmitted electrons decreases with smaller particle size due to fast removal of intermediate H2O2 (b). The graph also shows the mass transport rate found at the corresponding particle sizes. TEM images are suitable to show the attachment of small gold nanoparticles to platinum (c) and carbon nanoelectrodes (d). a: Reprinted with permission from [241]. Copyright (2003) American Chemical Society. b: Reprinted with permission from [231]. Copyright (2004) American Chemical Society. c: Reprinted with permission from [136]. Copyright (2010) American Chemical Society. d: Reprinted from [135] with permission from John Wiley and Sons.

For elucidating the relation between catalytic activity and size of particles it is necessary to have

a precise method to measure particle sizes. The effective electrode size after attachment of a

2 State of the Art


particle can be estimated by comparing the limiting currents for electrochemical reaction of

redox mediators from voltammograms before and after particle deposition.[135,244] The size is

estimated more precisely by exploiting redox properties or adsorption processes that are

specific to the material of the nanoparticle. For instance, the electric charge consumed for

hydrogen adsorption/desorption on platinum was used to determine the surface area and hence

the size of Pt nanoparticles.[231,241] In a similar way the charge for the formation and reduction of

a gold oxide layer can be used to measure the size of Au nanoparticles.[242,243]

3 Aim of the Work

35

3 Aim of the Work

The properties of materials and organisms are dictated by the complex workings of chemistry at

the micro- and nanoscale. Micro- and nanoelectrodes are effective yet simple means to shape

and understand the microscopic world. They can serve as small tools to trigger microscopic

chemical and physical changes to surfaces or small objects. Simultaneously, they may act as a

spectating probe to obtain analytical information from microscopic entities.

Reducing the dimensions of the electrochemical probes bears enormous potential to study even

smaller systems with higher resolution but comes at the cost of increasing technical difficulty to

fabricate, modify, maintain and use the electrochemical tools. In addition, interpreting analytical

information from these probes requires special attention. The merits of nanoelectrodes have

been recognized previously but it might be those difficulties that have made examples of their

successful applications for real-world analytical problems rare. This work tries to bridge the gap

between expectations and achievements of micro- and nanoelectrochemical methods. The

beneficial properties of micro- and nanoelectrodes – all a result of their small size – are

highlighted and exploited in different applications, ranging from surface patterning over

biosensing to electrocatalysis.

One part of this work addresses the capabilities of micrometric electrodes to act as a shaping

tool to write chemical patterns and create microscopic structures with high fidelity. Here, a

microelectrode guides the current to locally trigger electrochemical reactions on a surface.

Previous approaches have often misjudged the challenge to maintain the chemoselectivity of the

chemical writing when the patterning resolution is increased. Moreover, it is increasingly

difficult to obtain information about the chemical identity of the created modification as well as

the later attachment of molecules. Successful implementation of microscopic electrodes for

surface patterning requires the development of a reliable, electro-addressable surface chemistry

as well as carefully designing devices and parameters for the electrochemical writing process.

High-density microarrays for diagnostic biomolecule screening may emerge from pursuing this

strategy.

Furthermore, this work demonstrates the enormous analytical capabilities of small electrodes

used as sensors. In particular, these qualities are valuable for the chemical analysis of living

biological cells. The tremendous complexity of the cell still poses questions about its

functionality, let alone the manifold interactions between ensembles of cells. To detect separated

chemical processes or species and evaluate their influence on the whole system is a difficult

analytical task which can only be solved by sensors with sufficiently high spatial resolution to

3 Aim of the Work

36

target single cells. Cells communicate by releasing substances into their environment and

respond to released substances. Understanding these signaling pathway mechanisms requires

tools not only to detect biologically relevant substances in relation to cells but also map their

spatial distribution in space and follow the temporal dynamics of their release and uptake.

Moreover, to quantify toxic unstable substances only occurring for a short time inside the cell,

sensors that target the interior of the cell are necessary. All these requirements are met by

electrochemical sensors based on nanoelectrodes. Hence, the aim of this work is to establish

nanoelectrodes as non-invasive sensors for the analysis of single cells, analyte mapping and

intracellular measurements.

However, the small amount of chemical species originating from a single cell is not easy to

detect. In addition, the sensitive nature of cells requires non-invasive techniques. Hence, the

challenges addressed herein are to reduce the sensor dimensions while still ensuring high

sensitivity for the analytical method. Moreover, strategies for specific sensor modifications are

to be developed that allow chemical analysis with high selectivity in complex biological matrices.

In the last part of this work the objects of interest are even smaller. Electrochemical processes

occurring at single nanoparticles are investigated, which demands very sophisticated analytical

techniques. The constant search for materials for efficient conversion and storage of chemical

energy calls for a deep understanding of the factors that govern the special chemical properties

and catalytic activity of nanomaterials. However, methods looking at particle ensembles

complicate the distinction between different geometric and electronic effects that determine

their behavior. Nanoelectrodes may be used as extremely small probes to enter the realm of

nanomaterials. Hence, this work assesses the use of nanoelectrodes as a platform for the

systematic electrochemical characterization of single nanoparticles. The dual role of

nanoelectrodes, both to serve as a template for nanoparticle deposition and simultaneously as a

connection from the nanoparticle to the macroscopic world is expected to be of high importance.

In summary, the aim of the work is to exploit the advantageous properties of microscopic

electrodes as tools for various analytical and technological applications. Their successful

implementation for these critical applications is expected to enhance the scope of micro- and

nanoelectrochemical methods for surface patterning, biosensing and electrocatalysis in general.

4 Results and Discussion

4.1 Electrochemical Surface Patterning 37


4.1 Electrochemical Surface Patterning

Synthesis of TBDMS-HQ diazonium precursors, optimization of global electrode functionalization

and characterization as well as preliminary experiments for SECM patterning were developed and

described in the author’s Master thesis.[245] The scanning droplet cell was constructed by Kirill

Sliozberg, Ruhr-Universität Bochum. Parts of this section were published in ref. [246]: “Clausmeyer,

J.; Henig, J.; Schuhmann, W.; Plumeré, N.; ChemPhysChem 2014, 15, 151.” Experiments concerning

the patterning of nitrophenyl layers were performed by Lutz Stratmann, Ruhr-Universität Bochum.

Parts of the section were published in ref. [247]: “Stratmann, L.*; Clausmeyer, J.*; Schuhmann, W.;

ChemPhysChem 2015, 16, 3477-3482”.

* These authors contributed equally

4.1.1 Global electrode modification with p-hydroquinone layers

An electrochemical patterning strategy for the local activation and selective attachment of

biomolecules requires a well-defined surface chemistry that can be triggered electrochemically.

A layer of p-hydroquinone (HQ) moieties protected by the tert-butyldimethylsilyl (TBDMS)

protecting group meets these criteria (Figure 11 a). Global chemoselective electrochemical

activation (i. e. integral electrode surfaces deprotection) of protected quinones has been

demonstrated previously.[248–250] However, local chemoselective deprotection via

electrochemical methods was not reported due to the high chemical sensitivity of the quinone

groups. Moreover, redox-active quinone films have important patterning applications since they

allow for versatile and modular functionalization of surfaces via the electrochemical triggering

of interfacial Diels-Alder-cycloaddition[26,251] as well as 1,2-[252] and 1,4-addition.[253–255] The

electrochemically cleavable protecting groups, in particular based on the silylether

functionality,[256] introduce additional surface properties in the initial state.[27,28,248,250,257–259]

For the formation of the electroactive film, an approach based on the reduction of aryl

diazonium salts was selected owing to the excellent stability of the organic tether on a large

variety of conductive materials.[57,58] The bulky TBDMS group was chosen not only to protect the

quinone, but also to limit the formation of multilayers during electrografting.[260] The rigid linker

between the quinone and aminophenyl moieties avoids, upon tethering, undesired interaction of

the quinone with the surface or neighboring quinones.



The aryldiazonium salt was generated in situ in the electrochemical cell [261] and, upon cathodic

reduction, grafted to glassy carbon electrodes (GCE). In 0.1 M TBAHFP/acetonitrile, the amine

precursor was converted into its corresponding diazonium salt by addition of a small amount of

acid and tert-butylnitrite. The diazonium salt was electrochemically reduced in a cyclic

voltammogram between 0.4 and -0.1 V vs. SHE whereas the success of the surface modification

was evaluated by the passivation of the electrode surface.[57,58,246]

a

b

0.0 0.5 1.0 1.5

0

5

10

i /

µA

E / V vs. SHE

2

1

0.0 0.5

-1

0

1

c

0.0 0.2 0.4 0.6

-2

-1

0

1

220

310

1

i /

µA

E / V vs. SHE

2

Figure 11. Electrografted p-hydroquinone groups are deprotected, oxidized and functionalized with thiols under electrochemical control (a). CV for the electrochemical cleavage of the TBDMS protecting group in 50 mM acetate buffer pH 4.4, 0.1 M NaClO4. (b) Inset: CV characterization in 0.1 M phosphate buffer pH 7.0 after deprotection (solid line) and before deprotection (dashed line). Michael addition to the surface-confined benzoquinone in 250 µM 2-mercaptoethanol, 0.1 M phosphate buffer pH 7.0 (c). The numbers on the graph represent subsequent cycles. Scan rates 50 mV/s for (b) to (c) and 200 mV/s for (c).

After electrografting, the cleavage of the TBDMS group from surface-confined HQ is accom-

plished by anodic oxidation to the benzoquinone (BQ) under concomitant cleavage of the Si-O

bond[256] in aqueous electrolyte (Figure 11 b). During the first anodic scan of the cyclic

voltammogram (CV), the onset of the irreversible reaction takes place at a potential of 0.8 V vs.

SHE. Apart from a gradual increase in anodic current due to the oxidation of water, a current

peak at 1.2 V vs. SHE is seen. This signal is attributed to the direct oxidation of the hydroquinone

species and cleavage of the silylether protecting group.[256] After the first forward sweep,

symmetric current peaks at 0.6 V vs. SHE anodically and 0.2 V vs. SHE cathodically occur which

are attributed to the oxidation and reduction of the HQ/BQ couple, indicating successful

deprotection of the quinone. After transferring the modified electrode to neutral buffer, the



presence of the surface-confined deprotected quinone is confirmed by the reversible current

peaks in the CV at 0.34 V vs. SHE anodically and 0.15 V vs. SHE cathodically (Figure 11 b, inset).

The corresponding CV before electrochemical deprotection does not show any redox peaks.

From the charge transferred during the anodic peak, the surface coverage of active quinone was

determined to be (2 ± 1) 10-10 mol∙cm-2, which correlates with the theoretical value expected for

a monolayer assuming a spatial demand of 1 nm2 per molecule.

The peak separation (ΔE ≈ 200 mV) of the HQ/BQ couple deviates from the one expected for a

reversible surface confined redox couple (ΔE = 0 mV), however is typical of quinone systems

exhibiting an apparent two-electron transfer mechanism. Accordingly, slow electron transfer

kinetics of electrografted quinone derivatives have been observed previously on various

substrates[262–264] including glassy carbon.[265,266] The ability of the surface-tethered quinone

layer to undergo interfacial Michael addition reaction was evaluated using 2-mercaptoethanol as

a nucleophile. Upon addition of 2-mercaptoethanol as a model reactant, the current peaks

attributed to the surface-confined HQ/BQ couple shift in cathodic direction (Figure 11 c). The

shift in potential is consistent with an increased electron density in the aromatic system due to

the introduction of the sulfur-containing substituent.[253,254,267] This demonstrates that the

electrografted films of TBDMS-protected quinones are suitable for the reagentless

electrochemical activation and re-functionalization via tethering of Michael donors.

4.1.2 Potentiostatic patterning in the SECM

For patterning of redox-switchable surface moieties various electrochemical scanning probe

techniques were previously employed. Especially the direct mode of SECM seems suitable

because of its possibility for reagent-free surface modification. To activate the quinone-modified

glassy carbon substrate it is connected as the working electrode, a microelectrode serves as the

counter electrode and a reference electrode is placed in the electrolyte solution (Figure 12 a).

When applying a short potential pulse to the working electrode, the current for activation of the

sample is limited to the area underneath the micrometric counter electrode. However, the direct

mode is incapable of patterning of sensitive surface groups such as the quinone groups used in

this study: Attempts to cleave the TBDMS locally by applying a short anodic potential pulse in

the direct mode SECM result in massive corrosion of the carbon substrate. To achieve the local

deprotection of surface-confined HQ moieties on glassy carbon electrodes, potential pulses of

1.4 V were applied for 500 ms in the same electrolyte solution as used in Figure 11 b. As a result,

indentations with a depth of several µm were formed due to the corrosion of the carbon

substrate (Figure 12 b). AFM images show that the damage of substrate occurs irrespective of

the distance between the micrometric counter electrode and the length of the potential pulses in

a range of 3 - 20 µm and 5 – 500 ms, respectively.



a

b

c

0.5 1.0 1.5 2.0

-0.6

-0.4

-0.2

0.0

0.2

0.4

i /

mA

t / s

0.0

0.5

1.0

1.5

-100

-50

0

50

100

ECE

/ VEWE

/ V

Figure 12. Electrochemical patterning of quinone-modified glassy carbon electrodes in the direct mode leads to destruction of the sample surface. Scheme of the direct mode configuration (a). AFM image of a corroded area after applying a potential pulse of 1.4 V vs. SHE for 0.5 s with a 25 µm Pt electrode as counter electrode at a distance of 7 µm from the carbon substrate (b). Recording of the current and the potentials of the working electrode and the counter electrode during the direct mode patterning experiment (c).

Corrosion of the carbon substrate is most probably due to an etching process caused by reactive

species generated at the counter electrode tip during electrochemical water splitting. During the

local electroactivation, the large current necessary to charge the macroscopic sample is driven

through the microelectrode, whereas only electrolysis of the solvent at extreme potentials

provides sufficient current. Figure 12 c shows a recording of the current as well as the potentials

of the working electrode and counter electrode with respect to the reference electrode during a

500 ms potential pulse. Until the set value of 1.4 V is applied to the sample, a large current of up

to 0.4 mA is passed through the microelectrode. This is only possible when the potentiostats

applies a potential of -100 V to the counter electrode. Thus, when controlling the potential of the

sample electrode (potentiostatic patterning), the extreme potentials applied to the micrometric

counter electrode lead to corrosion of the carbon substrate. To avoid corrosion of the sensitive

carbon surfaces, two strategies are possible: 1) Limiting the current for the local activation of

the sample surface reduces the potential applied to the counter electrode and thus is expected to

avoid generation of etchants. 2) Using a larger and remote counter electrode while still

maintaining high spatial resolution will also avoid the destruction of the substrates. The

realization of the two strategies is shown in the following two paragraphs.

4.1.3 Galvanostatic patterning in the SECM

4.1.3.1 Patterning of TBDMS-protected p-hydroquinone layers

For potentiostatic patterning, i.e. when the potential of the sample to be locally activated is

controlled, the necessary current is only provided by a high rate of electrolysis of the solvent, at

which etchant species can be generated. Thus, limiting the current reduces the production of

undesired etchants at the microelectrode. The corrosion of the sensitive carbon surfaces is



avoided when the patterning is performed galvanostatically, i.e. under control of the current. To

evaluate the success of the local modification during electrochemical writing, feedback mode

SECM is used as a tool to read out the patterns. In the feedback mode experiments,

ferrocenedimethanol serves as the redox mediator that discriminates between the surface

carrying TBDMS-protected quinone groups and the quinone-terminated surface after

deprotection. A Pt microelectrode is polarized at a potential for diffusion-limited oxidation of

ferrocenedimethanol and the modified glassy carbon electrode is polarized at a potential for

reduction of the corresponding ferricinium cation.

0 1 2 3

0

1

2

3

4

5

i no

rm =

i/i

bulk

dtip-sample

/rtip

Figure 13. Feedback mode SECM with ferrocenedimethanol as the redox mediator distinguishes between TBDMS-protected quinone moieties and deprotected quinone moiteties. Approach curves to pristine TBDMS-HQ surface and after electrochemical deprotection. Etip = 0.56 V vs. SHE and Esample = 0.21 V vs. SHE. Redox mediator 1 mM ferrocenedimethanol, 0.1 M KCl, 25 µm Pt electrode.

When the tip is approached to the carbon substrate modified globally with TBDMS-protected

quinone groups, re-reduction at the substrate is inhibited. Thus, because diffusional access to the

microelectrode is obstructed as the probe comes closer to the sample surface, the current at the

microelectrode decreases (Figure 13, blue curve). A negative feedback of current is recorded. In

contrast, the deprotected quinone surface permits the recycling of the redox probe as the

microelectrode tip approaches and thus the tip current increases with decreasing tip-to-sample

distance (positive feedback, Figure 13, red curve).

Patterning of the TBDMS-HQ-modified electrodes is achieved by applying short current pulses to

the glassy carbon samples with the micrometric counter electrode positioned in close proximity.

Under galvanostatic control the currents may be decreased to avoid the generation of etchants at

the microelectrode. The potential of the working electrode during a current pulse is

inhomogeneously distributed over the whole surface with its highest magnitude centered at the

position under the microelectrode. The relation between current and working electrode

potential and its spatial distribution is unknown. Hence, the current magnitude that leads to a

sufficient potential to drive the anticipated removal of the protective group at the carbon surface

has to be determined empirically. The SECM allows for fast screening of the parameters leading



to the desired surface modification without destruction of the film or the underlying

substrate.[247] To optimize the conditions for the local cleavage of the TBDMS group the

magnitude of the direct mode current pulses is varied. The resulting local surface modifications

are visualized by means of FB mode SECM (Figure 14). The SECM image shows an array of

locally modified spots created by applying direct mode current pulses with magnitudes between

11.5 µA and 13 µA. The FB current over the local modifications increases due to recycling of

ferrocenedimethanol, suggesting the successful local removal of the TBDMS group.

a

b

Figure 14. Destruction-free patterning is achieved by applying short galvanostatic pulses for removal of the TBDMS group. FB mode SECM image after applying 11.5, 12, 12.5 and 13 µA pulses (from left to right) (a). AFM image of the rightmost spot from the SECM image (b). Patterning: pulse duration 0.5 s, tip-to-sample distance 5 µm. Imaging: Feedback mode as described in Figure 13.

The intensity and width of the spots increases with higher patterning current, indicating

increasing efficiency of the local surface-confined reaction. Local corrosion of the carbon

surfaces can be excluded: AFM images show no observable damage after local surface

modification (Figure 14 b). Thus, patterning by current pulses in the direct mode of SECM is

suitable for local modifications of sensitive surface groups. Corrosion of the sample is avoided by

limiting the current passed through the micrometric counter electrode.

4.1.3.2 Patterning of nitrophenyl layers

Galvanostatic patterning in the direct mode of SECM was also successfully applied for a different

type of electrografted functional groups, proving that the method is generic and can be adapted

for local modifications using diverse surface chemistry. Electrografting of nitrophenyldiazonium

salts yields well-characterized organic films with NO2-groups exposed at the surface.[268] Then,

via a second electrochemical reduction step, the surface-confined nitro groups can be converted

to hydroxylamino and amino groups upon applying a cathodic potential in acidic medium. As

previously shown,[269–271] the reduced amino groups can then be used as anchoring groups for

biomolecule immobilization. The SECM was used as a tool for local reduction of NO2 groups and

subsequent localized immobilization of proteins to create protein arrays (Figure 15 a). For the

electrografting of nitrophenyl groups glassy carbon plates were immersed in 1 mM

nitrophenyldiazonium tetrafluoroborate, 0.1 M H2SO4 and a cyclic voltammogram from 0.4 V

to -0.3 V vs. Ag/AgCl/ 3 M KCl was recorded.[268] Analogous to previous work on

4-nitrothiophenol self-assembled monolayers[270] the redox state of the nitro groups may be



changed locally using the SECM to generate patterns of immobilized proteins. As proton source

for the reduction of NO2 groups acetic acid (HAc) was chosen. At these relatively mild conditions,

proteins already immobilized on the array may retain their activity even when further areas are

locally modified subsequently.[49] The reduction of NO2 groups sets in at potentials more

negative than -0.5 V vs. Ag/AgCl/3 M Cl- and is most efficient at -0.9 V. Thus, in initial attempts

for the local modification of the surface, potential pulses of -0.9 V were applied in the direct

mode of SECM with the microcounterelectrode positioned in 4 µm distance from the

nitrophenyl-modified working electrodes. However, instead of the anticipated local reduction to

yield NH2 groups, the treatment leads to strong local carbon corrosion similar to the results as

shown in Figure 12.

a

b

Figure 15. Nitrophenyl groups grafted on glassy carbon are locally reduced to amino groups by galvanostatic pulsing in the direct mode of SECM. While potentiostatic patterning results in carbon corrosion, galvanostatic patterning yields the anticipated amino-surface functionalities (a). Amino groups are used for the attachment of alkaline phosphatase. Using pH-modulated SECM imaging, the identity of amino groups is unambiguously discriminated against pristine nitro-terminated films and corroded carbon resulting from destructive patterning. Depending on the pH value the electron transfer rate with ferrocyanide is altered (b).

Interestingly, the damage of the carbon substrate also occurs with a positively polarized counter

electrode: In the experiments described in the previous section (patterning of TBDMS-protected

p-hydroquinone layers), an anodic pulse is applied to the carbon working electrode, resulting in

highly negative potentials at the counter electrode. For the experiments aiming at the reduction

of NO2 groups, a negative potential pulse is applied to the carbon substrate and thus the

potential of the counter electrode is positive. Hence, etchants damaging the carbon substrate are

presumably generated in a cathodic reaction as well as in an anodic reaction.

The possible surface modification states after direct mode structuring are, in the case of

successful surface patterning, an amino-/hydroxylamino-terminated film or the unchanged

nitro-terminated tether. Additionally, the possibility of surface destruction and hence the local

desorption of the modifying film has to be considered (see above). It is known that electron



transfer with charged redox mediators across amino-terminated films is largely dependent on

the pH value.[272,273] The pristine nitrophenyl film blocks the electrode surface for redox

mediators irrespective of the charge or pH value, while blank glassy carbon generally allows

efficient electron transfer (Figure 15 b). Amino groups may be protonated or deprotonated

according to the pH value of the electrolyte. Amino-terminated layers do not allow fast electron

transfer for the oxidation of [Fe(CN)6]4- at pH 7, while electron transfer is efficient at pH 1. The

acceleration of electron transfer for the protonated amino-film is presumably to a favorable

interaction between the positively charged surface and the highly negatively charged

ferrocyanide anion. At neutral pH value, however the blocking properties of the film dominate.

The different electron transfer rates for the oxidation of [Fe(CN)6]4- are exploited to distinguish

the different surface modifications in a pH-modulated feedback mode SECM imaging experiment.

a

0 5 10

1

2

blank pH 1

blank pH 7

-NO2 pH 1

-NO2 pH 7

-NH2 pH 1

-NH2 pH7i n

orm

= i/i

bulk

dtip-sample

/rtip

b

c

d

Figure 16. Aminophenyl-terminated sample areas are identified by pH-modulated feedback mode SECM imaging. Approach curves to the differently modified substrate surfaces (see legend) show the differences in electron transfer rate with the redox mediator [Fe(CN)6]

3- (a). Images of a test sample (b)

containing areas with different surface functionalization (c, d). The NH2-modified area is identified by the change of properties upon protonation/deprotonation. FB mode in 1 mM K3[Fe(CN)6] in either 0.1 M HCl (pH1) or 0.1 M phosphate buffer (pH 7) with Etip = 0 V and Esample = 0.5 V (pH 1) or Etip = -0.1 V and Esample = 0.4 V (pH 7).

On glassy carbon samples carrying the different possible surface modification states SECM

approach curves were recorded (Figure 16 a). The redox mediator [Fe(CN)6]3- is reduced at the



microelectrode tip. As expected, the nitro-terminated surface prevents re-oxidation of

ferrocyanide produced at the microelectrode irrespective of the pH value, leading to a negative

feedback of current at both tested pH values (purple curves). On the other hand, the blank glassy

carbon sample always exhibits a positive feedback due to the efficient recycling of ferrocyanide

(black curves). Only the amino/hydroxylamino surface (cyan curves) changes the electron

transfer rate upon protonation/deprotonation. This leads to a positive feedback at pH 1 (dotted

line) while at pH 7 the blocking properties dominate and the feedback is negative (solid line). To

demonstrate the possibility of distinguishing between the different surface modification states

on a laterally inhomogenous sample, a test sample was prepared and investigated by feedback

mode SECM (Figure 16 b-d). A glassy carbon plate was dipped partially into 4-nitrophenyl

diazonium solution for electrografting of nitrophenyl moieties by means of CV from 0.4 to -0.3 V

vs. Ag/AgCl/3 M Cl-. The plate was then turned by 90 ° and dipped partially into sulfuric acid for

the electrochemical reduction of nitro groups during CVs between 0.75 and -0.9 V vs.

Ag/AgCl/3 M Cl-. Finally the sample consisted of four differently modified segments. Two

segments of blank glassy carbon, one of which was exposed to sulfuric acid and reducing

potentials. One segment was covered with the unmodified nitro-terminated film. The last

segment exhibited electrochemically reduced amino/hydroxylamino groups. Two constant

height SECM array scans at different pH values were performed using [Fe(CN)6]3- as redox

mediator, which is reduced at the SECM tip (Figure 16 c, d). In the two tip current images higher

cathodic current values represent a positive feedback current due to efficient recycling of the

redox mediator at the sample surface. Higher anodic current values (negative feedback)

correspond to a decreased electron transfer rate due to the presence of a blocking film at the

carbon surface. As expected from the behavior seen in the approach curves, at pH 1 only the

nitrophenyl-modified area exhibits negative feedback. At this pH value, aminophenyl moieties

are protonated and thus permit recycling of the negatively charged redox probe. In contrast, at

pH 7 the amino functionalities are largely deprotonated and thus the blocking properties of the

film hinder electron transfer with the redox probe. Hence, modulation of the pH value during

feedback mode imaging allows to unambiguously distinguishing between a blank GC surface, a

nitrophenyl- or an aminophenyl-terminated surface.

A nitrophenyl-modified electrode is patterned in the direct mode by applying galvanostatic

pulses with different current magnitudes at different positions of the microelectrode. The

current magnitude that leads to the sufficiently low potential to drive the anticipated reduction

of nitro groups at the carbon surface is determined empirically. The spots modified during the

galvanostatic pulses are characterized in the feedback (FB) mode of the SECM with [Fe(CN)6]3- as

mediator at different pH values. To read out the local surface modification, a SECM line scan is

performed across a series of spots which were modified by applying cathodic current pulses



with different magnitude (Figure 17). The current peaks in the line scan recorded at pH 1

indicate locally modified spots at positions where direct mode current pulses were applied. The

line scan at pH 7 (black curve) suggests that damage to the carbon sets in between an applied

Ipulse of -3.6 and -4.0 µA. At pH 7 only the removal of the tethered layer leads to a positive

feedback current. At lower pulse heights for galvanostatic surface patterning, electrochemically

reduced amino/hydroxylamino functionalities are obtained without apparent corrosion of the

carbon surface. In order to demonstrate the suitability of this surface patterning strategy for

biosensor applications and the fabrication of protein microarrays, alkaline phosphatase was

immobilized on the generated pattern as a model protein.

a

b

0 1000 2000 3000

0.5

1.0

1.5

2.0

2.5

i no

rm =

i/i

bu

lk

x / µm

1.2

1.4

1.6

i ge

ne

rato

r-co

llecto

r / n

A

-2.8 -3.2 -3.6 -4.0 -4.4

ipulse

/ µA

Figure 17. Galvanostatic SECM direct mode patterning allows local reduction of nitro groups to yield amino functionalities. Scheme for the different scans (a). SECM line scans across an array of locally modified spots generated by applying varying Ipulse for patterning (b). pH-modulated imaging (pH 1 blue, pH 7 black) indicates successful nitro reduction and the onset of carbon corrosion. Locally immobilized alkaline phosphatase is detected using the sample-generation/tip-collection mode. Line scan (red) in 1 mM pAPP, 0.1 M carbonate buffer pH 9.4 for oxidation of aminophenol phosphate at Etip = 0.3 V.

After structuring using galvanostatic pulses in the direct mode of SECM, the complete surface

was treated with sulfo-NHS-biotin and subsequently with avidin-conjugated alkaline

phosphatase. Alkaline phosphatase catalyzes the cleavage of the phosphate group from pAPP

(pAPP) and releases p-aminophenol (pAP). A line scan using the sample-generation/tip-

collection mode of SECM was performed in a solution containing p-aminophenol-phosphate. The

product of enzymatic cleavage pAP is redox-active and is detected by oxidation at the SECM tip.

The line scan across the patterned region (Figure 17, red curve) shows increased pAP oxidation

currents above the locally modified spots, indicating that alkaline phosphatase is predominantly

bound to the amino-terminated areas.



In summary, these results show that the local corrosion occurring during patterning in the direct

mode of SECM can be avoided by limiting the current for the local modification. Nitrophenyl

moieties are grafted to glassy carbon electrodes and the reduction of NO2 groups to

amino/hydroxylamino groups is performed locally using an SECM. However, the identity of the

resulting groups must be carefully assessed. In case of the conversion of NO2 groups to NH2

groups, pH-modulated feedback mode SECM imaging allows to distinguish between both surface

modification states as well as local corrosion of the carbon substrate. The locally generated

amino groups were exploited for the immobilization of an enzyme.

4.1.4 Patterning in the Scanning Droplet Cell

Alternative to patterning by applying galvanostatic pulses in the direct mode of SECM (previous

paragraph), highly chemoselective local modification is achieved in the scanning droplet cell

(SDC). Scanning droplet cells were originally developed for high-throughput screening in

corrosion research.[273,274] More recently their application was extended to the screening of

oxygen reduction catalysts[275,276] and materials for photoelectrochemical water splitting.[277]

a

b

c

d

-0.5 0.0 0.5 1.0 1.5

-2

0

2

4

6

2

i /

nA

E / V vs. SHE

1

0.0 0.5 1.0

-1

0

1

e

0.0 0.2 0.4 0.6

-2

0

2

50

1020

1

i /

nA

E / V vs. SHE

2

Figure 18. In the scanning droplet cell, the TBDMS protecting group from HQ layers is locally removed to generate a pattern of activated spots. The counter and reference electrode are located inside a capillary (a). Schematic longitudinal cross section of the employed PTFE capillary (b) and optical micrograph of the capillary orifice (c). CV for the local deprotection of the HQ groups in the same conditions as in Figure 11 (d). The inset shows the HQ/BQ redox signals (solid) and a local CV at a TBDMS-protected spot (dashed). Local Michael addition in 250 µM 2-mercaptoethanol, 0.2 M phosphate buffer pH 7.0. Scan rate 0.2 V/s.



In surface patterning, experimental setups similar to a SDC were employed for local deposition

of metals[278], metal oxidation[279,280] and deposition of conducting polymers.[281–285] Apart from

these examples, the SDC is well suited for local activation of modified surfaces. The SDC was

applied for the local electrochemical cleavage of TBDMS protecting groups from electrografted

p-hydroquinone moieties. To avoid corrosion of the carbon substrate, the protecting groups are

locally removed with high chemoselectivity to reveal spots of surface-tethered active quinone

moieties (Figure 18). In the SDC, the reference electrode and counter electrode are located

inside a capillary filled with electrolyte. When the capillary is in contact with the substrate, the

electrolyte droplet on the substrate surface dictates the size of the electrochemically addressed

area. By using soft capillaries made from polytetrafluoro-ethylene, damages to the surface are

excluded when touching the sample with the capillary. The capillaries have an orifice of only 100

µm diameter (Figure 18 c). The local deprotection is accomplished by applying the same

potential sweep as for integral deprotection (Figure 18 d). The CV for the deprotection in acidic

solution exhibits the same characteristics as the one recorded for global removal of the TBDMS

protecting group from grafted HQ moieties (compare to Figure 11). After the irreversible

oxidation of the HQ group under cleavage of the protective group, the signals attributed to the

HQ/BQ couple appear. After filling the capillary with pH 7 buffer solution and re-positioning it to

the deprotected spot, the reversible HQ/BQ signals are visible, whereas the CV recorded at a

TBDMS-protected spot shows no such signals (inset). The HQ/BQ redox signals provide direct

evidence for the anticipated local chemical modification and prove that the SDC allows for

chemoselective electrochemical patterning of the sensitive functional groups. Additionally, the

locally activated HQ groups can be re-functionalized via Michael addition with a thiol compound.

As previously described for the global Michael addition (Figure 11), the SDC is re-positioned on a

deprotected spot and HQ groups are oxidized in the presence of mercaptoethanol in the

capillary.

As expected, the current peaks attributed to the HQ/BQ couple shift towards more cathodic

potential. Hence, patterning in the SDC allows for site-selective immobilization of molecules on

the locally activated films. For the SDC, capillaries with different orifice sizes from 1 mm down to

100 µm can be used. In this study, the smallest orifice was used to achieve patterning with high

resolution. To assess the surface area of the wetted and thus electroactive spot on the TBDMS-

HQ-modified glassy carbon electrode, a potential step experiment with [Ru(NH3)6]3+ as redox

mediator was performed (see experimental part). The active surface area amounts to (8.6 ± 0.3)

10-5 cm2 which corresponds to a diameter of the droplet inside the capillary of 105 ± 2 µm and

thus is in good agreement with the dimensions of the capillary orifice.

In addition, the generated patterns of deprotected HQ groups surrounded by TBDMS-HQ can be

visualized by means of feedback mode SECM. The imaging contrast between the two surface



modification states is explained by the differences in heterogeneous electron transfer rate with

ferrocenedimethanol as redox mediator (also see Figure 13). The tip is scanned in constant

height mode over an array of spots of locally deprotected quinone resulting from patterning in

the scanning droplet cell (Figure 19 a). Accordingly, the SECM image of the area patterned in the

SDC exhibits clear circular features resulting from the modified spots. The diameter of the spots

is only slightly larger than the orifice of the SDC capillary. This is expected because SECM images

the diffusion layer of the modified spots which is larger than the spots themselves.

a

b

0 500 1000

1

2

in

orm

= i/i

bu

lk

x / µm

Figure 19. Generated patterns of deprotected HQ groups are visualized by means of SECM. Feedback mode SECM image of an array of deprotected spots generated in the SDC (a). Horizontal cross section through the same array (b). Etip = 0.56 V vs. SHE and Esample = 0.21 V vs. SHE. Redox mediator 1 mM ferrocenedimethanol, 0.1 M KCl, 25 µm Pt electrode, scanning height 5 µm.

In conclusion, non-destructive surface patterning is achieved using the scanning droplet cell for

the local electrochemical modification of surface functionalities. In contrast to the direct mode of

SECM, the counter electrode is large and remote from the substrate surface. Thus, degradation of

the sensitive sample surfaces by etchants generated at the counter electrode tip is excluded.

Moreover, the electrochemical data, namely the CVs for the electrochemical cleavage of the

TBDMS group, the redox signals for the surface-confined hydroquinone/benzoquinone couple

and the monitoring of the Michael addition of thiols to the quinone-terminated surface are direct

evidence for the anticipated local surface modifications.

The identity of the etchants responsible for carbon corrosion remains unknown. Possibly, the

very corrosive OH radical plays a role for the etching process. Previous studies for destructive

patterning in the SECM showed evidence that OH radicals are produced cathodically as well as

anodically. For instance, excessively high potentials applied to the microelectrode led to the

polymerization of vinylic monomers in solution which hints at the radical nature of the

etchants.[286] Also when cathodic potentials were applied to the microelectrode in the presence



of oxygen, incomplete reduction O2 led to the formation of OH radicals and to corrosion of

organic films.[287]

In the future, the spatial resolution and patterning density of the SDC could be significantly

increased by using nanopipettes instead of the micrometric PTFE capillaries employed in this

work. Strategies for the non-contact distance-control of the nanopipettes and formation of a

nanometric liquid meniscus on the sample surface have been described.[282,283,288,289] These

nanopipette based scanning probe methods, in conjunction with elaborated surface chemistry

for the immobilization of biomolecules[290] will create powerful electrochemical tools for the

fabrication and read-out of bionanoarrays.[1]


4.2 Nanoelectrode Fabrication and Characterization 51

4.2 Nanoelectrode Fabrication and Characterization

Carbon nanoelectrodes are fabricated according to a method first proposed by Takahashi et

al.[99] To create sharp, needle-type electrodes first nanopipettes are pulled from quartz glass

capillaries using a Sutter P-2000 laser puller. The size of the carbon nanoelectrodes is dictated

by the size of the capillary orifice, which along with the shape of the taper can be controlled by

adjusting the parameters of the puller. The outer radius of the glass insulator is as well dictated

by the pulling parameters and the specifications of the glassware used. The ratio between outer

radius of the whole probe and the inner radius (RG value) remains largely constant during the

pulling process. Using glassware with typical dimensions of 1.2 mm outer diameter and 0.9 mm

inner diameter, the ratio between outside radius and the active disk electrode after pyrolysis is

around 1.3, resulting in very small overall dimensions of the probe. To fabricate carbon

nanoelectrodes, conductive carbon is deposited inside the pulled quartz glass capillaries by

pyrolysis of a butane/propane mixture (Figure 20).

Figure 20. Laser-assisted pulling of quartz glass capillaries and subsequent pyrolysis of butane/propane gas in inert argon atmosphere inside the resulting nanopipettes yields carbon nanoelectrodes.

The pulled capillary is filled with the carbon precursor gas, inserted into a second unpulled

capillary that is filled with an inert argon atmosphere and heated to approx. 1200 °C using a

butane torch flame. As a result, the pyrolytic carbon clogs the nanopipette and forms a disk-

shaped electrode at the tip of the pipette. This fabrication procedure is a very fast and simple

route to carbon nanoelectrodes. Only inexpensive materials and equipment are necessary (see

experimental section) and electrodes are fabricated with nearly 100 % efficiency. The overall

time to produce one functional electrode ranges between 1 and 2 minutes. The nature of the

electrode materials was characterized by means of Raman spectroscopy. Because of the

difficulty to analyze nanometric sample areas, the spectra were not acquired from the

nanometric disk-shaped tip of the electrode but from the material deposited on the inside walls

of the nanopipettes after breaking open the capillary.[291] From the high intensity of the D band

relative to the G band as well as the absence of G’ and 2D bands it was concluded that the



electrode material is graphite-like, however with a substantial amount of defects in the graphitic

structure.

-0.4 -0.2 0.0

-600

-400

-200

0

i /

pA

E / V vs. Ag/AgCl

-0.5 0.0

-5

0

Figure 21. Carbon nanoelectrodes are fabricated with tunable electrode radii. Cyclic voltammograms in 5 mM [Ru(NH3)6]Cl3, 0.1 M KCl for selected electrodes with radii of 2, 11, 68, 141, 271 nm calculated according to equation (1). Inset: Enlargement of the CV for the smallest electrode.

The quality and size of the electrochemically active part of the carbon electrodes is assessed

from cyclic voltammograms in ferrocenemethanol or [Ru(NH3)6]3+ (Figure 21). The flat current

plateaus in the cyclic voltammograms show that the reduction of [Ru(NH3)6]3+ is fast and only

limited by diffusional mass transport to the electrode. From these steady-state currents, the

radius of the disk-shaped electrodes is estimated according to equation (1).[292]

𝑖𝑠𝑠 = 4.64 𝑛 𝑐 𝐷 𝐹 𝑟 (1)

iss is the steady-state current, n the number of transferred electrons, c the concentration of redox

species in the bulk solution, D its diffusion coefficient, F the Faraday constant and r the radius of

the electrode. Equation (1) is valid for flat, disk-shaped electrodes surrounded by a coplanar

insulator and a RG value of 1.5.

Figure 22. SEM images show the high aspect ratio and disk-shaped geometry of carbon nanoelectrodes. Two different electrodes are shown. Electrochemically estimated radii were 60 nm (left electrode) and 4 nm (right electrode). Scale bars 200 nm.



As the CVs for selected electrodes (Figure 21) show, the radius of the carbon nanoelectrodes can

be tuned in a range from a few nm to about 300 nm. A good correlation is observed comparing

the electrochemically estimated electrode radii with electrode dimensions observed in SEM

images of different electrodes (Figure 22). In addition, the SEM images show the pointy shape of

the electrodes that are surrounded by only a thin layer of insulating quartz glass, justifying the

assumptions made for the size determination according to equation (1). However, the image of a

small electrode exhibiting only an apparent electrochemically determined radius of 4 nm (right

image) demonstrate the limitations of SEM as a characterization tool for structures in the lower

nm regime. The resolution of the SEM is not sufficient to reliably determine the dimensions at

the apex of the nanoelectrode.


4.3 Amperometric Nanosensors in Biological Applications 54

4.3 Amperometric Nanosensors in Biological Applications

Experiments at brain slice samples were performed in collaboration with Paolo Actis, Sergiy Tokar

and Ainara López Córdoba, Imperial College London, who also provided cell samples. Parts of the

section were published in ref. [291]: “Actis, P.; Tokar, S.; Clausmeyer, J.; Babakinejad, B.;

Mikhaleva, S.; Cornut, R.; Takahashi, Y.; López Córdoba, A.; Novak, P.; Shevchuck, A. I.; Dougan, J.

A.; Kazarian, S. G.; Gorelkin, P. V.; Erofeev, A. S.; Yaminsky, I. V.; Unwin, P. R.; Schuhmann, W.;

Klenerman, D.; Rusakov, D. A.; Sviderskaya, E. V.; Korchev, Y. E.; ACS Nano 2014, 8, 875–884”.

Ainara López Córdoba, Imperial College London, provided DRG neurons. Macrophages were

cultured in collaboration with Melanie Mark and Miriam Marquitan, both Ruhr-Universität

Bochum. Experiments on macrophage cells were performed by Miriam Marquitan and partially

presented in her master thesis.[293] Parts of this section were published in ref. [294]: “Clausmeyer, J.;

Actis, P.; López Córdoba, A.; Korchev, Y.; Schuhmann, W.; Electrochem. Commun. 2014, 40, 28–

30.”

4.3.1 Platinized nanoelectrodes for oxygen measurements

Measuring the oxygen concentration around cells is useful to evaluate the respiratory activity

and the viability of cells.[295] The electrochemical detection of O2 requires electrochemical

sensors with a high sensitivity for the oxygen reduction reaction. However, it is known that

carbon electrode materials generally possess high overpotentials and slow electron transfer

rates for the reduction of O2. Thus, carbon nanoelectrodes are modified by depositing a small

amount of platinum on the electrode tip. It is important that the small overall dimensions of the

electrode are maintained. The cyclic voltammogram in a solution containing the platinum

precursor [PtCl6]2- shows a curve shape typical for a nucleation and growth mechanism (Figure

23 a): Only small cathodic currents are observed in the first cathodic sweep until the potential is

sufficiently negative to form Pt nuclei. In the subsequent anodic sweep larger cathodic currents

are observed, corresponding to the facilitated growth of Pt particles once nuclei are formed. In

addition, the enhanced current after deposition of a Pt particle may be attributed to the

reduction of protons. In the second cycle, more Pt is deposited on the electrode. To limit

uncontrolled growth of the Pt deposit, the potential is swept to only -0.5 V vs. Ag/AgCl/0.1 M Cl-.

As shown by cyclic voltammograms in air-saturated buffer solution containing

ferrocenemethanol as a redox probe (Figure 23 b), the amount of deposited Pt is sufficient to

reduce the overpotential for the oxygen reduction reaction while maintaining the small

electrode dimensions. The curve recorded after deposition of platinum shows a significantly

increased onset potential and higher currents for the oxygen reduction reaction whereas the

anodic diffusion-limited current corresponding to the oxidation of ferrocenemethanol remains

unchanged indicating mainly unchanged electrode size.



a

-0.5 0.0

-300

-200

-100

0

2nd

scan

i /

pA

E / V vs. Ag/AgCl

1st scan

b

Figure 23. Deposition of platinum on carbon nanoelectrodes allows electrochemical detection of oxygen. Cyclic voltammograms (0.5 V/s) in 2 mM H2PtCl6, 0.1 M HCl (a) and 1 mM ferrocenemethanol, air-saturated PBS (b) before and after deposition of platinum.

The platinized carbon nanoelectrodes are then used to probe the oxygen consumption of a living

brain slice taken from the rat hippocampus. The nanoelectrode is polarized at –0.6 V vs.

Ag/AgCl/0.14 M Cl- and the sensor is approached to the tissue in a stepwise manner (Figure 24

a). Upon approach of the sensor, the cathodic current corresponding to the oxygen reduction

decreases due to the local depletion of oxygen caused by the respiratory activity of living

neurons in the brain slice. The nanoelectrode is then advanced further to penetrate into the

tissue, which leads to a further decrease of the current signal. With the electrode placed inside

the tissue, it is not only the hindered diffusional transport of oxygen to the sensor but the active

consumption of oxygen by the neurons that leads to its depletion. With a dead brain slice, linear

sweep voltammograms are recorded at different relative positions of the nanoelectrode with

respect to the tissue (Figure 24 b). Due to the absence of respiratory activity only a minor

decrease of the cathodic current is observed when the electrode is placed inside of the tissue. In

contrast, in a living brain slice, the sensor records a large concentration gradient between the

inside of the tissue and bulk solution. Because the decrease in the oxygen reduction current in

the tissue is a measure of metabolic cell activity, these findings show the utility of these

nanoprobes to monitor metabolic activity in brain slices. The nanometer size of the

nanoelectrode allows measurement of oxygen consumption deep inside the brain slice with

minimal damage and perturbation to the biological enivronment. Moreover, platinized carbon

electrodes are suitable to detect a number of ROS and RNS including H2O2, ONOO-, NO•, and

NO2-.[174–176] Despite the cells of interest are located together with many other cells in a tissue,

the small overall dimensions of the nanoelectrodes allow to perform electrochemical

determination of ROS species at the single-cell level.



a

b

-0.5 0.0

-400

-300

-200

-100

0

-0.5 0.0

Live Brain Slice

i /

pA

E / V vs. Ag/AgCl

Dead Brain Slice

Figure 24. Oxygen consumption in a live tissue slice from rat hippocampus is monitored using platinized nanoelectrodes. Vertical oxygen concentration profile measured at -0.6 V vs. Ag/AgCl/0.14 Cl

- at different

positions of the nanoelectrode with respect to the tissue (a). Linear sweep voltammetry curves recorded with the nanoelectrode positioned 300 µm away, near the surface and 100 µm inside of the tissue in a dead brain slice and a living brain slice.

The nanoelectrode can be precisely inserted into an individual neuron within the brain slice to

monitor intracellular molecules. Cyclic voltammograms are measured continuously at 0.4 V/s as

the nanoelectrode is manually approached to the neuron (Figure 25 a). In the moment when the

cell membrane is penetrated, the stacked voltammograms show higher anodic currents at

potentials above 0.4 V. The anodic current is ascribed to the oxidation of various ROS and RNS

species, as observed by Amatore et al.[174–176] In another experiment, the nanoelectrode is

polarized at + 0.85 V vs. Ag/AgCl/0.14 M Cl-, a potential where all ROS/RNS species are oxidized,

and the amperometric current is monitored (Figure 25 b).

a

b

0 20 40 60

0.0

0.5

1.0

1.5

retraction

i /

pA

t / s

penetration

Figure 25. Platinized nanoelectrodes are inserted into individual neurons in a tissue to perform intracellu-lar electrochemical measurements. Stacked cyclic voltammograms with the voltammetric current repre-sented in false colors (a). The cell membrane is punctured after 25 s. Amperometric current at 0.85 V vs. Ag/AgCl/0.14 M Cl

- during penetration and retraction of the sensor (b).

In the moment of penetration, a current spike occurs, followed by a stable current plateau. After

withdrawal of the nanoprobe, the current jumps back to the initial baseline. The nearly constant

current recorded when the nanoelectrode is inside the cell corresponds to a steady flux of all



intracellular species that are oxidizable at the high anodic potential. Apart from the relevant

H2O2, ONOO-, NO•, and NO2- species also other interferents occurring in the cell, e.g. antioxidants

like ascorbic acid, uric acid or glutathione can be oxidized at this potential and thus contribute to

the overall analytical signal measured after penetration. Amatore et al. demonstrated that the

individual currents for oxidation of secreted H2O2, ONOO-, NO•, and NO2- can be deconvoluted by

applying a potential step profile to the sensing electrode.[173–175] By comparing and subtracting

currents measured at different detection potentials, the amount of the individual species

relevant to oxidative stress was calculated. For intracellular measurements, truly selective ROS

measurements are more challenging because of the complex intracellular matrix that contains

additional interferents. Additionally, the change in electrode capacitance upon cell penetration

may complicate the interpretation of such potential step experiments. Instead, to further

investigate reaction cascades in the context of oxidative cell stress, a more advanced sensor

design is necessary to create sensors capable of selectively detecting particular ROS species.

4.3.2 Prussian Blue-modified nanoelectrodes for the detection of H2O2

To identify the contribution of specific compounds to the overall oxidative stress, the

nanoelectrodes need to be modified with a sensing layer that facilitates the electrochemical

conversion of the analyte while protecting the sensor from interference by other species. PB is a

well-known selective electrocatalyst for the reduction and oxidation of hydrogen

peroxide.[170,182] PB can be electrochemically deposited by reduction of [Fe(CN)6]3- in the

presence of Fe3+ and K+ which leads to the precipitation of KFeIIIFeII(CN)6. For the

electrochemical sensing of hydrogen peroxide, PB can either be oxidized to form Berlin Green

which acts as the electrocatalyst for H2O2 oxidation[296] or reduced to Prussian White (PW) to

detect H2O2 cathodically.[297,298] For measurements in complex biological environments, the

detection by reduction of H2O2 is advantageous because at the mild potentials (around 0 V vs.

Ag/AgCl) necessary for detection, no interference by undesired electrochemical reduction of

oxygen takes place. Moreover, no other substances occurring in biological systems such as

ascorbic acid or neurotransmitters are known to be reduced at this detection potential.

Figure 26. Prussian Blue is oxidized to form Berlin Green or reduced to form Prussian White. Both the oxidized and the reduced form can act as electrocatalysts in sensors for hydrogen peroxide.



However, the stability of PB films, especially in neutral and alkaline media, has remained critical

because iron species are complexed by OH- and leeched out of the material. Common strategies

to improve the longevity of the sensor devices on macroscopic electrodes are heat treatment[298],

entrapment of PB in carbon inks/pastes[299] and coating with polymer films[297]. Microsensors for

H2O2 were based on electrodes fabricated from carbon microfibers or metal wires[117,184,185,300,301].

However, functionalization of nanoprobes to comprise a selective electrochemical sensor

renders challenging due to their small size. The mechanical and chemical stability of films

deposited on nanoelectrodes deviates drastically from the one observed on micro- or

macroelectrodes. For the deposition on carbon nanoelectrodes this limitation can be overcome

by electrochemically etching a nanocavity into the carbon electrode. Subsequently, a PB film is

electrodeposited into the cavity. To generate the cavity, the electrode material is etched

anodically in alkaline medium by cycling the potential to 2 V.

a

-0.4 -0.2 0.0-3

-2

-1

0

1

2

i /

nA

E / V vs. Ag/AgCl

b

c

Figure 27. Nanocavities are etched into the tip of carbon nanoelectrodes. Cyclic voltammograms in 5 mM [Ru(NH3)6]Cl3, 0.1 M KCl at an unetched electrode, after 5 etching cycles and after 15 etching cycles (a). Schematic pictures at different stages of etching (b). Etching was performed in 0.1 M KOH, 0.01 M KCl by cycling the potential between 0 and 2 V vs. Ag/AgCl/0.01 M Cl

- with 0.2 V/s. Electrode radius 153 nm. SEM

images of a different nanoelectrode before and after etching (c).

At the high anodic potential applied for etching, among other processes such as oxidative water

splitting and presumably oxidation of Cl- anions, the carbon electrode material corrodes and

thus considerable amounts of carbon are removed from the nanoelectrode. The results of the

etching process can be followed by cyclic voltammetry with a soluble redox probe, e.g.



[Ru(NH3)6]3+ (Figure 27 a). Demonstrated on an electrode with a radius of 153 nm, the initial

steady-state cathodic current for [Ru(NH3)6]3+ reduction decreases after applying 5 etching

cycles to the electrode. This behavior was theoretically predicted and experimentally observed

by Sun and Mirkin.[302] At a recessed microelectrode, the diffusion-limited steady-state currents

are decreased because the unidirectional diffusion inside of the recessed electrode is

substantially slower than the radial diffusion normally observed at planar micro- or

nanoelectrodes (Figure 27 b). Moreover, based on numerical solutions of the mass transport

laws, the depth of the recessed electrode can be estimated from the diminished steady-state

current. For the electrode shown in Figure 27, the steady-state current after 5 etching cycles

decreased to 30 % of the initial value, corresponding to a recess depth of ca. two times the

electrode radius, i.e. about 500 nm.[302] For all recessed electrodes made in this fashion, the

steady-state current after etching was at least diminished by 50 %, corresponding to a minimum

recess depth of one electrode radius. After longer periods of etching, a different behavior is

observed. The cyclic voltammograms for the electrodes after 15 etching cycles show nearly

symmetrical oxidation and reduction peaks with a small peak separation of 36 mV, centered

around the formal potential for the reduction of [Ru(NH3)6]3+. This observation indicates the

formation of a nanocavity within the carbon nanoelectrode which is surrounded by quartz glass.

The nanocavity electrode has structural similarities with a leaking thin layer cell:[303] Upon

sweeping the potential, the complete amount of soluble redox mediator inside the cavity is

oxidized/reduced leading to a depletion of the current after full conversion and thus, the peak

shape of the curve. The formation of a cavity is confirmed from scanning electron microscopy

(SEM) images of electrodes before and after etching. After etching, the contrast between the

cylindrical hole and the glass sheath seems higher than the contrast between the carbon disk

and the glass, suggesting the successful removal of carbon material.

Prussian Blue is then deposited electrochemically into the recessed electrode or nanocavity by

sweeping the potential to -0.4 V in the presence of [Fe(CN)6]3- and Fe3+ (Figure 28). When

Prussian Blue is deposited on an unetched, coplanar electrode, the cathodic current plateau

corresponding to the reduction of [Fe(CN)6]3- and Fe3+ gradually increases from cycle to cycle

(Figure 28 a). This finding is attributed to a growth of the Prussian Blue film that gradually

increases the effective surface area of the electrode and thus leads to an increasing rate of

deposition. After deposition the Prussian Blue film is electrochemically activated by extensive

cycling in acidic solution containing K+ ions. During this activation process K+ is incorporated

into the crystal lattice which increases the electrochemical reversibility and the stability of the

material.[182] However, the Prussian Blue film quickly dissolves or detaches during the activation

when Prussian Blue is deposited on an unetched nanoelectrode (Figure 28 b). The CV shows the

current peaks corresponding to the redox cycling between the oxidized Prussian Blue form and



the reduced Prussian White. The decrease of the redox peaks indicates the loss of Prussian Blue

from the electrode. After typically around 100 activation cycles, the film is completely removed

from the electrode.

a

-0.5 0.0 0.5

-200

-100

0

E / V vs. Ag/AgCl

i /

pA

b

-0.5 0.0 0.5

-200

-100

0

100

140

50

i /

pA

E / V vs. Ag/AgCl

1

c

-0.5 0.0 0.5

-200

-100

0

100

i /

pA

E / V vs. Ag/AgCl

d

-0.5 0.0 0.5

-200

-100

0

100

200

11050

E / V vs. Ag/AgCl

i /

pA

1

e

-0.5 0.0 0.5

-4

-2

0

2

4

i /

nA

E / V vs. Ag/AgCl

f

-0.5 0.0 0.5

-2

0

2

140

50

i /

nA

E / V vs. Ag/AgCl

1

Figure 28. Deposition (a, c, e) and electrochemical activation (b, d, f) of Prussian Blue on an unetched (a, b), slightly recessed (c, d) and nanocavity electrode (e, f). Deposition in 1 mM K3[Fe(CN)6],

1 mM FeCl3, 0.1

M HCl, 0.1 M KCl at 0.2 V/s. Initial electrode sizes r = 144 nm (a, b), r = 114 nm (c, d) and 152 nm (e, f).



In contrast, when Prussian Blue is deposited into a slightly recessed nanoelectrode generated by

electrochemical etching, the film remains largely stable during the electrochemical activation

(Figure 28 d). The deposition of PB in the recessed nanoelectrode (Figure 28 c) features a

current plateau for the reduction of [Fe(CN)6]3- and Fe3+ that first increases from cycle to cycle

and then reaches a stable value in the last deposition cycles. Presumably, the growth of the film

is self-limited when the film thickness increases and prevents further reduction of [Fe(CN)6]3-

and Fe3+. During the activation step (Figure 28 d), the intensity of the PB/PW redox peaks

decreases during the first cycles but stabilizes after about 100 cycles. The initial decrease is

attributed to a detachment of excess material from the electrode. However, the deposition of PB

inside the recession of the etched nanoelectrode drastically increases the stability of PB. The PB

is trapped inside the recession which prevents its dissolution or mechanical detachment upon

redox cycling. The observed formal redox potential of the PB/PW transition is 0.42 V vs. SHE.

The amount of PB is increased when the material is deposited inside a deep, etched nanocavity

in the carbon nanoelectrode (Figure 28 e and f). During deposition the CV shows PB/PW redox

peaks with increasing intensity. In the activation step, the peak intensities are largely unchanged

during potential cycling and show a typical value of 2 nA as opposed to 200 pA observed for

deposition in a slightly recessed electrode. The decrease of the peak separation ΔEp observed in

Figure 28 d and f indicates the improvement of electron transfer rate due to the electrochemical

activation.

For the amperometric detection of H2O2 at PB-modified electrodes the composition of the

surrounding medium influences the performance of the H2O2 sensor. As the reduction of PB to

PW involves the incorporation of K+ into the sensing film, the effect of the K+ concentration on

the reduction of PB was tested. Figure 29 a displays cyclic voltammograms recorded in

physiological buffer solution (pH 7.4) with varying concentrations of K+. The redox peaks for the

reduction of PB and oxidation of PW shift towards more anodic potentials when the K+

concentration is gradually increased from 4 mM to 894 mM. In addition, peak sharpening is

observed at higher K+ concentration. The formal reduction potential for the PB/PW is extracted

from the CVs according to E = ½ (Ep,a + Ep,c), with Ep,a and Ep,c the anodic and cathodic peak

potential, respectively. As expected from the Nernst equation, the formal potential depends

linearly on the logarithm of the K+ concentration (Figure 29 b). However, a linear fit of the

experimental data shows a slope of 80 mV dec-1 which is higher than the value of 59 mV

expected from the Nernst equation. Most likely, the deviation from the theoretical value is due to

non-unity stoichiometric coefficients for the involvement of electrons and K+ during the

reduction of PB to PW (see also Figure 26). The fitted intercept of 181 mV vs. Ag/AgCl is an

approximation of the standard reduction potential of PB. Due to the dependence of the peak

potentials on the K+ concentration the PB-modified nanoelectrodes are suited as pseudo-



potentiometric sensors for K+, i. e. the redox potential could be measured indirectly with the

voltammetric current response as the sensor signal. Considering that the depolarization of the

cell membrane potential during action potentials at neurons is mainly caused by a flux of K+

through ion channels in the membrane, such a sensor could be used to monitor the fluctuations

in K+ concentration in and around individual neurons to investigate physiological changes

during cellular communication on the single-cell level.

a

b

-3 -2 -1 0

-0.05

0.00

0.05

0.10

0.15

0.20

E /

V v

s.

Ag

/Ag

Cl

log (c / mol l-1)

E = 0.08 V (log c) + 0.181 V

R2 = 0.97

Figure 29. The K

+ concentration influences the observed formal reduction potential for the reduction of

PB to PW. Cyclic voltammograms at a PB-modified nanoelectrode in external K+ solution (pH 7.4) with

various amounts of K+ added (a). Extracted formal potential ½ (Ep,a + Ep,c) as a function of the logarithm of

K+ concentration (b).

This work exploits the use of PB-modified nanoelectrodes for the amperometric detection of

H2O2 to evaluate oxidative cell stress on the single-cell level. For the detection reaction, the

cathodic reduction of H2O2 is chosen, as interferences by other oxidizable substances are largely

avoided.[299] To determine the suitable potential range for amperometric detection in

physiological medium, CVs in phosphate buffer (pH 7) at a PB-modified nanoelectrode were

recorded in the presence and absence of H2O2 (Figure 30 a). Whereas the curve recorded in the

absence of H2O2 (blue curve) shows the expected peak shape, the CV in a solution containing

1.8 mM H2O2 (red curve) exhibits a flat cathodic current plateau that corresponds to the

electrocatalytic reduction of H2O2 at diffusion-limited rate. The onset of the steady-state current

coincides with the cathodic peak for the reduction of PB to PW which confirms the role of PW as

the active electrocatalyst. The range of the current plateau indicates that H2O2 can be detected at

mass transport-limited rate at potentials more cathodic than 0.1 V vs. Ag/AgCl/0.1 M Cl-. For all

experiments involving PB-modified electrodes as H2O2 sensors, detection potentials slightly

more negative than the cathodic peak appearing in the CV were chosen. Figure 30 b shows the

current response for the reduction of hydrogen peroxide at a constant potential of -0.05 V when

the H2O2 concentration is increased stepwise. The large current fluctuations are due to

instrumental and electrochemical noise introduced when the Faraday cage containing the cell

was opened, H2O2 solution was added and the solution was agitated. However, the



chronoamperogram exhibits stable current plateaus with increasing intensity as more H2O2 is

added. The calibration curve of the current values vs. the H2O2 concentration exhibits a linear

trend for concentrations between 10 µM and 3 mM (Figure 30 c). The limit of detection (LOD),

defined as the analyte concentration at which the sensor response exceeds 3 standard deviations

of the sensor response in the absence of analyte, is typically 10 µM and is largely dictated by the

instrumental noise when measuring small currents below the pA range. The root-mean-square

noise for the shown measurements was typically 50-100 fA whereas the sensor response at the

LOD was a few hundreds of fA.

a

-0.2 0.0 0.2 0.4

-10

0

10

i /

pA

E / V vs. Ag/AgCl

b

500 1000 1500 2000

-150

-100

-50

0

i /

pA

t / s

c

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0

50

100

150

200

-i /

pA

c / mmol l-1

0.01 0.1 1

1

10

100

Figure 30. PB-modified nanoelectrodes detect H2O2 with high sensitivity. CVs in 0.05 M phosphate buffer pH 7.0, 0.1 M KCl before and after addition of 1.8 mM H2O2 (a). Scan rate 0.05 V/s. Amperometric detection of H2O2 at -0.05 V vs. Ag/AgCl/0.1 M Cl

- upon stepwise addition of H2O2 standards (final concentrations 1

µM to 2.8 mM) (b). Calibration of the H2O2 reduction currents from (b) with respect to the H2O2 concentration (c). A logarithmic representation is shown in the inset. Recessed nanoelectrode with r = 108 nm in (a) and nanocavity electrode with r = 152 nm (b).

The concentration of H2O2 in cells under normal conditions is estimated below 1 µM.[169]

However, oxidative stress is associated with H2O2 levels in excess of 10 µM. Especially when

considering that most previous measurements determined the H2O2 concentration as an average

over extended periods of time, the transient H2O2 levels during oxidative bursts might be higher.

Hence, PB-modified nanosensors are sufficiently sensitive to detect H2O2 in oxidative stress

situations or during stimulated H2O2 production.[173,175] The upper detection limit is limited by

the stability of the sensor. At H2O2 concentrations substantially higher than 3 mM the sensor



response rapidly decreases due to dissolution of the PB film in the presence of excess amounts of

H2O2. To compare the sensitivity of the sensors, the slope in the linear range of the calibration

curves was normalized by the surface area of the active nanoelectrode. For electrodes where

Prussian Blue was deposited into a slight recession after gentle etching (see also Figure 27 and

Figure 28) the average sensitivity for H2O2 is 13 ± 8 A mol-1 l cm-2. Sensors where PB was

deposited in deep nanocavities showed significantly higher sensitivity of 50 ± 30 A mol-1 l cm-2.

Because the latter sensors exhibit thicker PB films, the amount of the PB seems to be a critical

parameter for the performance of the sensors. Compared to previous studies reporting PB-based

H2O2 determination the present sensor has the smallest dimensions and one of the highest

values for the sensitivity.[182,304] However, the etching creates a channel inside the glass sheath

that has to be passed by the analyte before reaching the PB film inside the cavity, resulting in a

loss of sensitivity and increase of the response time due to slow planar diffusion inside the

cavity. As predicted for recessed electrodes[302] the steady-state currents observed for H2O2

reduction are only slightly diminished with respect to the maximum theoretical value expected

for ideal planar disk-shaped geometry. Taking the average sensitivity of 50 ± 30 A mol-1 l cm-2

and a typical electrode radius of 150 nm, the resulting calculated current is only three times

smaller than the ideal value obtained from the equation iss = 4.64∙r∙F∙c∙D (with

D = 1.7∙10−5 cm2 s−1).[294,305]

a

0 5 10

0.0

0.5

1.0

i /

i 0

t / h

b

Figure 31. Long-term stability of H2O2 sensors during constant detection in 2.9 mM (a) and 0.2 mM H2O2

(b). Currents for reduction H2O2 normalized by initial currents at t = 0 for PB deposited on a slightly recessed nanoelectrode and in a nanocavity electrode. Detection in 0.05 M Phosphate buffer pH 7.0, 0.1 M KCl at -0.05 V.

As the chemical stability of PB in neutral or alkaline solution has proven to be a critical

parameter for many sensor designs, the long-term stability for different sensors and different

conditions was tested at constant operation of the sensors. At neutral pH and in the presence of

millimolar concentrations of H2O2, the initial sensor response rapidly decreases for both slightly

recessed and nanocavity electrodes (Figure 31 a). After 5 hours of operation, only 50 % and

30 % of the initial current signal are retained for recessed and nanocavity electrodes,



respectively. When less H2O2 is present (200 µM), both sensor types produce a stable current

signal during the first hour and still exhibit 80-90 % of sensitivity after 5 hours of constant H2O2

reduction. The stability is sufficient for typical experiments aiming at the detection of H2O2 in or

around single cells. These experiments have a duration of typically less than 2 hours and do not

require constant analyte detection during the entire experiment.

4.3.3 H2O2 measurements at single living cells

PB-modified carbon nanoelectrodes are suitable to target single cells to evaluate the role of H2O2

in the context of oxidative cell stress. To demonstrate the feasibility of intracellular H2O2

detection, dorsal root ganglia (DRG neurons) were used as a cell model. Prior to the cell

experiments, a one- or two-point calibration for H2O2 was performed in the cell medium

(external K+ solution). Since the linearity of the calibration curves in a wide concentration range

was already demonstrated (Figure 30), this quick calibration method is a good compromise

between the quantification of H2O2 and the overall duration of the experiments using living cells.

To measure the intracellular H2O2 concentration, the PB-modified sensors were mounted on a

micromanipulator in an angle of about 45° with respect to the Petri dish containing the cultured

cells in buffer solution. Using an optical microscope, the sensors were advanced to single

neurons under optical control until touching or nearly touching the cell surface. Then, the sensor

was moved 1 µm along the angular direction to impale the cell membrane while simultaneously

monitoring the current with a suitable detection potential applied to the electrode. Figure 32 a

shows optical micrographs of the nanosensors and a cell before and after successful penetration.

Due to the small overall size of the sensors, cell impalement was achieved with nearly 100 %

efficiency. After retraction of the sensor, the morphology of the cell is unchanged, indicating that

the cells are still alive after the penetration experiments. In control experiments bare carbon

nanoelectrodes without PB modification were inserted into cells while applying different

detection potentials (Figure 32 b, c). Irrespective of the applied potentials (ranging from -0.15 V

to + 0.1 V) no current signals are observed upon cell impalement. In contrast, large cathodic

current spikes with a duration of about 2 seconds are observed for inserting PB-modified

nanosensors into cells (Figure 32 d, e). Assuming that the observed current is only due to

electrocatalytic conversion of H2O2, the hydrogen peroxide concentration in the moment of

penetration reaches a few hundreds of µM or even the millimolar range. Two distinct typical

shapes for the chronoamperograms are observed. In some experiments, a large cathodic current

spike is followed by an anodic signal (Figure 21 d). The origin of the latter signals is unknown. At

the applied potential of -0.05 V no anodic oxidation of H2O2 at PB-modified electrodes is known.

Another typical curve shape does not show such anodic signal (Figure 32 e). Instead, the initial

cathodic current peak slowly decays and approaches 0.



a

b

0 20 40

-1.0

-0.5

0.0

0.5

1.0

i /

pA

t / s

c

0 20 40

-1.0

-0.5

0.0

0.5

1.0

i /

pA

t / s

d

0 10 20 30 40 50

-100

-50

0

200

100

0

c (

H2O

2)

/ µ

M

i /

pA

t / s

4 6 8 10

-100

-50

0

200

100

0

e

80 100 120

-50

0

4.5

0.0

c(H

2O

2)

/ m

M

i /

pA

t / s

75 80

-50

0

4.5

0.0

Figure 32. H2O2 nanosensors are inserted into DRG neurons to perform intracellular measurements. Optical micrographs of the nanosensors before and after penetration of the cell and after retraction of the sensor (a). Control experiments for inserting unmodified, bare carbon nanoelectrodes into neurons at an applied potential of -0.05 V (b) and -0.15 V (c). Cathodic current response of two different sensors upon impalement of two different cells (d, e) at a detection potential of -0.05 V (d) and -0.15 V (e). Red and blue arrows indicate the moment of cell penetration and retraction of the sensor, respectively. The insets show an enlargement of the current upon penetration. The right axes show the measured H2O2 concentration calculated from the calibrations. Sensor sizes 77 nm (b, c), r = 110 nm (d) and 106 nm (e).

When inserted into the cell, the sensor suddenly faces a chemical environment which is different

from the medium solution outside of the cell. Besides many other constituents of the cytosolic

matrix, an important difference in the composition of the intracellular and the extracellular

medium is the K+ concentration. The K+ gradient is the major contributor to the Nernstian cell

membrane potential. The K+ concentration inside of the cell is above 100 mM while outside only

a few mM K+ are present. As it was shown previously (Figure 29), the redox state of the PB at a

given applied potential depends on the K+ concentration. Hence, it is conceivable that the signals

observed during insertion of the PB-modified sensors are partially caused by a jump in the K+

environment of the sensor that could lead to a change in the concentration ratio PB/PW and

hence a faradaic current. To evaluate the effect of the different chemical environments inside

and outside of the cell membrane, CVs were recorded with the sensor inserted into the cell and



after retraction from the cell (Figure 33 a). The two curves show the redox peaks for PB

reduction and PW oxidation with very similar peak potentials and peak currents. Considering

that the K+ concentration in the cytosol and in the external solution are around 140 mM and

4 mM, respectively, the redox peaks recorded inside the cell are expected to shift anodically by

0.12 V when assuming the K+ sensitivity displayed in Figure 29. In contrast, the observed formal

potential E = ½ (Ep,a + Ep,c) of the PB inside the cell is nearly identical to the one observed with

the sensor located outside the cell (- 0.017 V and -0.008 V vs. Ag/AgCl/0.14 M Cl-, respectively).

Hence, the difference between the intracellular and the extracellular environment does not

strongly affect the redox state of the PB film and thus is unlikely to create any measurement

artifacts during the intracellular measurement of H2O2.

a

-0.5 0.0 0.5 1.0

-100

-50

0

50

100

i /

pA

E / V vs. Ag/AgCl

b

-0.5 0.0 0.5 1.0

-500

0

500i /

pA

E / V vs. Ag/AgCl

Figure 33. Influence of cell penetration experiments on the state of the PB film on nanoelectrodes. CVs at a PB-modified sensor inside and outside (in ext. K

+ solution) of a DRG neuron (a). CVs in 0.1 M HCl, 0.1 M

KCl before and after a series of penetration experiments.

However, during the cell penetrations the sensitivity of the sensor decreases. Figure 33 b shows

CVs for the redox cycling of PB and PW before and after a series of penetration experiments.

During these measurements, the amount of PB in the nanoelectrode sensor decreases, as

indicated by the decrease of peak intensities and peak areas observed in the CV. Repeated H2O2

calibrations after cell penetration experiments show that the sensors are still sensitive to

hydrogen peroxide, however exhibit a sensitivity that is decreased by an average of 50 %. The

loss of sensitivity can be caused by a loss of the amount of PB as well as cell debris and

membrane fragments partially passivating the modified electrodes.

In conclusion, PB-modified carbon nanoelectrodes are well suited for intracellular

measurements to assess the H2O2 production of cells during oxidative stress situations. The

small sensor size allows efficient penetration of cells and minimizes damage caused to the cells.

The cathodic current spikes observed during cell impalement are due to the reduction of

endogeneous H2O2. However, more careful evaluation of the H2O2 concentrations observed

inside the cell is necessary. In particular, the sensor calibration is performed in the medium



outside of the cell. Thus, the validity of the calibration for measurements in the cytosol, which

has a different and very complex composition, has to be assessed critically. Because it is difficult

to mimic the complex cytosolic matrix artificially, the focus of future investigations should be on

methods aiming at a sensor calibration inside the living cell. In the presented experiments,

surprisingly high transient H2O2 concentrations up to the millimolar range are observed

immediately after cell penetration. Despite the sensors have relatively small dimensions, it is

conceivable that these signals are at least partially caused by the mechanical disturbance caused

by puncture of the cell membrane. This behavior was – despite using larger electrochemical

probes – observed previously.[176] Thus, more thorough investigation is necessary to link the

measured H2O2 production to specific physiological conditions that favor the production of ROS

species. To give unambiguous evidence for the correlation between observed sensor signals and

the cellular mechanisms to produce ROS and H2O2 in particular, oxidative cell stress can be

induced artificially using a number of reagents or the cells’ natural protection mechanisms can

be specifically inhibited. Macrophages are known to kill bacteria as part of the immune response

to pathogens. Bacteria are engulfed into phagosomes and attacked by excess amounts of O2- and

NO which are released into the phagosomes by activity of NADPH oxidase and nitric oxide

synthase.[157] Murine macrophages have been previously used to study ROS and RNS production

by means of voltammetric methods.[176] To stimulate the macrophage cells, lipopolysaccharides

from E. coli (LPS) have been used previously.[173,175] Macrophages recognize the

lipopolysaccharide fragments as a potential pathogen and start an inflammatory response

primarily by increased expression of NOS which leads to enhanced production of NO.[306]

However, LPS stimulation also leads to an increased release of superoxide which is closely

related to H2O2 levels via the activity of superoxide dismutase (Figure 5). [160,307]

a

0 50 100

-8

-6

-4

-2

0

2

134

101

67

34

0

-34

c(H

2O

2)

/ µ

M

i /

pA

t / s

b

0 100 200

-5

0

5

10

84

0

-84

-167

c (

H2O

2)

/ µ

M

i /

pA

t / s Figure 34. Immunostimulated macrophages release H2O2 into the extracellular environment. Cell penetration experiments after stimulation with 5 µg/ml LPS. The arrows indicate the moments when the sensor is positioned close to the cell, penetration and retraction of the sensor.

Hence, macrophages were incubated with LPS and PB-modified nanoelectrodes were used to

evaluate the increased production of H2O2. Figure 34 shows two typical current-time curves



recorded when the H2O2 sensor is positioned in proximity to an LPS-stimulated macrophage cell

followed by puncture of the cell membrane. Notably, already in the beginning of the curve when

the sensor is in contact with the cell membrane or positioned only a few µm away from the cell

cathodic currents are recorded which are attributed to extracellular H2O2. From the

corresponding sensor calibrations the concentration of released H2O2 in direct proximity of the

cell surface is calculated to be in the range of several tens of µM. Some experiments (5 of 12)

show cathodic peaks after cell penetration which are attributed to the reduction of H2O2 in the

cell (Figure 34 a). However, another typical curve shape (7 of 12) exhibits one or more anodic

peaks after inserting the sensor into the cell (Figure 34 a). To elucidate the origin of these signals

requires further investigation.

a

b

40 60 80 100

-2

-1

0

65

32

0

c(H

2O

2)

/ µ

M

i /

pA

t / s

c

100 120 140 160

-8

-6

-4

-2

0

2

258

194

129

65

0

-65

c(H

2O

2)

/ µ

M

i /

pA

t / s

d

no reagent 3-AT

0

2

4

6

(n=7)

t 1/2 /

s

(n=8) Figure 35. Inhibition of catalase in macrophage cells draws out the duration of H2O2 outbursts. Optical micrograph of the cell penetration. Current response of a H2O2 sensor during penetration of a macrophage cell after incubation with 20 mM 3-AT to inhibit catalase (b) and without adding an inhibiting reagent (c). Comparison of cathodic peak half-life between catalase-inhibited and non-treated cells (d). The column height represents the median and whiskers show the 25

th and 75

th percentiles.

Cells have a number of ROS scavenging systems to protect themselves from a detrimental

overabundance of ROS. For instance, hydrogen peroxide in the cytosol is efficiently eliminated

by catalase which accelerates the disproportionation of H2O2. Specific inhibition of catalase has

been previously achieved using 3-amino-1,2,4-triazole (3-AT).[308] When cells are incubated in



presence of millimolar concentrations of 3-AT, the inhibitor covalently binds to catalase and

leads to an accumulation of H2O2 and consequently modifications of proteins, lipids and DNA in

the cell.[148,149] Thus, 3-AT can be used to evaluate the cell’s natural protection mechanisms

against excess amounts of H2O2. Also, for a more thorough assessment of the sensor response

during ROS production by the cell, macrophage cells were chosen as the cell model. Figure 35

shows the results of intracellular H2O2 measurements in cultured murine macrophages. The

current-time traces show similar shapes as seen for DRG neurons. In the moment of cell

impalement, a cathodic current spike is observed, followed by a decay of the current signal

(Figure 35 b). After addition of 3-AT to the cell medium, and thus after inhibition of catalase, the

decay of the cathodic peak is substantially retarded (Figure 35 c). Figure 35 d shows a

comparison of the peak half-lifes, i.e. the times after the penetration until the cathodic current

peaks have decreased by 50 %. After inhibition of catalase, the average half-life is 1.4 s whereas

cells not treated with 3-AT exhibited a half-life of 0.8 s. This behavior is attributed to the cell’s

diminished capability to annihilate H2O2 when a major part of its natural protection mechanisms

against ROS species is partially dysfunctional. These results demonstrate the utility of PB-

modified nanoelectrodes to evaluate the antioxidant capabilities of cells. In the future, this

technique may allow to correlate dysfunctionalities of protective mechanisms against an

overabundance of ROS with the development of pathogenic conditions. Because single cells are

targeted, the fate of individual cells in the development of diseases could be monitored.


4.4 Potentiometric Sensors on Nanoelectrodes 71

4.4 Potentiometric Sensors on Nanoelectrodes

Experiments were performed by Anna Muhs, Ruhr-Universität Bochum in a student project.

The results shown above demonstrate the capabilities of nanoelectrodes for the study of single

living cells. Nanoelectrodes are specifically modified by depositing electroactive materials on

their surface to improve the resulting sensor’s sensitivity and/or selectivity for certain analytes.

Until now, these electrochemical probes are amperometric sensors, i.e. the faradaic current

generated for the oxidation or reduction of an analyte is measured as a function of the analyte

concentration. As the dimensions of the active sensor decrease to the nanoscale and small

analyte concentrations are to be detected, amperometric sensors are limited by the capabilities

of state-of-the-art electronics current amplifiers, that is, the electronic noise of the instruments

becomes larger than the signal. The development of more powerful current amplifiers and

optimization of the measurement environment such as improved electromagnetic shielding and

isolation from vibration and thermal fluctuations may go some way in pushing forward the

reliable detection of small currents. However, it is unlikely that these strategies will allow to

measure only a few electrons passing nanometric electrodes in a faradaic detection reaction. For

instance, a current of 1 fA still corresponds to roughly 6000 electrons transmitted per second.

Hence, strategies to overcome this sensitivity limit have to be developed to boost the utility of

nanoelectrodes in sensing applications.

Potentiometry is known to allow chemical analysis with superior sensitivity. The analytical

signal is directly depending on the activity of the analyte, not the total amount. Moreover, given

that the sensor is small, virtually no analyte is consumed. This ensures that potentiometric

sensors are very powerful tools for detecting small analyte concentrations in small volumes,

which is particularly interesting for detection inside cells. For example, potentiometric sensors

were capable of detecting 100 pM Ca2+, Ag2+ and Pb2+ in only 3 µl sample volume which

corresponds to a total amount of 300 amol.[309] Previous designs of potentiometric sensors

mainly employ ion-selective membranes that permit the transfer of a specific analyte while

blocking access of other species to the electrode.[310–314] The concentration gradient built up at

these sensor interfaces creates the difference in electric potential that is described by the Nernst

equation. The pH electrode, where protons diffuse into a porous glass membrane to build up a

potential gradient is the most widespread realization of this concept. This principle was also

applied for micrometric sensors. For instance, glass pipettes with an opening of a few

micrometer or smaller and filled with selective ionophores have found widespread use in pH[315]

or Na+ and K+ sensing.[202,316,317] Alternatively, all-solid-state potentiometric sensors have been

proposed using metal electrodes at which the analyte specifically adsorbs at the interface.[318] It

was demonstrated that glass nanopipettes filled with carbon and subsequently platinized can



potentially serve as potentiometric probes to measure the membrane potential upon cell

penetration.[319] The applications for chemical detection are, however, restricted to

measurement of the potential of a solution containing large concentrations of the simple model

redox pair ferricyanide/ferrocyanide and the capabilities for sensitive and dynamic detection of

small analyte concentrations are unrecognized.

No faradaic reactions were involved in the sensing scheme. However, potentiometric sensors

can be extended to exploit faradaic processes: The open circuit potential of an electrode

immersed in solution is dictated by the sum of all redox active compounds in contact with the

electrode. However, only the redox reactions showing the fastest reaction kinetics at that given

electrode surface force their redox potential upon the electrode. For an electrochemical sensor

designed to selectively detect only one analyte the electrode surface has to be modified such that

the overpotential for one particular reaction has is decreased to allow fast kinetics while the

electrode has to be made insensitive for other processes. While this was the main goal in most of

the extensive research in the development of amperometric sensors in the past decades, the

concept has found little realization in potentiometric sensors yet.

By depositing redox active species on an electrode surface, a sensor for potentiometric detection

of faradaic processes can be created. The potential of the sensing electrode is then dictated by

the redox active moieties in the mediating layer. The sensitivity of such a potentiometric sensor

for faradaic processes can be controlled by adjusting the amount of redox active species in the

film. A small amount of analyte can change the potential of the electrode drastically if the

electrode is modified by only a small amount of redox active species. The mediating layer needs

to cover the sensor surface completely to exclude interferences. Hence, small electrodes are

particularly well suited to construct sensors with superior sensitivity because a small amount of

redox active moieties is sufficient to cover the entire sensor surface. This is in contrast to

amperometric sensors where sensitivity issues are encountered in particular for smaller

sensors.

Prussian Blue-modified nanoelectrodes are well suited to demonstrate this principle in the

following considerations and experiments. PB meets the above stated criterion of selectivity. The

reduction of the analyte H2O2 is favored over other possible redox reactions of all species in the

solution. According to the Nernst equation, the open circuit potential EOCP of the PB-modified

electrode is dictated by the ratio of activities of the oxidized form PB and the reduced form PW

in the film:

𝐸𝑂𝐶𝑃 = 𝐸0 +

0.059 𝑉

𝑧𝑙𝑜𝑔

𝑎𝑃𝐵

𝑎𝑃𝑊

(2)



E0 is the standard reduction potential for the reduction of PB to PW, z refers to the number of

transferred electrons and a denotes activities of the redox active species. Equation (2) can be

expressed in terms of the amounts of PB and PW in the film because the activity coefficients in

the standard state are unity and the volume of the film is assumed to be constant.

𝐸𝑂𝐶𝑃 = 𝐸0 +

0.059 𝑉

𝑧𝑙𝑜𝑔

𝑛𝑃𝐵

𝑛𝑃𝑊

(3)

The presence of H2O2 will change the amounts of PB/PW in the film with a first order reaction

kinetics law with respect to the H2O2 concentration (see also Figure 26):

𝑑𝑛𝑃𝐵

𝑑𝑡= 𝑘 [𝐻2𝑂2]

(4)

𝑑𝑛𝑃𝑊

𝑑𝑡= −𝑘 [𝐻2𝑂2]

(5)

[H2O2] is the bulk concentration of hydrogen peroxide and k is the apparent rate constant for the

oxidation of PW caused in the presence of H2O2. k is determined by not only the electron transfer

rate of H2O2 oxidation at the PB-modified electrode but also contains the mass transport rate of

H2O2 to the electrode which itself is a function of the electrode geometry. The integrated forms of

the rate laws are:

𝑛𝑃𝐵(𝑡) = 𝑘𝑡[𝐻2𝑂2] + 𝑛𝑃𝐵(𝑡 = 0) (6)

𝑛𝑃𝑊(𝑡) = −𝑘𝑡[𝐻2𝑂2] + 𝑛𝑃𝑊(𝑡 = 0) (7)

If the sensor is sufficiently small with respect to the volume of the electrochemical cell, the

consumption of H2O2 is negligible and its concentration can be regarded as constant. Hence, a

new rate constant K is defined as the apparent turnover of PW in the presence of H2O2.

𝑛𝑃𝐵(𝑡) = 𝐾𝑡 + 𝑛𝑃𝐵(𝑡 = 0) (8)

𝑛𝑃𝑊(𝑡) = −𝐾𝑡 + 𝑛𝑃𝑊(𝑡 = 0) (9)

Equations (8) and (9), inserted in equation (3) express the temporal dependence of the open

circuit potential of the electrode:

𝐸𝑂𝐶𝑃(𝑡) = 𝐸0 +

0.059 𝑉

𝑧𝑙𝑜𝑔

𝐾𝑡 + 𝑛𝑃𝐵(𝑡 = 0)

−𝐾𝑡 + 𝑛𝑃𝑊(𝑡 = 0)

(10)

Not the OCP itself but the change of OCP with time at different H2O2 concentrations is taken as

the analytical signal of the sensors. In the beginning of such a detection experiment, the OCP is

far cathodic of the standard redox potential of PB/PW, so that only PW is present:

𝑛𝑃𝐵(𝑡 = 0) = 0 (11)

𝑛𝑃𝑊(𝑡 = 0) = 𝑛𝑡𝑜𝑡𝑎𝑙 (12)

ntotal is the overall amount of PB/PW deposited on the electrode. Hence, equation (10) simplifies

to equation (13).



𝐸𝑂𝐶𝑃(𝑡) = 𝐸0 +

0.059 𝑉

𝑧𝑙𝑜𝑔

𝐾𝑡

−𝐾𝑡 + 𝑛𝑡𝑜𝑡𝑎𝑙

(13)

According to Faraday’s law of electrolysis, the total amount of PB/PW can be estimated from

experimental data from the charge transmitted during the voltammetric peak for the reduction

of PB. Figure 36 a and b show CVs for a sensor with a large PB deposit (ntotal = 6 ∙ 10-14 mol) and a

smaller deposit (1 ∙ 10-16 mol).

As it can be seen from equation (13), the sensor works analogous to a dosimeter that returns the

OCP of the electrode as the accumulated response to exposure to H2O2. In Figure 36 c-e the

calculated course of the open circuit potentials are plotted for different rates of oxidation of PW

by H2O2. The curves start at a fully reduced state, i.e. PW is the predominant species in the film.

Then, as initially PW is converted into PB, the OCP of the sensor increases with time.

a

0.0 0.5

-5

0

5

i /

nA

E / V vs. Ag/AgCl

b

0.0 0.5

-20

-10

0

10

i /

pA

E / V vs. Ag/AgCl

c

0 50 100

-0.2

0.0

0.2

0.4

0.6

10 pA

100 pA

1 pA10

-17 mol/s

10-16

mol/s

10-15

mol/s

E /

V v

s.

Ag

/Ag

Cl

t / s

10-14

mol/s1 nA

d

0 50 100

-0.2

0.0

0.2

0.4

0.6

10 fA10

-19 mol/s

100 fA10

-18 mol/s

10 pA

1 pA10

-17 mol/s

E /

V v

s.

Ag

/Ag

Cl

t / s

10-16

mol/s

e

0 50 100

-0.2

0.0

0.2

0.4

0.6

100 aA10

-21 mol/s

1 fA10

-20 mol/s10 fA

10-19

mol/s100 fA10

-18 mol/s

E /

V v

s.

Ag

/Ag

Cl

t / s Figure 36. The sensitivity of potentiometric PB sensors increases with decreasing the total amount of PB/PW deposited on the electrode. Experimental CVs of sensors with a large amount (ntotal = 6 ∙ 10

-14 mol)

(a) and a small amount of PB/PW (1 ∙ 10-16

mol) (b) in 0.1 M HCl, 0.1 M KCl. Theoretical OCP curves according to equation (13) at different turnover rates of PW and different total amounts of PB/PW. ntotal = 5 ∙ 10

-14 mol (c), 1 ∙ 10

-16 mol (d) and 1 ∙ 10

-18 mol (e).

The highest turnover rate in Figure 36 c of 10-14 mol/s corresponds to a current of 1 nA that

would be observed at the same turnover rate at an amperometric H2O2 sensor. Consequently, the

lowest turnover rate in Figure 36 e corresponds to 100 aA. The sensitivity of potentiometric PB



sensors increases with decreasing amount of ntotal. Figure 36 c and d show the theoretical OCP

curves for the large and small PB/PW amounts experimentally determined in Figure 36 a and b.

For the large PB sensor, marked differences in the OCP curves are only observed between

turnover rates from 10-16 to 10-14 mol/s whereas the smaller sensor may distinguish between

H2O2 concentrations leading to turnover rates between 10-18 and 10-16 mol/s. Even higher

sensitivity can be obtained when further decreasing the amount of electroactive material on the

sensor surface. For a sensor carrying a PB amount approx. 100 times smaller than the one

shown in Figure 36 b, a turnover rate of as little as 10-21 mol/s (ca. 600 molecules per s) is

sufficient to change the OCP in a reasonable amount of time and thus to induce a noticeable

sensor response. The predicted behavior is confirmed by experiments. Figure 37 shows

experimental OCP curves recorded at PB-modified electrodes in acidic solution containing

different concentration of H2O2. During each experiment, the sensors were first polarized to a

potential of -0.4 V vs. Ag/AgCl/3 M Cl-. This potential is substantially more cathodic than the

formal potential of the PB/PW couple (0.2 V vs. Ag/AgCl/3 M Cl-). Hence, predominantly the

reduced form PW was present in the initial stage of the experiment. After the initial charging of

the sensor, the electrochemical cell was switched off at t = 0 and the change of OCP was

monitored.

a

0 20 40 60

-0.4

-0.2

0.0

0.2

0.4

E /

V v

s.

Ag

/Ag

Cl

t / s

no H2O

2

0.5 mM

1 mM

1.5 mM

2 mM

2.5 mM

b

0 20 40

-0.4

-0.2

0.0

0.2

E /

V v

s.

Ag

/Ag

Cl

t / s

no H2O

2

5 µM

50 µM

500 µM

Figure 37. The OCP vs. time curves observed at PB-modified nanoelectrodes change with the H2O2 con-centration. Experimental curves measured at two different sensors with ntotal = 2 ∙ 10

-15 (a)

ntotal = 8 ∙ 10-15

mol (b) in 0.1 M HCl, 0.1 M KCl with different concentrations of H2O2.

The experiment was repeated in solutions with different concentrations of H2O2. As expected,

the OCP-time curve recorded in the absence of H2O2 is flat and the curves become steeper when

the H2O2 concentration is gradually increased to 2.5 mM (Figure 37 a). Hence, the observed

change in OCP is largely due to oxidation of PW to PB by H2O2. To assess the sensitivity of the

potentiometric sensor, the OCP is measured at smaller concentrations of H2O2 (Figure 37 b).

Compared to the blank measurement, the slope of the OCP curve is markedly higher in 50 µM

and 500 µM H2O2. However, in the presence of only 5 µM the OCP does not change faster as



compared to the behavior seen in the absence of H2O2. Thus, the sensitivity of the sensor does

yet not allow to detect H2O2 concentrations smaller than around 50 µM. It is the fast change of

the OCP in the background measurement without H2O2 that precludes the distinction of the

curves and thus the detection of small analyte concentrations. This is attributed to the effect of

the strongly reducing potential of -0.4 V applied for initial charging of the sensor. At this

cathodic potential, presumably parasitic reduction reactions such as the reduction of O2 may

occur and thus change the redox state of the PB/PW sensor even in the absence of H2O2. To

optimize the sensing conditions, the OCP measurement was tested with different starting

potentials for the charging of the sensor (Figure 38). When the starting potential is below the

formal redox potential of the PB/PW couple (-0.3 V and 0.05 V in Figure 38 a and b,

respectively), the OCP response shows a rapid increase in the beginning of the experiment for all

tested concentrations and also in the background measurement in the absence of H2O2. This is

due to the above mentioned high driving force for parasitic cathodic reactions. When the initial

potential is close to or higher than the formal potential of the surface-confined redox species

(0.2 V and 0.275 V), the OCP in the background measurement is stable (Figure 38 b and c). In the

absence of H2O2 no oxidation of PW takes place and thus the redox state of the PB-modified

nanoelectrode remains unchanged. When H2O2 is added, the OCP still changes rapidly. Thus, by

tuning the starting potential of the potentiometric measurement, the background signal can be

suppressed which allows more reliable detection of small analyte concentrations.

a

0 50 100

-0.2

0.0

0.2

E /

V v

s.

Ag

/Ag

Cl

t / s

b

0 50 100

0.0

0.2

0.4

0.6

E /

V v

s.

Ag

/Ag

Cl

t / s

c

0 50 100

0.2

0.4

0.6

E /

V v

s.

Ag

/Ag

Cl

t / s

d

0 50 100

0.4

0.6

0.8

E /

V v

s.

Ag

/Ag

Cl

t / s

Figure 38. OCP measurements at PB-modified sensors in absence of H2O2, 500 µM and 1 mM H2O2 and 0.1 M HCl, 0.1 M KCl at different starting potentials of -0.3 V (a), 0.05 V (b), 0.2 V (c) and 0.275 V (d).

In conclusion, a potentiometric detection scheme for the measurement of redox active analytes

is a promising alternative to amperometric detection. The key difference and main advantage

compared to amperometric sensors is that the sensitivity of the faradaic potentiometric sensor

increases with decreasing sensor size. By modifiying nanoelectrodes with redox active materials



that dictate the OCP of the nanoelectrode, the sensor works analogous to a dosimeter. The

sensor can be recovered by applying a potential to restore the intial redox state. A repeated

sequence of potentiostatic recovery of the sensor followed by OCP measurement will allow

continuous detection of analytes. The principle of a potentiometric sensor for the detection of

faradaic reactions is demonstrated in this work using PB as the sensitive film for H2O2

measurements. Further optimization and characterization of the PB-based potentiometric

nanosensors has to follow before the sensor can be used in a real analytical application.

However, the presented method is generic. Any type of redox active molecules or materials that

i) have a reversible redox transition and ii) can be deposited on a nanoelectrode surface are

suitable for this detection scheme. This may empower nanoelectrodes for the detection of very

small analyte concentrations with unprecedented sensitivity.


4.5 Field Effect Transistor Sensors on Nanoelectrodes 78

4.5 Field Effect Transistor Sensors on Nanoelectrodes

Experiments were performed in collaboration with Yanjun Zhang and other researchers from the

Division of Medicine at the Imperial College London, who also provided cell samples. Parts of the

following section are also published in ref. [320]: “Zhang, Y.*; Clausmeyer , J.*; Babakinejad, B.*;

López Córdoba, A.; Ali, T.; Shevchuk , A.; Takahashi , Y.; Novak , P. Edward , C.; Lab, M.; Gopal, S.;

Chiappini, C.; Anand, U.; Magnani, L.; Coombes, C.; Gorelik, J.; Matsue, M.; Schuhmann, W.;

Klenerman, D.; Sviderskaya, E.; Korchev, Y.; ACS Nano 2016, In Press, DOI:

10.1021/acsnano.5b05211”


4.5.1 pH-sensitive polypyrrole FETs on dual carbon nanoelectrodes

The sensitivity of amperometric nanosensors is limited by the technical ability to measure small

currents below the pA range. Field effect transistor sensors overcome this problem by indirectly

measuring the electric potential change upon interaction of the FET with the analyte. The

potential modulates a large and easy-to-measure current signal passing through the transistor

channel. Due to the intrinsic amplification, the current has a high signal-to-noise ratio and thus

allows highly sensitive measurement and potentially even the detection of single molecules.

However, the complex fabrication and large sensor dimensions of state-of-the-art FET sensors

often preclude their application to measurements in small confined volumes especially for the

analysis of cells.

To overcome this problem, dual carbon nanoelectrodes are used as templates to form a FET

sensor that can be freely moved in space and has a high spatial resolution to target single cells. A

quartz Θ-capillary is pulled into a double-barrel nanopipette using a laser puller. Then, pyrolytic

decomposition of butane deposits conductive carbon, and produces two individually

addressable carbon nanoelectrodes, separated by a few nanometer thin glass wall.[99,103] The two

electrodes are of the same quality as single barrel carbon nanoelectrodes, however have a

semicircular shape. A CV with ferrocenemethanol as the redox mediator shows steady-state

current plateaus for fast ferrocenemethanol oxidation. Both electrodes show approximately the

same current, indicating similar size.



0.0 0.2 0.4 0.6

0

5

10

i /

pA

E / V vs. Ag/AgCl

Figure 39. CV at a dual carbon electrode (electrode 1, electrode 2) in 1 mM ferrocenemethanol, 0.1 M KCl, 0.2 V/s.

Equation (1), normally used to estimate the size of single barrel nanoelectrodes from the

limiting steady-state current, is only valid for circular electrodes surrounded by a coaxial

insulator. Hence, applying this equation to dual carbon electrodes can only give a rough

estimation of their size. For instance, the steady-state currents observed in Figure 39

correspond to a radius of 25 nm for each electrode assuming circular geometry. To generate a

field effect transistor, a semiconducting material is deposited on the dual carbon electrode to

comprise the channel of the transistor. To demonstrate the possibility of fabricating a functional

FET sensor on the tip of a dual carbon electrode, a polypyrrole (PPy) nanojunction is used as the

transistor channel. PPy is a conducting polymer with properties similar to a p-type

semiconductor. The polymer is deposited by an electrochemically initiated radical

polymerization. Upon applying anodic potential the pyrrole monomer is oxidized and forms first

dimers, then oligomers and finally PPy. Electric conduction occurs when PPy is oxidized, so that

positive charge carriers are introduced into the polymer backbone which then transport

electrical current.

Figure 40. Mechanism of electropolymerization and electrical conduction of PPy.



FET biosensors based on conducting polymers, especially PPy, are advantageous because of the

ease of electropolymerization and functionalization, good biocompatibility and chemical

stability.[212,321] PPy has given rise to various FET sensor designs.[213–215,322,323] For instance, single

PPy nanowires were electrochemically grown between the drain and source electrodes[215] or

synthesized chemically and placed between the contacts.[323] Varying the width-to-length ratio of

the nanowires confirmed that the sensitivity of the resulting FET devices is inversely

proportional to the width of the channel. In this work, PPy is anodically electropolymerized from

an aqueous acidic solution containing the pyrrole monomer and ClO4- ions. Both electrodes are

contacted simultaneously with a two-channel patch clamp amplifier and a potential sweep from

-0.3 to 0.6 V vs. Ag/AgCl/no Cl- is applied to oxidize pyrrole on both electrodes. Successful

formation of a conductive junction between the two contacts is indicated by a sudden jump of

recorded current (see also Figure 42). The polymer is deposited as a thin film exclusively on the

apex of the dual carbon nanoelectrode, as shown by scanning electron microscopy (Figure 41).

PPy constitutes the transistor channel while the two carbon electrodes serve as the drain and

source contacts. The formed PPy transistor channel has a diameter of about 200 nm. A series of

SEM images each taken after gradually removing the PPy deposit by FIB allows to estimate the

thickness of polymer film. About 100 nm of material are removed until the underlying carbon

nanoelectrodes become visible (Figure 41 d).

a

b

c

d 1 2

3 4

Figure 41. Depositing PPy forms a nanometric transistor channel of about 200 nm diameter on the needle-like dual carbon nanoelectrodes. Schematic of the FET (a), low magnification (b), high magnification SEM image (c) of the same transistor and series of SEM images taken after gradual FIB milling to assess the thickness of the PPy deposit (d). Scale bars 20 µm (b) and 200 nm (c, d).

In all experiments shown, the FET nanoprobe is easily interfaced using a two-channel patch

clamp amplifier or potentiostat. The both carbon barrels are connected at the two channels of

the amplifier (working electrodes) and drain-source voltage VDS is applied as an offset between



them (Figure 42 a). As current passes through the PPy channel, the current measured

individually at the two electrodes has nearly equal magnitude but opposite sign. This current is

the drain-source current IDS (or -IDS for the other electrode). The gate voltage VG is applied with

respect to the ground electrode of the current amplifier. An external Ag/AgCl wire in the

electrolyte solution is used as the gate electrode. The IDS-VDS curves show that IDS is linearly

dependent on VDS and can be reversibly switched by applying different gate voltages with

respect to an external Ag/AgCl electrode (Figure 42 b). When the potential (VG) is above 0 V, the

PPy channel is fully oxidized and becomes conductive due to the insertion of positive charge

carriers into the polymer backbone. At gate voltages below 0 V, the PPy becomes increasingly

reduced. Hence, the junction becomes insulating and the IDS vanishes. At VG of -0.6 V, the PPy

channel is fully insulating. Thus, by applying different VG, the PPy channel of the transistor can be

opened or closed.

a

b

0.00 0.05 0.10

0

50

100

1500.2 V0 V

-0.2 V

-0.4 V

I DS /

nA

VDS

/ V

-0.6 V

0 5 10

0

10

20

Figure 42. The PPy-FET is interfaced using a dual channel patch clamp amplifier (a) and shows typical transistor behavior. The current measured at the two electrodes has opposite polarity and represents IDS. VDS is applied between the two barrels, VG is applied with respect to an external Ag/AgCl electrode. IDS

depends linearly on the VDS. Recording at different VG from -0.6 V to 0.2 V vs. Ag/AgCl/0.1 M Cl- in 0.1 M

HCl (b).

Moreover, the drain-source current is strongly dependent on the pH value, turning the PPy

nano-FET into a sensitive pH sensor. When VG is swept dynamically while keeping a constant

small VDS of a few mV (IDS-VG curve), opening and closing of the transistor is observed (Figure 43

a). The IDS-VG curve also shows that the conductance shows a maximal value at a particular VG

and then decreases due to overoxidation of PPy.[215,323] The interaction of protons with the PPy

nanojunction has two effects: Firstly, a general increase of conductance is observed which leads

to higher values for IDS. The dependence of conductivity on pH is attributed to the

protonation/deprotonation of the pyrrolic nitrogen in the PPy nanowire. Protonation results in

the formation of delocalized radical cations and is accompanied by an increase in

conductivity.[215,323] Secondly, a shift in the IDS-VG curve is observed due to the electrical potential

shift induced by proton accumulation/depletion that adds to the externally applied gate voltage.

This phenomenon is the field effect. The shift is seen clearest in the IDS-VG curves normalized by



their peak current values (Figure 43 c). Extracting the position of the peaks on the VG scale

reveals a voltage shift of 51 mV per pH unit, close to the value of 59 mV predicted for Nernstian

behavior (Figure 43 d). Alternatively, pH measurements can be performed reliably by extracting

IDS by averaging over the full IDS-VG curve (Figure 43 b). For all following presented results, IDS

was measured in this fashion.

a

-0.4 -0.2 0.0 0.2 0.4

0.0

0.2

0.4

0.6

I DS /

nA

VG / V

pH2.4

pH4.5

pH7.0

pH7.5

b

2 3 4 5 6 7 8

0.0

0.2

0.4

I DS /

nA

pH

c

-0.4 -0.2 0.0 0.2 0.4

0.0

0.5

1.0

1.5

I DS /

ID

S,p

eak

VG / V

pH 2.4

pH 4.5

pH 7.0

pH 7.5

d

2 3 4 5 6 7 8

-0.1

0.0

0.1

0.2

VG

,peak /

V

pH Figure 43. The PPy-FET is a pH nanosensor. From IDS-VG curves (a) changes in pH value can be measured as change of average drain-source current (b). The IDS-VG curves normalized by their peak currents also show a shift of peak voltage (c) which can be taken as a measure for the pH as well (d). Measurements at VDS = 5 mV. VG was changed with 0.4 V/s.

The pH-sensitive PPy-FET can be used to monitor pH changes in the physiologically relevant

range from pH 5 to pH 7.5 in real time (Figure 44 a). Each data point corresponds to the average

IDS from a full VG sweep between -0.6 V and 0.3 V. The pH is changed by immersing the FET into

phosphate buffered solutions with different pH values. IDS responds to the changes in acidity of

the environment in a response time of a few seconds, indicated by the stable current plateaus

after changing the solution. When the pH after this calibration is changed back to the initial

value, the sensor response follows the pH change, indicating reversibility of the measurement.

The sensor response is linear with respect to the pH in the range between 5 and 7.5 (Figure 44

b). The sensitivity towards protons is expressed as 1/G0 ∙ ΔG/ΔpH, with the conductance G =

IDS/VDS and G0 the conductance at pH 7. Note that this measure is equivalent to 1/IDS,0 ∙ ΔIDS/ΔpH



and thus independent of VDS. To compare all pH sensors, normalization by G0 (or IDS,0) is

necessary because of the variation of the conductance between probes that is due to small

variations in the size of the electrodes and the size and quality of the deposited PPy. However,

the variation in the normalized sensitivity for pH is relatively small. The sensitivity is -0.5 ± 0.1

pH-1. Quantitative comparison of the sensitivity with values reported in the literature is difficult

because the values depend on specific experimental details. For instance, Shirale et al. report

similar sensitivities of up to 0.4 pH-1 for single PPy nanowires depending on the aspect ratio of

the PPy transistor channel.[323] However, the values were extracted at different VG and

normalized by IDS at a different pH value (pH 10) compared to this study.

a

0 100 200 300 400

5

10

15

20

257.554.75

5.96

6.95

I DS /

nA

t / s

7.55

b

5 6 7 8

4

6

8

10

12

14

16

I DS /

nA

pH

Figure 44. PPy-FET nanosensors monitor pH changes in the physiologically relevant range in real time. IDS as the sensor response to changing the pH in the phosphate-buffered media (a). IDS values depend linearly on the pH in the range between 5 and 7.5 (b).

In contrast to amperometric sensors or conventional ion-selective sensors like the standard pH

electrode, FET devices can also be applied as gas sensors and chemical noses.[222] This is because

the analytical signal IDS does not require electron or ion transfer at the electrochemical interface

between the electrode and the electrolyte solution. Instead, current also passes through the dry

transistor channel. Applying a gate voltage with respect to a gate electrode immersed in

electrolyte solution is not strictly necessary for the operation of the transistor sensor. Figure 45

shows the utility of the PPy-FET nanosensors for detection of H+/OH- from molecules in the gas

phase. The probe was pulled out of the solution, leaving the gate electrode disconnected. Yet, IDS

can pass the transistor channel. H+-donating/accepting compounds are detected when the FET

sensor is moved in proximity to small open vials filled with solutions of concentrated ammonia

and acetic acid (Figure 45). The two volatile compounds interact with the transistor at the solid-

gas interphase. When the FET probe is brought close to the container filled with NH3, IDS

decreases as expected from the response to alkaline pH seen for calibration curves in the liquid

phase (Figure 44). After retraction of the sensor, the signal recovers. Moving the sensor close to

the vial filled with acetic acid induces an increase of IDS due to the release of protons into the PPy

channel.



0 200 400 600

2

4

6

I DS /

nA

t / s

NH3

NH3

H3CCOOH

NH3

Figure 45. PPy-FET probes detect H+-donating/accepting compounds from the gas phase. IDS as a

response to moving the sensor close to vials containing NH3 or H3COOH (indicated by horizontal bars).

4.5.2 pH measurements at cells

Cancer cells are characterized by a reversed pH gradient across the cell membrane, that is, the

extracellular proton concentration is higher than the one found inside the cell.[324] Malignant

cells switch to glycolysis as their main metabolic pathway, which leads to the production of

acidic metabolites such as pyruvate in the cytoplasm. Also, active proton pumps are expressed to

a larger extent. Moreover, cancer cells may produce lactate through the anaerobic metabolic

pathway even in the presence of oxygen.[325,326] Identifying malignant cells by their characteristic

acidification is of high value for diagnostics and fundamental research related to drug design.

For instance, the degree of extracellular acidification tends to correlate with tumor

aggressiveness. Hence, drugs that limit the extent of proton extrusion, for instance by inhibiting

proton pumps, are promising alternatives for cancer treatment.[327] Another family of new anti-

cancer drugs exploits the local pH characteristics for controlled pH-triggered release of the

active compounds to target only malignant cells and thus reduce the side-effects of cancer

treatment.[328]

To measure the true local pH around cells, analytical methods with a high spatial resolution are

necessary. Classical H+-sensitive glass membranes have been adapted to fit micropipettes.[329]

Micropipettes filled with selective ionophores have been widely used to measure the pH value

and concentrations of other cations in biomedical research and neurophysiology.[310–314] Like a

conventional pH electrode, the microsensors measure the electric potential at the interface of

the ionsensitive membrane. Based on this principle, a number of electrochemical as well as

optical pH microsensors is commercially available, for instance from the companies PreSens,

Unisense or World Precision Instruments. However, the diameter from a few µm to up to

hundreds of µm allows measurements in tissues and in vivo but precludes controlled access to

single cells. As an alternative, microelectrodes made from antimony[318] or palladium[330] allowed



potentiometric pH sensing and imaging of pH gradients. “Pseudo-potentiometric” sensors are

built by adsorbing redox active molecules on the electrode whose redox transition involves the

consumption or release of H+ or OH-. The sensor signal is the peak potential of the corresponding

peaks in fast-scan voltammograms, as demonstrated for quinone species grafted to carbon

microfiber electrodes.[263]

To reduce the sensor dimensions, a nanopipette-based sensor suitable for intracellular pH

measurements was proposed recently.[331] The opening of the glass nanopipette was modified

with chitosan. Upon protonation, the modified orifice changed its surface charge which resulted

in a change of ion current passing through the nanopipette opening. Recent examples for pH

sensors based on carbon nanoelectrodes exploit a voltammetric detection scheme.

Nanoelectrodes were modified with pH-sensitive materials whose redox potentials shift in the

presence of different proton concentrations. The variation in redox potential is detected by the

shift of the corresponding voltammetric peaks. The principle was demonstrated for the

deposition of iridium oxide on the tip of nanoelectrodes (see also Figure 4 b).[142] Also the

adsorption of the organic compound syringaldazine proved suitable to comprise a pH-

responsive nanosensor.[332] These methods exhibited sufficiently high spatial resolution and low

response times to produce high-resolution images of micrometric sources of OH-.

PPy-FET nanosensors on dual carbon electrodes are suitable to perform pH measurements in

small confined volumes in biological samples. Due to the probes’ high sensitivity and small tip

dimensions it becomes possible to follow small pH changes (≈ 0.1 pH units) when investigating

the local pH around cancer cells. Reliable local pH measurements in the microenvironment of

cells would allow to identify individual cancer cells by their metabolic characteristics. For

instance, melanocytes are pigment-producing cells in the human skin and also the cells that

become cancerous in malignant melanoma, skin cancer. Compared to normal melanocytes,

melanoma cells have a higher metabolic activity and thus can acidify their extracellular

environment because of high glucose uptake rates, increased glycolysis, and the accumulation of

lactic acid.[326,333–335] Figure 46 a shows the pH value calculated from recorded IDS values when

vertically moving the PPy-FET nanoprobe into a cluster of cultured melanoma cells. As expected

from the highly active cells, a pH decrease from 7 to 6.3 is detected when the sensor is inserted

into the cell cluster. Retracting the sensor by 220 µm away from the cells restores the sensor

response to the value corresponding to the pH in the bulk medium. To compare the local pH

values around cancerous melanoma with the one found at healthy melanocytes, a calibrated

probe was also inserted into a cluster of melanocytes (Figure 46 b). In contrast to melanoma, no

local acidification was detected. Thus, the FET sensor is suitable to distinguish cancerous from

normal cells and by this could help to investigate an individual cell’s fate in the development of

cancer.



However, in the above mentioned experiments, prior to insertion of the sensor into the cell

cluster, the cluster was artificially formed by moving and gathering cells using the FET probe as

a manipulation tool. Yet, this treatment is necessary, because there is no noticeable sensor

response when the probe approaches a single isolated melanoma cell (Figure 46 c). The pH-

sensitivity of the probe is yet insufficient to detect the local pH change generated from a single

cancerous cell. Presumably, significant amounts of extracellular protons are only accumulated if

cells are located in a confined space obstructing the rapid diffusion of protons away from their

source. A single cancer cell is a micrometric source of protons exhibiting a hemispherical

diffusion layer so that released H+ may efficiently escape from the cell. This might be the reason

why the FET sensor does not detect protons released from the single cell. Similar observations

and considerations were made in previous studies conducted with different sensing strategies.

Thakur et al. reported an electrochemical detection strategy to probe the extracellular pH

change of cancer cells and claimed that at least 5 cancer cells were necessary to induce a

significant sensor reponse.[334] Also with regard to the application of flat, chip-like FET sensors

for metabolic studies it was recognized that the acidification is easier to detect if occurring in a

confined space between the cell and the detecting sensor.[224]

a

b

c

Figure 46. The high-aspect ratio FET probe can measure the extracellular acidity of cancer cells. The sensor is moved in and 220 µm out of a cluster of melanoma cells (a), a cluster of normal melanocytes (b) and close to a single melanoma cell (c). Arrows indicate the vertical movement of the probe.

4.5.3 PPy-FET nanobiosensors for ATP measurements

Adenosine triphosphate (ATP) is not only a molecule used by organisms to store and transport

chemical energy but also a co-neurotransmitter abundant in all nerves in the peripheral and

central nervous systems.[336,337] ATP has manifold and often opposing effects on different cell

types, depending on the ATP receptors expressed in the cells. Two families of ATP receptors are

responsible for particular signaling cascades: P2X receptors are ion channels for instance

partially responsible for the contraction of smooth muscle cells. P2Y receptors activate different

signaling cascades inside the cell via G proteins. For instance, for the control of muscle tone in



blood vessels, increased ATP levels lead to contraction of smooth vascular muscles via P2X

receptors but stimulate the release of nitric oxide from endothelial cells which dilates vessels.

Thus, ATP signaling is closely related with ischemic disorders, i.e. a restriction of oxygen and

glucose supply due to blocked blood vessels. In relation to cancer, ATP causes ambivalent

responses as well. Some P2Y receptors mediate cell proliferation and thus favor tumor

development while certain P2X receptors promote anti-proliferation and cell death.[337]

Moreover, ATP mediates the reception of pain, mechanosensitivity and sensitivity for O2 in the

brain.

Thus, analytical tools to detect and quantify the ATP release from cells in response to different

stimuli may help to identify communication mechanisms based on ATP. The classical method to

quantify the ATP release from cells, however from a whole cell population and without spatial

resolution, are luciferase assays[338–341]. Luciferase, a protein extracted from fireflies, converts its

substrate luciferin into a luminescent product, oxyluciferin, in the presence of O2 and ATP

whereas the luminescence serves as the analytical signal. The method shows very high

sensitivity towards ATP (picomolar detection limits) but the assay performs ex situ and thus is

not suitable to monitor the ATP release in real time. Most importantly, these methods obtain the

information from the bulk solution and hence only report the diluted analyte concentrations.

Alternatively, amperometric microsensors have been proposed for ATP detection in vivo.[336,342–

345] Mixtures of enzymes, namely adenosine deaminase, nucleoside phosphorylase and xanthine

oxidase are entrapped in a polymer film on Pt microelectrodes and the final product of the

enzymatic decomposition, H2O2 is detected by anodic oxidation.[343] A similar method used

glycerol kinase and glycerol-3-phosphate oxidase for the enzymatic cleavage of ATP that results

in the formation of H2O2.[342] These sensors with a minimal size of 25 µm were sufficiently small

to quantify the ATP release in response to O2 deprivation[336] or inflammation[344] in the brain.

Another interesting sensing approach used a cell itself as the sensor to quantify the ATP release

from other cells.[346] A PC12 cell which expresses P2X receptors and thus responds to ATP by

opening ion channels was patched to a micropipette. The whole-cell ion current through ATP-

dependent ion channels served as the sensor response. By positioning the sensor close to

cardiac myocytes, the ATP release from the single heart cells could be measured when the

cardiac myocyte faced ischemic, hypoxic (low O2 supply) or hypotonic (low osmolarity)

conditions. Migita et al. proposed an ion-selective FET (ISFET) responding to ATP.[347] The

enzyme apyrase was deposited in a polymer gel on the tantalum oxide gate of a FET. When

apyrase hydrolyzes ATP, the resulting change in phosphate and proton concentration induced a

sensor response on the FET. However, due to the thick polymer film as well as interferences

with other ionic species, the sensor yet lacked adequate response time, sensitivity and

specificity.



The pH-sensitivity of the nanometric PPy-gated FET based on dual carbon nanoelectrodes

presented in this work can be exploited for numerous detection schemes to detect analytes

other than protons if a chemical conversion of the analyte is translated into a pH change. It is

well known that the enzyme hexokinase catalyzes the addition of phosphate from ATP to

glucose, which releases one proton per molecule ATP in the glycolysis reaction (Figure 47 a).

However, neither the analyte and reactants nor the products of cleavage are redox active. To

overcome this issue, some amperometric biosensors use a competition for glucose by ATP-

dependent hexokinase activity and activity of glucose oxidase (GOX).[345,348] The sensors detect

the decrease in H2O2 oxidation current originating from GOX-catalyzed conversion of glucose

when hexokinase removes glucose in the presence of ATP. Instead, to demonstrate the

possibility of converting the pH sensitivity of the PPy FET nanosensor into sensitivity for ATP,

hexokinase is added to the solution and the FET sensor records the pH change caused by the

cleavage of ATP (Figure 47 b). The probe is placed in a Petri dish containing melanoma cells and

an excess of glucose as the co-substrate of the enzymatic reaction. When hexokinase is added to

the dish, the enzyme cleaves the ATP released from the cells into the solution. As a result, IDS

increases due to the shift of the pH in the bulk solution caused by activity of hexokinase.

a

b

0 100 200 300 400

30

35

I DS /

pA

t / s

Hexokinase

Figure 47. Hexokinase releases stochiometric amounts of protons when cleaving ATP and phosphoryla-ting glucose (a) which is detected by the pH-sensitive FET probe (b). The probe is immersed in a dish with melanoma cells and medium containing 20 mM glucose. Hexokinase is added to the bulk solution.

For more precise quantification of the amount of ATP that is released from the cells and to

exclude the large change of pH in the solution, hexokinase is immobilized on the transistor

channel. The PPy-modified probe is first immersed in a solution of 25 % glutaraldehyde to

facilitate protein immobilization to the interface. Then the FET pipette is immersed into a

solution of hexokinase to attach the protein. The attachment mechanism is unknown. Possibly,

the pyrrolic nitrogen of PPy attacks glutaraldehyde which may later capture hexokinase by the

formation of imines with amino groups on the protein shell. Presumably, glutaraldehyde at high

concentration may also undergo aldol condensation reactions to form polyglutaraldehyde on the

sensor which may assist in attaching hexokinase. However, the difficulty in probing the surface



chemistry of nanoelectrodes hampers the further investigation of the attachment mechanism.

The FET with hexokinase immobilized to the PPy channel detects ATP with high sensitivity

(Figure 48). As expected, IDS increases with increasing ATP concentration due to the local

production of protons by hexokinase. The probe is capable of detecting ATP concentrations

down to a LOD of 10 nM (i.e. the signal at 10 nM is larger than the blank signal + 3 standard

deviations). In the concentration range from 10 nM to 3 µM, IDS linearly changes with the

logarithm of ATP concentration. The sensitivity in the linear range extracted as 1/G0 ∙ ΔG/Δlog

cATP, amounts to 0.3 ± 0.2 per decade of ATP concentration (G=IDS/VDS and G0 is the conductance

in the absence of ATP). Due to the unity stoichiometry between ATP and produced protons, the

sensitivity for pH of 0.5 ± 0.1 pH-1 (see above) represents the maximum possible sensitivity for

ATP when every molecule ATP in proximity to the FET translates into H+. The ATP sensitivity of

0.3 ± 0.2 dec-1 is nearly as high as the pH sensitivity, which indicates that the immobilization of

hexokinase is relatively efficient. For concentrations higher than 3 µM, the calibration deviates

from the linear trend. Unmodified bare PPy probes did not record any increase in IDS when ATP

standards were added.

a

b

-2 -1 0 1 2

0.15

0.20

0.25

I DS /

nA

log (cATP

/ µM)

R2=0.95

Figure 48. Immobilizing hexokinase at the tip of the FET probe creates a highly sensitive biosensor for ATP. Detection scheme (a) and calibration curve for ATP in unbuffered cell medium containing 20 mM glucose (b).

4.5.4 ATP detection at cells

Due to its needle-like shape and maneuverability, the FET biosensor enables to measure the

extracellular ATP gradient in three dimensions around living cells (Figure 49). After initial

testing of the sensitivity by adding 10 µM ATP, the ATP sensor is vertically approached to

melanoma cells (Figure 49 a). As the probe slowly approaches the cells, IDS increases due to the

release of ATP from the cells. Withdrawing the FET decreases the measured IDS. The sensor

responds quickly to the movements as it is moved back and forth in the ATP concentration

gradient originating from the cells. Additionally, due to the high ATP release rate a general

increase of the ATP concentration is observed with time. Precise quantification of ATP in this

concentration regime is difficult because of the non-linearity of the sensor response at high



concentrations (Figure 48 b). Nevertheless, the magnitude of the concentration gradient can be

estimated. Slow, stepwise vertical approach to the cells reveals that more than 10 µM ATP are

present at a distance of 1 mm away from the cells (Figure 49 b). Even higher concentrations,

between 30 µM and 70 µM are measured in direct proximity to the cells. Because of the non-

linearity of the sensor response, the graph shows the IDS current levels from calibration

experiments as horizontal bars. A bare PPy nano-FET probe not modified with hexokinase on the

other hand does not record any changes in IDS when approached to the cells and thus indicates

that the pH value in the solution is constant (Figure 49 d). This together with the control

experiment presented in Figure 47 demonstrates that the sensor response of the hexokinase-

modified probe is selective to the detection of ATP and no interference by other species in the

complex medium surrounding the cells is observed.

a

3

4

5

I DS /

nA

to melanoma cells

no cells+ 10 µM ATP

cell surface

d = 750 µm

120 s

b

1000 500 0 -500

0.8

0.9

1.0

70 µM

30 µM

I DS /

nA

distance / µm

10 µM

c

d

1000 500 0

7.0

6.5

6.0

pH

distance / µm

3.5

4.0

4.5

5.0

I DS /

nA

Figure 49. Spatial and temporal ATP gradients released by melanoma cells are measured by the PPy FET nanosensor. First, the sensitivity is checked by adding ATP to the cell dish. The signal follows the sensor movement upon repetitive vertical approach and withdrawal from the cell. Additionally, a rise of the ATP level is observed with time. The measured ATP concentrations can reach to several tens of µM (b). Optical micrograph of the sensor positioned close to a melanoma cell. Control approach to cells with a non-modified PPy FET probe (d).

In a similar fashion the FET nanosensor is used to determine the ATP gradient originating

from a single cardiomyocyte (Figure 50 b). Here, submicromolar levels are found in the distance

and several µM are recorded close to the cardiomyocyte. Again, it is important to note that no pH



change or other interference was detected with the FET probe not modified with hexokinase.

Additionally, the probe proves to be a valuable tool to monitor ATP release as a response to cell

stress caused by mechanical stimulation (Figure 50 c). When the cell is touched with the probe, a

sudden increase in IDS is recorded due to the ATP release. Similarly, hypo-osmolarity in the

surrounding medium causes an osmotic shock to cells. When the osmolarity of the medium is

decreased by 20 % by adding water to the dish containing cells, the ATP sensor positioned in

close proximity of the cell detects elevated levels of locally released ATP as a response to the

osmotic stress (Figure 50 c).

a

b

1000 500 0 -500

2.4

2.6

2.8

3.010 µM

1 µMI DS /

nA

distance / µm

0.3 µM

c

2.6

2.8

3.0

3.2

+ 20 % water

10 µM

I DS /

nA

1 µM

touching

300 s

d

0.2

0.4

0.6

0.8

I DS /

nA

180 s

penetration

Figure 50. Localized ATP measurements are performed on a single cardiomyocyte. Optical micrograph (a). The cardiomyocyte produces a vertical gradient from its proximate microenvironment into the bulk solution (b). Additional ATP release is induced by touching the cell with the sensor to impose mechanical stress and adding water to the solution to create osmotic stress (c). The probe allows for penetration into the cardiomyocyte (d).

Cardiac myocytes, melanoma and many other cells have also been reported to release ATP in

response to mechanical disturbance and osmotic stress.[337,346,349] Owing to its small dimensions,

the FET probe may finally be used for intracellular measurements (Figure 50 d). Inserting the

sensor into a cardiomyocyte results in a jump of IDS by virtue of the sudden shift in potential

applied to the PPy channel when the probe faces the additional negative contribution of the

membrane potential. Shortly after, a gradual increase in IDS is recorded, presumably as a result of



the high intracellular ATP level. However, more thorough investigation of the intracellular

signals will be necessary before quantitative measurements can be made inside cells. In

conclusion, these examples of the applications of PPy-FET sensors based on dual carbon

nanoelectrodes demonstrate that this class of sensors shows excellent perspectives for sensitive

chemical analysis of physiological processes at the level of a single cell. Compared to

amperometric nanoelectrodes, FET nanodevices allow real-time monitoring of physiological

information with higher sensitivity due to their intrinsic amplification capabilities and high

signal-to-noise ratio. However, their fabrication requires complex protocols to place the sensing

nanomaterial between the source and drain contacts. To reduce electrochemical noise, these

contacts then have to be well isolated. Moreover, the large electrode size make their local access

to biological samples such as single cell a continuing challenge.[224,350,351] Nanometric dual carbon

electrodes are fabricated at low cost and provide an effective platform with which to create

novel FET nanobiosensors by generating a semiconductor link between two adjacent electrodes

exposed at the tip of the spear-shaped nanopipette.

The extracellular ATP concentration is a key biochemical constituent of the microenvironment of

both tumours and cardiac cells. For instance, it is well known that hypoxia causes ATP release

acting via purinoceptors.[337,352] Cardiac myocytes, melanoma and many other cells have also

been reported to release ATP in response to mechanical disturbance and osmotic stress.

[337,346,349] The PPy-FET probes can be functionalized to make an ATP nanobiosensor through

binding hexokinase to the PPy channel. The resulting nano-FET probe is capable of detecting

ATP concentrations down to 10 nM. A linear sensor response is observed in the concentration

range from 10 nM to 30 µM, with a sensitivity of 0.3 ± 0.2 dec-1. The sensitivity may be even

further increased by optimizing the binding efficiency and activity of the ATP-detecting

enzyme.[207] Due to its small dimensions and its spear-like design the nanobiosensor can

measure spatial concentration gradients originating from cell and follow changes of these

gradients in time. The measurement is not affected by any interferents in the complex biological

environment. ATP levels released from melanoma cells easily reach tens of µM. Similar

extracellular ATP concentrations have been observed previously from neonatal cardiac

myocytes.[346] Even higher levels, in the hundred micro-molar range, have been found in the

human melanoma microenvironment.[339]

In localized measurements, the distance of the detecting device to the probed cells is closely

related to the measured analyte concentration. In order to achieve absolute measurements,

precise distance control is necessary. The use of multiple-barrel nanopipettes[104] to approach

the nanobiosensor to about 100 nm from the cell surface under the feedback control of SICM or

under shear force control in SECM is envisaged. ATP release can be measured in close proximity

to the cell without disturbance and with sub-cellular resolution enabling high resolution



chemical imaging. However, yet the response time of a few seconds precludes the use of the

sensors in fast scanning probe protocols and thus needs further optimization. Such real-time

local detection of ATP release and its gradient at the single-cell level could be beneficial in the

understanding of cancer cell metabolism in heterogeneous tumor populations. In this work, the

possibility to insert the nanometric FET device into a cell for performing intracellular

measurements is demonstrated. In the future, after carefully assessing possible interferences in

the complex cytosolic matrix, applications may be expanded to the detection of substances

whose presence is restricted to the intracellular space.

Additionally, dual carbon nanoelectrodes could be used as a platform to construct new FET

sensors by employing others of the myriad of nanomaterials exhibiting semiconducting

properties giving rise to viable FET sensors.[209,212] Not only different materials for the transistor

channel may be tested but also specific functionalization of those materials may widen the scope

of such needle-type FET sensors. For instance, as it was shown previously, DNA[219,353,354] or

PNA[210] capture probes may be bound to the sensitive transistor to achieve selective detection

of DNA or proteins in or around single cells. This will be of high diagnostic value for biomedical

investigation.


4.6 Single Nanoparticle Electrochemistry 94

4.6 Single Nanoparticle Electrochemistry

Some experiments were performed by Denis Öhl, Ruhr-Universität Bochum, during a student

project. Parts of the following section are published in ref. [355]: “Clausmeyer, J.; Masa, J.; Ventosa,

E.; Öhl, D.; Schuhmann, W.; Chem. Commun. 2016, 52, 2408–2411”.

4.6.1 Electrocatalyst studies

The oxygen evolution reaction (OER) plays an important role in electrolyzers for

electrochemical water splitting. Energy is stored in the form of H2 which is produced at the

cathode and serves as a fuel for later consumption in fuel cells as well as a feedstock for a

number of industrial syntheses.[356] However, the efficiency of the overall water splitting

reaction is limited by the kinetics of the OER in alkaline (4 OH- O2 + 2 H2O + 4 e-) as well as

acidic (2 H2O O2 + 4 H+ + 4 e-) conditions. Hence, while the hydrogen evolution occurs at

relatively low overpotential, the OER still leads to a substantial energy loss during water

splitting. Catalyst materials for the OER need to be optimized to ensure energy storage as H2

with minimal energy losses. Designing new materials requires a deep understanding of the

mechanisms leading to enhanced electrocatalytic activity. Especially the knowledge about the

activity of materials for electrochemical water oxidation in relation to structure and size of

particles is still limited. It is the demand to understand the nature of the enhanced elec-

trocatalytic activity of nanostructured materials that motivates the development of suitable

analytical tools to characterize these challenging samples. For nanomaterials and in particular

catalytically active nanoparticles, the extraordinary (electro)catalytic activity results from a

number of different effects taking place at the nanoscale: The high number of coordination-

unsaturated surface and edge atoms leads to unique properties for the adsorption and

desorption of reactants and products which can affect the overall turnover rate for catalytic

conversion. Additionally, the fact that electrons are confined in a small particle volume results in

special electronic properties that may favor or disfavor catalytic reactions.[228] Finally, the small

particle dimensions result in fast transport of reactants, intermediates and products to and away

from the reactive interface which ensures high reaction rates but may also change the selectivity

of the catalytic reaction.[231] The catalytic activity of nanoparticles is a complex function of these

factors and only the deconvolution of these effects will help to identify the lead concepts for the

development of new highly active materials applied in energy and chemical conversion

devices.[230] However, the investigation of the individual contributions of different effects

governing the catalytic activity is hampered by the polydispersity of nanoparticle samples.

Nanoelectrodes allow to analyze single nanoparticles without averaging over large statistical en-

sembles. Nanoparticles can be directly deposited on or attached to nanoelectrodes. This strategy

ensures to study the catalytic reactions at single particles under high mass transport rates.



Deposition of electroactive materials on nanoelectrodes was previously used for studying the

activity of individual nanoparticles towards reactions relevant for electrochemical energy

conversion such as the HER[135,357] and the ORR[136,231] However, systematic studies of the

dependence of catalytic activity on particle size are rare. In particular, never was the OER

subject of experiments to evaluate the electrocatalytic activity at single nanoparticles.

State-of-the-art OER catalysts are transition metal oxides and hydroxides with different metallic

compositions of Ir, Ru, Ni, Co and Fe.[358–360] Despite catalysts based on Ir and Ru show better

performance and high stability in acidic electrolyzers, Ni, Co and Fe-based materials find wide

application due to the high abundance and low cost of these non-noble metals. Perovskites, a

family of materials with the general structure ABO3 (A is a large lanthanide or alkaline earth

cation, B is a transition metal cation) exhibit similarly high OER activities.[361] Despite different

mechanisms for the OER have been proposed, there is general agreement that according to the

Sabatier principle the adsorption energies of intermediate OH and OOH groups are crucial to the

catalytic activity. Those materials which show intermediate adsorption strength for the

adsorbed species are particularly active. Ni(OH)2 has proved to be a suitable catalyst for

electrochemical water oxidation[358,362] and finds application in industrial alkaline

electrolyzers.[356] Especially, some nanostructured materials show superior performance

compared to their bulk counterparts.[363–368] Ni(OH)2 can assume two distinct crystal structures,

the α and β-phase. While β-Ni(OH)2 has a well-defined structure consisting of two-dimensional

Ni slabs with OH-groups pointing into the intersheet space, α-Ni(OH)2 is a general term for a

group of poorly defined crystalline structures with incorporated H2O and ions.[369,370] Upon

oxidation, the active catalyst for the OER, NiOOH is formed (equation (14)). In NiOOH, the

distances between Ni atoms are smaller but intersheet distances are larger. Upon potential

cycling α-Ni(OH)2 is converted to γ-NiOOH while β-Ni(OH)2 is oxidized to β-NiOOH. Moreover, at

high potentials, often a phase transformation from the β-β cycle to the α-γ cycle takes place. For

the OER, γ-NiOOH is believed to have the higher catalytic activity.[371] For instance, hollow

nanospheres of α-Ni(OH)2 supported higher current densities and had lower overpotentials and

lower Tafel slopes as compared to β-Ni(OH)2 nanoplates and nanoparticles.[363] However, in

previous studies it was difficult to distinguish the effects of the morphology of the nanomaterials

from the role of the crystal phase. For instance, particularly high OER activity was reported for

β-Ni(OH)2 nanoplates, despite their less active crystal phase.[372] Apart from structural aspects,

also the electrical conductivity of the materials is crucial for the OER reaction rates.[360,373] At

high current densities, the ohmic drop occurring in the material reduces its energy efficiency.

Because electrons as well as chemical species need to be transported between the solution and

active sites, the performance of a given catalyst depends on the morphology and thickness of the

catalyst film which determine accessibility to the active centers. Especially poorly defined



porous architectures complicated the identification of activity trends for OER catalysts because

of the difficulty to compare surface areas, the number of electrochemically active sites, and

electron/mass transport properties.[356]

Carbon nanoelectrodes are suitable to investigate the electrocatalytic activity of individual

Ni(OH)2 particles for the OER. The single particles are good model systems because particle sizes

and mass transfer properties are well defined. Ni(OH)2 is deposited cathodically on the carbon

nanoelectrodes from a solution of 5 mM NiCl2 at potentials between -1 V and -1.2 V vs.

Ag/AgCl/3 M Cl-. Presumably, the material is deposited as metallic nickel but undergoes

spontaneous transformation into NiO and Ni(OH)2.[360] Different electrolysis times are tested to

control the amount of deposited Ni(OH)2 and thus particle sizes. However, only little correlation

was found between the electrolysis time and the Ni(OH)2 amount. However, the amount of

deposited material and thus the particle sizes can be precisely measured by both SEM and an

electrochemical method. SEM images of various single nanoparticle electrodes show nearly

spherical Ni(OH)2 particles located at the tip of carbon nanoelectrodes. Due to the small

dimensions of the carbon nanoelectrode, the particles are larger than the tip diameter (Figure

51). The particle sizes range from 20 nm to 500 nm.

Figure 51. Ni(OH)2 is deposited as spherical nanoparticles on the tip of carbon nanoelectrodes. The particle dimensions are in good agreement with the electrochemically estimated particle radii. Scale bars are 500 nm (first three from left) and 200 nm (small inset on the right). The electrochemically estimated radii are 446, 227, 186 and 55 nm from left to right.

The composition and structure of OER catalysts is crucial for the electrochemical activity.[359,366]

The elemental composition of the particles was tested by energy-dispersive X-ray spectroscopy

(EDX) (Figure 52). The EDX spectrum shows strong signals for the Ni K and L transitions as well

as signals attributed to Si, O and C, the constituents of the SiO2-insulated carbon electrode. Apart

from small signals attributed to Al, which are presumably originating from the aluminum sample

holders and residual K, no other elements are detectable.



Figure 52. EDX spectrum showing the elemental composition of a single Ni(OH)2 particle (r ≈ 1µm).

Apart from electron microscopy, the particle size can be easily estimated electrochemically. The

amount of material on the carbon nanoelectrode surface is estimated from the charge

transmitted for the oxidation of Ni(OH)2 to NiOOH using a simple model.

(14)

𝑛𝑁𝑖𝑂𝑂𝐻 = 𝑞𝑁𝑖𝑂𝑂𝐻/𝐹 (15)

nNiOOH is the amount of Ni(OH)2/NiOOH, q the charge for oxidation of Ni(OH)2 and F the Faraday

constant. A one-electron transfer is assumed. The volume of a deposited nanoparticle is

proportional to the NiOOH oxidation charge:

𝑉 =

𝑞𝑁𝑖𝑂𝑂𝐻𝑀

𝐹𝜌

(16)

M is the formula weight of Ni(OH)2 and ρ its density. The radius r is calculated assuming that

particles have spherical shape:

𝑟 = √3

4𝜋𝑉

3

(17)

Accordingly, the surface area of the particle is the surface area of the corresponding sphere.

Figure 53 shows steady-state voltammograms for the OER at single Ni(OH)2 nanoparticles of

different sizes (r = 365, 83 and 20 nm). The redox peaks seen in the insets at potentials between

1.3 V and 1.5 v vs. RHE correspond to the redox reaction of the Ni(OH)2/NiOOH couple. The

redox peaks are well separated from the onset of OER allowing precise measurement of the

transmitted charge. The particle sizes measured in the SEM images are in good agreement with

the radii estimated from the electrochemical measurement, allowing fast characterization of the

single nanoparticle electrodes with the electrochemical method. For instance, the



electrochemically estimated radii of the particles shown in Figure 51 are 446, 227, 186 and

55 nm. The quantification of the deposited catalyst amount and thus of the particle size is the

prerequisite for analyzing large datasets of nanoparticle activity with respect to their size.

In the steady-state voltammograms shown in Figure 53 the OER sets in at potentials higher than

1.5 V vs. RHE. The steady-state voltammograms show a steady increase of anodic current for the

OER. Due to the high mass transport rates at nanoelectrodes, the electrocatalytic reaction is not

limited by diffusion, indicated by the absence of a current plateau. As a result, the OER is

performed at high current densities of up to 10 A cm-2.

a

b

1.2 1.4 1.6 1.8 2.0

0

10

20

30

i /

nA

E / V vs. RHE

0

1

2

J /

A c

m-2

1.4 1.6

0.0

0.5

c

1.2 1.4 1.6 1.8 2.0

0

200

400

600

800

i /

pA

E vs. RHE / V

1.3 1.4

0

10

0.0

0.2

0.4

0.6

0.8

J /

A c

m-2

d

1.2 1.4 1.6 1.8 2.0

0

200

400

600

800

i /

pA

E / V vs. RHE

0

5

10

15

J /

A c

m-2

1.45 1.50

0.0

0.5

1.0

Figure 53. OER is catalyzed by individual Ni(OH)2/NiOOH particles deposited on nanoelectrodes. Steady-state voltammograms (0.01 V/s) in 0.1 M KOH for the OER at particles with 365 nm (b), 83 nm (c) and 20 nm (d). The insets show the current peaks associated with the Ni(OH)2/NiOOH redox couple which are used for the estimation of the particle size.

For a catalytic reaction, the turnover frequency (TOF) indicates the rate of generation of product

normalized by the number of active sites of the catalyst. The TOF is thus often taken as a

measure of the specific activity of a catalyst. For the OER at NiOOH electrodes, the TOF is

calculated according to equation (18), whereas iOER is the current for the OER at a given

overpotential.



𝑇𝑂𝐹𝑂𝐸𝑅 =

𝑖𝑂𝐸𝑅

4𝐹𝑛𝑁𝑖𝑂𝑂𝐻

(18)

Knowledge of the amount of Ni(OH)2 in each particle allows calculating the TOF. Single

nanoparticle electrodes can be driven to high turnover frequencies at high potentials of 1.88 V

vs. RHE (overpotential η = 0.65 V). Since no additives like binders or conductive additives as

typically used to formulate porous electrodes are necessary, degradation of those components at

the high anodic potentials is excluded. Also, in contrast to thick porous films and powder

electrodes, the particles are directly contacted by the nanoelectrodes to reduce the influence of

electric resistance in the film. Figure 54 shows the TOFs at the individual nanoparticles as a

function of their radius. At single particles, relatively high variability of the TOF is observed,

which may reflect information concerning the heterogeneity of individual particles that is

normally lost when studying large ensembles of particles. Nevertheless, the analysis of the TOF

with respect to the particle size shows a general decrease of the TOF with increasing particle

radius.

10 100 1000

1

10

100

1000

TO

FO

ER /

s-1

r / nm

Figure 54. The turnover frequencies for the OER decrease with increasing particle size. TOFs are extracted from steady-state voltammograms in 0.1 M KOH at a potential of 1.88 V vs. RHE (η = 0.65 V).

Since the TOF is considered an intrinsic material property, this observation may be used to

support the claim that smaller Ni(OH)2 particles are intrinsically more active concerning the

OER in comparison to larger particles. The increased TOF at smaller particles is however not a

true nano-effect as termed by Compton,[232] i.e. is not a kinetic acceleration due to the size of the

nanoparticle. The variation of the TOF can rather be explained by a geometric effect that

compromises the estimation of the number of active sites. Due to the higher surface-to-bulk

ratio of the smaller particles the estimation of the number of active centers is more precise for

these particles. For large particles, NiOOH centers in the bulk of the particle do not contribute to

the evolution of oxygen and thus decrease the apparent TOF. This problem was previously

recognized for differences in the thickness of thin catalyst films.[356] Additionally, at larger

particles electrons have a longer path between the carbon electrode and the surface of the



particles. Because NiOOH has a relatively low electronic conductivity,[361,374,375] a substantial

potential drop may occur in large particles due to the electric resistance. The resulting

overpotential could be an additional explanation for the lower TOFs at large particles. Similar

findings were made by Zou et al. who investigated the TOF for the OER as a function of the film

thickness of Fe(oxy)hydroxide films.[373] The authors observed a decrease of TOFs with

increasing mass loading of the electrodes and ascribed it to the growing influence of the limited

electrical conductivity when the films become thicker.

An increased specific activity at smaller particles can however be excluded by comparing the

electrode kinetics at small and large particles. A common descriptor of electrode kinetics is the

Tafel slope which is extracted from a linear fit of an E vs. log J plot (or log J vs. E) assuming

Butler-Volmer kinetics:

𝐽 = 𝐽0 {exp (

𝛼𝑧𝐹(𝐸 − 𝐸0)

𝑅𝑇) − exp (

(1 − 𝛼)𝑧𝐹(𝐸 − 𝐸0)

𝑅𝑇) }

(19)

J0 is the exchange current density, α the charge transfer coefficient and E0 the equilibrium

potential, all other symbols have their usual significance. When the overpotential for the forward

or backward reaction becomes sufficiently large, the other exponential term becomes negligibly

small and the Butler-Volmer equation assumes Tafel form. The Tafel slope is only dependent on

the reaction mechanism but not on the surface area, mass loading or number of active sites on

the electrode. Hence Tafel slopes are a good descriptor of intrinsic activity of a material.[360]

Tafel analysis in the low overpotential region of the OER at single Ni(OH)2 particles reveals that

the electrode kinetics are largely invariant with the particle size. Figure 55 a shows Tafel plots

for a small and a large nanoparticle. Note that J is plotted against E and thus, the slope observed

in the graph is the reciprocal of the Tafel slope. Both curves exhibit similarly low slopes in the

low overpotential region between 1.57 and 1.66 V vs. RHE (η = 0.34 and η = 0.43 V) followed by

larger values at higher potentials. While the final current densities at high overpotential vary

between particles, the Tafel slopes extracted in the low overpotential region range around the

same values for all analyzed particles (Figure 55 b). Comparing the values for the Tafel slope

over the whole population of individually analyzed particles yields an average slope of 0.05 ±

0.01 V dec-1. This value is in agreement with values reported in the literature for highly active

OER catalysts.[363,365,366] The Tafel slope is a parameter describing the electrode kinetics of the

electrochemical reaction. Thus, these findings indicate that the electron transfer kinetics for the

OER do not depend on the particle size of the Ni(OH)2 particles. The similar Tafel slopes for all

particles do not explain the observed differences in TOF (Figure 54). At small particles the true

number of active sites can be estimated with higher precision because of the increased fraction

of active centers located in direct contact with the electrode and the electrolyte solution. These



findings highlight that the TOF is generally underestimated due to the difficulties in determining

the number of active sites, especially at large particles or thick porous catalyst films on

macroscopic electrodes. Also, in thick film or powder electrodes, electronic conductivity may

have a detrimental influence on the reaction rate, which might be already seen at larger

nanoparticles in this study.

a

1.6 1.8 2.0

-3

-2

-1

0

1

log

10 (

J /

A c

m-2)

E vs. RHE / V

b

10 100 1000

0.04

0.06

0.08

0.10

0.12

Ta

fel slo

pe

/ V

de

c-1

r / nm Figure 55. Electron transfer kinetics for the OER at NiOOH nanoparticles do not depend on particle size. Tafel plots for a large nanoparticle (r = 365 nm) and a small particle (r = 20 nm) (a). Analysis of Tafel slopes in the low overpotential region between 1.57 and 1.66 V vs. RHE as a function of the particle radius.

A strong effect of extensive potential cycling (conditioning) is observed for the activity of

Ni(OH)2 nanoparticles for the OER. Conditioning of Ni(OH)2 is often performed to induce a

change in the crystal phase of the material which alters the electrochemical activity for the

OER.[369] Whereas some authors report a catalytic activation by conditioning,[366,371,375] a

deactivating effect[363,368] is observed for the single nanoparticles in this study. After potential

cycling in 0.1 M KOH in the range 1 V - 1.8 V vs. RHE for at least 500 cycles, the conditioned

particles show a higher onset potential for the OER (Figure 56 a) as compared to as-grown

particles. Also, the measured Tafel slopes in the range between 1.68 and 1.88 V vs. RHE are

significantly higher than for as-grown particles (Figure 56 b). For most particles after

conditioning the Tafel slopes range between 0.15 and 0.2 V dec-1. However, some particles,

predominantly small ones, show very slow electron transfer kinetics with Tafel slopes up to

0.6 V dec-1. Correspondingly, due to the slow electrochemical reaction, the TOF after

electrochemical conditioning are lowered (Figure 56 c). Interestingly, the smaller nanoparticles

with radii below 100 nm seem to be more strongly influenced by this deactivation. Even though

this effect contradicts the common notion that small particles are more active in catalytic

reactions, this observation is a true nano-effect. The specific catalytic activity for the OER,

determined by the reaction kinetics, depends on the particle size, however in an undesired way.

The nature of the change in material properties when the Ni(OH)2 particles are subjected to

potential cycling in alkaline solution still needs to be further investigated.



a

1.6 1.8 2.0

-4

-3

-2

-1

0

log

10 (

J /

A c

m-2)

E vs. RHE / V

b

10 100 1000

0.0

0.2

0.4

0.6

Ta

fel slo

pe

/ V

de

c-1

r / nm

c

10 100 1000

0.1

1

10

100

1000

TO

FO

ER /

s-1

r / nm

Figure 56. Potential cycling in KOH deactivates Ni(OH)2 nanoparticles for the OER. Conditioned particles show a higher OER onset potential (a), larger Tafel slopes (a, b) and lower turnover frequencies (c) than as-deposited particles. Particle radii 111 nm for the as-deposited and 71 nm for the conditioned particle in (a). Tafel slopes for conditioned particles extracted in the range 1.68 V to 1.88 V.

The change of OER activity could be due to a transformation of the crystal phase. Many authors

report that Ni(OH)2 synthesized by cathodic deposition is predominantly α-Ni(OH)2.[369,376] Upon

oxidation, α-Ni(OH)2 forms γ-NiOOH. Upon overcharging in aqueous alkaline solution α-Ni(OH)2

transforms into β-Ni(OH)2 which reacts to β-NiOOH upon oxidation. There is generally little

consent in the literature concerning the most active phase for the OER. Previously β-NiOOH was

believed to be the more active OER catalyst.[365,368] Novel insights show that the α-Ni(OH)2 → γ-

NiOOH couple is responsible for the high catalytic activity.[363,371] Also the role of impurities

incorporated into the Ni(OH)2 lattice has been discussed recently.[377] Trotochaud et al. have

proposed that NiOOH is only active for the OER in the presence of Fe impurities.[375] By potential

cycling in electrolyte solution containing Fe impurities, the activity for OER changed drastically.

Because of their high surface area, nanoparticles are particularly prone to quickly take up or

release ions or water molecules from the electrolyte solution.

However, without the possibility of further structural characterization of the Ni(OH)2 particles,

the observed transformation remains elusive. The most commonly used methods for

characterization of the crystal structure, x-ray diffraction (XRD) techniques, are not applicable to

the analysis of single nanoparticles. Thus, the exploitation of Selected Area Electron Diffraction



(SAED) for single nanoparticles on nanoelectrodes is anticipated. Because the technique is

performed in a Transmission Electron Microscope (TEM), first studies reporting TEM images of

nanoparticles in such a configuration[135,136] may be an indicator of the feasibility of SAED

characterization. As the electrochemical activity of individual particles could not only be

correlated to particle size but also to structure, an additional tool for structure characterization

would largely enhance the scope of the method.

4.6.2 Study of energy storage materials

Despite its utility as a catalyst for the OER, the Ni(OH)2/NiOOH redox couple has been widely

used in highly dispersed materials for energy storage in aqueous batteries and pseudo-

capacitors. While batteries store energy in faradaic redox reactions or intercalation processes in

the bulk of the material, electrochemical capacitors store energy by charging the electrochemical

double layer at the material surface. Pseudo-capacitors are devices that exploit faradaic

reactions which are confined to species only located at the surface or near-surface. This

conceptual difference has important implications for the speed of charge release or uptake. Due

to fast charge transfer at the surface, capacitors and pseudo-capacitors cater for high power

densities. Batteries generally have a higher energy density but are limited in charge/discharge

rate due to slow diffusion in the bulk material.[378] The general paradigm is that surface or near-

surface reactions, i.e. pseudo-capacitive contributions to the charge storage become more

important with decreasing material dimensions. Ni(OH)2 is frequently referred to as a battery

material[369,376,379–383] but also as a pseudo-capacitor material.[364,384–386] Brousse et al. highlighted

the misidentification of the two concepts in the literature for various materials.[387]

Nanoelectrodes are a good tool to investigate the predominant charge storage mechanism at

nanoparticulate energy storage materials. The nanoelectrode is in direct contact with the

nanoparticle which minimizes the detrimental effect of electrical resistance that otherwise

occurs in thick porous film or powder electrodes. In addition, when analyzing the diffusion of

charge carriers in bulk materials, the influence of diffusion in the electrolyte can be excluded

because diffusional mass transport at the small electrode dimensions is fast and non-limiting.

Voltammetric analysis of the Ni(OH)2 → NiOOH + H+ + e- transition reveals a linear dependence

of the corresponding anodic peak currents with respect to the scan rate (Figure 57). The trend

line of the log ipa vs. log v plot shows a slope close to the theoretical value of 0.5 expected for a

diffusion-controlled process, as opposed to a value of 1 for a surface-confined species (i.e. a

pseudo-capacitive process). Hence, it is the diffusion of protons inside the bulk of the Ni(OH)2

nanoparticle that limits the overall oxidation reaction. Due to the analogy to a macroscopic

electrode with diffusion-limitation in the electrolyte, the Randles-Sevcik equation (20) can be



used to describe the voltammetric peak currents at the nanoelectrode with diffusion-limitation

in the particle bulk.[380]

𝑖𝑝 = 0.4463(𝑧𝐹)

32𝐴𝑐𝐷

12(𝑅𝑇)

12 ∙ 𝑣

12

(20)

ip is the voltammetric peak current, A is the surface area of the electrode (in this case of the

particle), v is the scan rate and all other symbols have their usual significance.

a

1.0 1.2 1.4 1.6 1.8

-20

0

20

i /

pA

E / V vs. RHE

b

-2.0 -1.5 -1.0 -0.5 0.0

-12

-11

log

10 (

i pa /

A)

log10

(v / V s-1)

pseudo-capacitive

slope = 1

linear fit

slope = 0.48

diffusion

slope = 0.5

Figure 57. The oxidation of Ni(OH)2 nanoparticles is limited by proton diffusion in the particle bulk. Cyclic voltammograms at a single Ni(OH)2 nanoparticle (r = 75 nm) electrode at varying scan rates from 0.01 to 0.5 Vs

-1 in 0.1 M KOH (a) and analysis of anodic peak currents as a function of the scan rate (b).

The proton diffusion coefficients at single Ni(OH)2 nanoparticles are calculated from the

Randles-Sevcik equation using the electrochemically estimated particle surface area of the

spherical particles assuming stoichiometric proton release. The nominal bulk concentration of

protons in Ni(OH)2 is ρ/M and amounts to 44.7 mol l-1. The calculated diffusion coefficients

range around 2 x 10-11 cm2 s-1 for particles smaller than r = 75 nm. Slower apparent diffusion is

observed for larger particles. This might be due to the contribution of poor electronic

conductivity of Ni(OH)2 which becomes more important for long electron paths inside larger

particles. There is a large variation of values for the proton diffusion coefficient in Ni(OH)2

reported in the literature. The values range from 10-11 to 10-7 cm2 s-1 [374,376,379,380,382] which might

reflect the challenge in finding appropriate models due to the difficulty in investigating thick

porous film electrodes. Also, at macroscopic electrodes the slow diffusion in the electrolyte

solution as well as the internal electric resistance in film or powder electrodes might interfere

with the precise measurement of the diffusion coefficient.

In conclusion, studying the electrochemistry of single nanoparticles deposited on carbon

nanoelectrodes has several advantages: 1) Extremely fast mass transport rates are obtained

which by far exceed those accessible in rotating ring-disk electrode experiments.[231] 2) The

particles are directly contacted with low resistance. In addition, possible degradation of non-

active components at the high potentials necessary for the OER can be excluded. 3)



Characterization of individual particles allows precise knowledge of particle size and thus to

deduce size-activity relations. Using these advantages, it is found that the oxidation of Ni(OH)2, a

typical reaction representative for charge storage in aqueous batteries and supercapacitors, is

entirely limited by proton diffusion inside the particle bulk even for relatively small particles.

Capacitive behavior due to charging of the electrochemical double layer or pseudo-capacitive

contributions due to faradaic reactions at the surface or near the surface are insignificant.

For the electrocatalytic evolution of oxygen the size-dependent catalytic turnover rate of

individual Ni(OH)2 particles is investigated. Variations of the TOF with particle size are

attributed to imprecise estimation of the number of catalytically active centers for and the

increasing detrimental influence of the electric resistance in larger particles. In contrast to the

notion that small particle size results in high catalytic activity, constant reaction kinetics are

observed for particles with radii between 20 nm and 500 nm. Conditioning of Ni(OH)2 particles

by potential cycling leads to a deactivation of the material for OER, indicated by higher Tafel

slopes. A size-specific loss of activity is found. The deactivation is more prominent for small

Ni(OH)2 particles. Analysis of single active nanoparticles using nanoelectrodes will develop to be

a powerful tool to elucidate more size-specific phenomena in catalysis at the nanoscale and thus

will help in understanding size-structure-activity relations. The capabilities of the method will

be further increased when spectroscopic methods for the structural characterization of the

nanoparticles become available. SAED is anticipated as a method to give complementary

structural information about the single nanoparticles.

5 Conclusions and Outlook

106


Sophisticated micro- and nanoelectrochemical tools are required in many fields of research

ranging from biomedicine to electrocatalysis. The present work shows the development and

application of electrochemical methods based on micro- and nanoelectrodes for controlled local

modification of surfaces as well as the electrochemical analysis of micro- and nanoscopic

entities.

Electrochemical methods are attractive alternatives to the established methods for micro- and

nanopatterning. Carbon surfaces modified with different electrochemically addressable surface

functional groups are locally activated toward the immobilization of biomolecules. In the direct

mode of SECM current pulses are applied for the local cleavage of TBDMS protecting groups

from surface-confined p-hydroquinone as well as for the local reduction of NO2 groups to yield

NH2 groups. The locally activated spots on the inert surface are suitable to capture and

immobilize model molecules such as alkaline phosphatase. Moreover, SECM proves useful to

image the generated micropatterns and gives evidence for the anticipated local surface reaction

as well as for the successful immobilization of target proteins. As an alternative strategy, the

scanning droplet cell is proposed for efficient triggering and control of the electrochemical

surface modifications. The technique yields unambiguous proof for the chemoselectivity of the

local activation and has the potential for further improvement of the patterning resolution.

As a versatile platform to build electrochemical nanosensors for various applications, carbon

nanoelectrodes are proposed. The needle-type electrodes are made by pyrolytic decomposition

of carbon precursor gas inside pulled quartz nanopipettes. The fabrication procedure ensures a

high success rate and control of the electrode geometry. The nanoelectrodes are well suited for

the analysis of single living cells as well as for the study of electrochemically active

nanoparticles. Their pointy shape and small overall dimensions allow precise positioning in

three dimensions and highly localized measurements to target single cells. Analytical methods to

probe the physiological state of a single living cell are advantageous to evaluate the influence of

various chemical and physical stimuli on cell function and monitor individual cell fate pathways

in the context of pathological conditions. Moreover, malignant cells may be identified by their

characteristic metabolic fingerprints.

To evaluate oxygen metabolism rates as well as the detrimental effect of reactive oxygen species

on cell viability, amperometric sensors based on modified carbon electrodes are used to probe

cells. Carbon nanoelectrodes modified with Pt black show high sensitivity for oxygen and

reactive oxygen species. The nanosensors’ capability for intracellular measurements is


107

demonstrated by inserting the electrodes into single neurons in a tissue. The total sum of

reactive oxygen and nitrogen species is detected inside the cells by anodic oxidation at high

potentials.

More elaborated sensor designs are proposed to increase the selectivity of the detection in

complex biological matrices. By deposition of Prussian Blue, the carbon electrodes are sensitized

to the reduction and oxidation of hydrogen peroxide. The mild potential applied for the cathodic

reduction of H2O2 largely excludes interferences by other oxidizable substances. However, to

assure chemical and mechanical stability of the PB film, the electrocatalyst needs to be deposited

and buried into etched nanocavities at the tip of the carbon electrodes. The resulting

nanosensors are capable of detecting H2O2 with a dynamic detection range between micromolar

and millimolar concentration and are sufficiently stable to be used in experiments for single cell

analysis. Extracellular H2O2 is detected after stimulation of ROS production. Upon inserting the

PB-modified nanoelectrodes into dorsal root ganglia neurons and macrophage cells the sensors

record cathodic current spikes corresponding to the reduction of intracellular H2O2. When a

specific inhibitor of catalase is added to the medium, the cells need substantially more time to

recover from the oxidative burst because the cells’ natural protection mechanism against H2O2 is

weakened by inhibition of catalase. These observations highlight the utility of the H2O2-sensitive

probes for the evaluation of the antioxidant capabilities of cells and might help to associate their

dysfunctionality with malignant phenotypes. More experiments involving reagents to

specifically stimulate the production of ROS will help to establish the method and may finally

lead to the application of the technique for the identification of pathogenic conditions at the

single-cell level.

The currents for the faradaic detection reactions of these amperometric nanosensors are below

the pA range. The sensitivity of the sensors is limited by the performance of the potentiostats

and current amplifiers used. As a more sensitive alternative, potentiometric sensors for the

detection of faradaic processes are proposed. A thin but dense film of redox active species

immobilized on the nanoelectrode, for instance the PB films introduced in this work, dictates the

Nernstian potential of the electrode. The analyte undergoes reactions with redox centers in the

film and thus changes the redox state of the sensor, which results in a measurable change of the

electrode open circuit potential. In contrast to amperometric sensors, the sensitivity of this

potentiometric sensor increases with decreasing dimensions of the redox active film on the

electrode. First experiments for PB-modified electrodes in the context of H2O2 detection show

promising results for the enhancement of sensitivity at electrochemical nanosensors.

An additional strategy for improving the analytical performance of nanosensors is proposed:

The intrinsic high sensitivity of field effect transistor sensors is combined with the high spatial


108

resolution of nanopipette-based methods. A FET sensor is created by depositing polypyrrole on

dual carbon nanoelectrodes. The two carbon electrodes serve as drain and source contacts while

the PPy nanojunction comprises the sensitive transistor channel. The device shows typical

reversible modulation of the drain-source current by changing the redox state of the conducting

polymer. The FET sensors at the tip of the dual nanopipettes are sensitive to pH changes. The

needle-type design of the probe and the small overall dimensions allow to measure the pH value

in microenvironments around cells. First results show that the method could be used to identify

cancerous cells by their characteristic acidification of the environment.

Moreover, the pH sensitivity of the FET nanosensors is converted into sensitivity for ATP. By

attaching hexokinase to the transistor channel, the FET detects protons released from the

enzymatic cleavage of ATP. The FET probe can specifically measure ATP released from

melanoma and cardiomyocyte cells. Due to the high localization the sensor can follow

concentration gradients in space and records dynamic changes in the ATP level. Single cells are

targeted and the ATP release upon mechanical and osmotic stimulation is measured. Finally, the

small dimensions allow puncturing the cell membrane and perform intracellular measurements.

The described nanosensor using PPy as the transistor channel is a first example of the

capabilities of FET sensors based on double barrel pipettes. Other materials could be deposited

on dual carbon electrodes to create FET sensors with different properties and possible analytes.

Moreover, the FET channel could be decorated with biological recognition elements or capture

probes to enlarge the scope of the method for sensing of biomolecules such as DNA or proteins

with high specificity. For chemical imaging and three-dimensional mapping of analyte

concentrations the combination of the proposed FET sensors with Scanning Ion Conductance

Microscopy and Scanning Electrochemical Cicroscopy are envisaged. By using multi-barrel

capillaries additional carbon nanoelectrodes or nanopipettes could be employed for controlling

the tip-to-sample distance using SECM or SICM, respectively.

Another interesting application of nanoelectrodes is in the investigation of the electrochemical

activity of single nanoparticles. Electrochemical reactions at nanoparticles are conducted at high

mass transport rates and without the detrimental influence of electrical resistance in thick

porous films or ionic resistance. Herein, the oxygen evolution reaction is studied for the first

time at individual nanoparticles. The electrode serves as a template for the deposition of

nanoparticles from Ni(OH)2, an efficient catalyst for electrochemical water oxidation. The

electrochemical activity for the OER is correlated with the particle size, which is precisely

estimated from the charge transferred for the oxidation of Ni(OH)2. Turnover frequencies for the

OER decrease with increasing particle size. This is however not a “nano-effect”, i.e. an increase of

the reaction rate due to the small particle size. Intrinsic electron transfer kinetics, as described

by Tafel slopes are invariant with particle size in the investigated range between 20 and 500 nm.


109

Ni(OH)2 is also investigated regarding its good properties as an energy storage material in

aqueous batteries and supercapacitors. The investigation of the charge storage in the

oxidation/reduction of the Ni(OH)2/NiOOH couple shows that this process is entirely limited by

the diffusion of protons inside the bulk material, even at relatively small nanoparticles. This

observation contradicts some reports that claim Ni(OH)2 is a pseudo-capacitive material, i.e. that

charge is stored in surface or near-surface reactions.

Investigating the electrochemical properties of individual nanoparticles at nanoelectrodes in

non-ensemble measurements is advantageous because the electrochemical behavior can be

directly correlated to particle size. This will help to understand the relationship between

geometry and size of electrocatalytically active particles. Moreover, because the particles are

stably attached to the electrodes, additional structural characterization might be possible. For

instance, selected area electron diffraction in the transmission electron microscope conducted

on the single particles will lead to a deeper understanding of the factors governing the catalytic

activity of nanostructured materials.

6 Experimental Procedures

110


6.1 Syntheses

Synthesis of ((2-bromo-1,4-phenylene)bis(oxy))bis(tert-butyl-dimethylsilane)

Figure 58. Synthesis of ((2-bromo-1,4-phenylene)bis(oxy))bis(tert-butyldimethylsilane).

Bromohydroquinone (2 g, 10.6 mmol) and triethylamine (3.3 ml, 23.3 mmol) were dissolved in

chloroform (40 ml). With ice cooling, tert-butyldimethylchlorosilane (3.51 g, 23.3 mmol) was

slowly added to the stirred solution. After 15 min the mixture was allowed to warm up to room

temperature and stirred overnight. The mixture was poured on ice and the product was

extracted with chloroform (100 ml). The organic phase was washed sequentially with 0.1 M HCl

(100 ml) and saturated K2CO3 solution (200 ml). After drying over Na2SO4, the solvent was

removed under reduced pressure. After characterization, the crude product (3 g), which

contained still a fraction of the unreacted alcohol, was further reacted with

tert-butyldimethylchlorosilane (1.5 g, 9.9 mmol), and triethylamine (1.4 ml, 9.9 mmol) in

chloroform (20 ml) followed by extraction, washing and evaporation of the solvent, applying the

same procedure as described above. The crude product was then purified by silica gel column

chromatography (12 cm height, 5.5 cm diameter) with hexane as the mobile phase. The pure

compound was isolated as a white solid in 55 % yield (2.45 g, 5.87 mmol).

1H NMR (400 MHz, CDCl3, 25 °C): δ(ppm)= 0.18 (s, 6 H; CH3), 0.22 (s, 6 H; CH3), 0.97 (s, 9 H; CH3),

1.04 (s, 9 H; CH3), 6.65 (dd, 3J(H,H)=9 Hz, 4J(H,H)=3 Hz, 1 H; CH), 6.73 (d, 3J(H,H)=9 Hz, 1 H; CH),

7.02 (d, 3J(H,H)=3 Hz, 1 H; CH). 13C{1H} NMR (100.6 MHz, CDCl3, 25 °C): δ(ppm)= -4.38, -4.11,

18.30, 18.49, 25.81, 25.95, 115.04, 119.68, 120.31, 124.74, 147.22, 150.16. MS (EI+):

m/z = 418.4, 416.4 (M+).

Synthesis of 4-((2,5-bis((tert-butyldimethylsilyl)oxy)phenyl)ethynyl) aniline

((2-bromo-1,4-phenylene)bis(oxy))bis(tert-butyldimethylsilane) (1.5 g, 3.6 mmol) and

diisopropylethylamine (3.5 ml, 20.1 mmol) were dissolved in anhydrous THF (4.5 ml) in a

Schlenk flask under argon atmosphere. In a second Schlenk flask, 4-ethynylaniline (421 mg,

3.6 mmol), bis(triphenylphosphine)palladium(II) dichloride (83 mg, 0.12 mmol) and copper(I)

iodide (14 mg, 0.07 mmol) were weighed in. The liquid phase and the solids were combined and


111

the mixture was stirred for 2 days at 60 °C. The reaction was monitored by silica gel thin layer

chromatography with 3:1 hexane/ethyl acetate as the mobile phase. After completion of the

reaction, the mixture was diluted with ethyl acetate and decanted so that a black, sticky residue

remained in the reaction vessel. The remainder was triturated and sonicated in

dichloromethane. The remaining solid was filtered off and the dichloromethane and the ethyl

acetate phase were combined. The solvent was removed under reduced pressure. The crude

product was triturated in a small amount of ethyl acetate and purified from the liquid phase by

silica gel chromatography (15 cm height, 5.5 cm diameter) with 3:1 hexane/ethyl acetate as the

mobile phase. The product was isolated as a yellow oil in 17 % yield (275 mg, 0.61 mmol).

1H NMR (400 MHz, CDCl3, 25 °C): δ(ppm)= 0.18 (s, 6 H; CH3), 0.23 (s, 6 H; CH3), 0.98 (s, 9 H; CH3),

1.04 (s, 9 H; CH3), 3.79 (s, 2 H; NH2), 6.62 (d, 3J(H,H)=8 Hz, 2 H; CH), 6.66-6.70 (m, 2 H; CH), 6.91

(d, 4J(H,H)=2 Hz, 1 H; CH), 7.32 (d, 3J(H,H)=9 Hz, 2 H; CH). 13C{1H} NMR (100.6 MHz, CDCl3,

25 °C): δ(ppm): -4.32, -4.17, 18.30, 18.44, 25.85, 25.97, 84.88, 93.49, 113.36, 114.90, 116.84,

120.45, 120.90, 124.02, 132.97, 146.60, 149.43, 150.73. MS (FAB+): m/z = 453.3 (M+).

Figure 59. Synthesis of 4-((2,5-bis((tert-butyldimethylsilyl)oxy)phenyl)-ethynyl)aniline.

Synthesis of nitrophenyldiazonium tetrafluoroborate

5 mmol 4-nintroaniline were dissolved in 4 ml 50 % (v/v) aqueous tetrafluoroboronic acid. At

0 °C 5 mmol sodium nitrite, dissolved in a small volume of water, were slowly added to the

stirred mixture. The yellow precipitate was filtrated and washed with cool methanol and

diethylether and then dissolved in acetonitrile. The solid was precipitated with diethylether,

filtrated and washed with ether again. Yield 71 %. 1H-NMR (200 MHz, CD3CN) δ = 8.76 ppm (m,

part A of an AA’BB‘ system, 2H); 8.63 ppm (m, part B of an AA’BB‘ system, 2H).


112

6.2 Global Electrode Modification and Characterization

6.2.1 Surface modification with TBDMS-protected p-hydroquinone groups

Electrode modification with TBDMS-protected p-hydroquinone groups, electrochemical and

chemical deprotection and characterization of the electrode surfaces is described in detail

elsewhere.[245,246] Electrochemical experiments for global modification/characterization of

electrodes using this particular surface chemistry were carried out using a Reference 600

potentiostat (Gamry Instruments). All electrochemical measurements were carried out in a

three-electrode cell with a coiled Pt wire as the counter electrode, a Ag/AgCl (3 M KCl) reference

electrode for aqueous systems and a Haber-Luggin capillary with a Ag/Ag+ (0.01 M AgClO4)

reference electrode for organic solvents. The latter was calibrated with ferrocene as an external

standard. Interconversion of potentials was accomplished according to Pavlishchuk et al.[388] As

working electrodes 20 x 10 x 2 mm Sigradur® G glassy carbon plates (HTW) were employed and

polished on a polishing wheel with 3 µm, 1 µm, 0.3 µm and 0.05 µm alumina slurries and

ultrasonicated in water. Experiments in organic solvent were performed in a custom-made air-

tight cell under dry and oxygen-free conditions. Before transferring into a new solution, all

electrodes were thoroughly rinsed with acetonitrile and ultrapure water.

For electrografting of quinone layers, the diazonium salt precursor 4-((2,5-bis((tert-

butyldimethylsilyl)oxy)phenyl)ethynyl)aniline (4.8 mg, 0.01 mmol) was dissolved in 0.1 M

TBAHFP/acetonitrile (10.5 ml) in the electrochemical cell described above. After adding first

0.1 M TFA/acetonitrile (10.5 µl, 1 µmol) followed by t-butylnitrite (3.7 µl, 0.03 mmol) it was

stirred for 15 min until the electrodes were sequentially functionalized. For electroreduction of

the in situ generated diazonium salt the potential of the glassy carbon electrodes was swept

between 0.7 V and -0.7 V vs. SHE with 0.05 V/s for typically three cycles until the CV displayed

complete passivation of the electrode surface. The electrodes were rinsed with excess amounts

of acetonitrile and water.

6.2.2 Surface modification with nitrophenyl groups

All experiments, except direct mode SECM patterning, were conducted in a three-electrode

configuration with a Pt wire as counter electrode and a Ag/AgCl/3 M KCl reference electrode.

For conventional electrochemistry a PGSTAT12 (Metrohm-Autolab) was employed as

potentiostat. Sigradur® G glassy carbon plates (HTW) were polished on polishing cloth with

3 µm, 1 µm, 0.3 µm and 0.05 µm alumina slurries and ultrasonicated in water and ethanol. For

the electrografting of nitrophenyl groups the glassy carbon plates were immersed in 1 mM

nitrophenyldiazonium tetrafluoroborate, 0.1 M H2SO4 and a cyclic voltammogram from 0.4 V

to -0.3 V (3 cycles, 0.1 V/s) was recorded. The glassy carbon plates were ultrasonicated in water.


113

For the global reduction of the nitro groups cyclic voltammograms were recorded between

0.75 V and -0.9 V in 0.1 M H2SO4 (3 cycles, 0.1 V/s).

6.3 SECM Patterning and Imaging

6.3.1 Fabrication of microelectrodes

Borosilicate glass capillaries (length 100 mm, outer diameter 1.5 mm, inner diameter 0.75 mm),

(Hilgenberg) were pulled with a gravity puller equipped with a tungsten coil. A piece of 25 μm

platinum wire (Goodfellow) was inserted into the capillaries until it was stuck in the pulled-out

end of the capillary. The wire was fixed by melting the surrounding glass with the tungsten coil

while applying vacuum to the capillary. The platinum wire was electrically contacted with a

copper wire and conductive epoxy glue, which was then hardened at 120° C for 30 min. The

platinum disk was uncovered by grinding on P 1200 emery paper. The electrodes were then

polished with 3 μm and 1 μm fine emery paper (3M) and rinsed with water. The RG value was

4-5. Prior to use the electrodes were freshly polished and the roughness and cleanliness were

tested by means of cyclic voltammetry in 5 mM [Ru(NH3)6]Cl3, 0.1 M KCl.

6.3.2 Surface modification with TBDMS-protected p-hydroquinone groups

Scanning electrochemical microscopy was conducted on a home-built device consisting of a PG

100 bipotentiostat (Jaissle Elektronik), PS-30 step motors and controller (OWIS) and a

NanoCube® P-611.3S piezo scanner with a E-664 LVPZT amplifier (Physik Instrumente)

connected to a PC via AD/DA cards (Measurement Computing). Experiments for which the

potential of the counter electrode was monitored were performed using a Model 273

potentiostat (Princeton Applied Research). Galvanostatic patterning was conducted using a

PGSTAT 302N potentiostat (Metrohm Autolab). 25 µm glass-shielded Pt disk electrodes with a

ratio of the outer diameter of the glass sheath to the diameter of the Pt disk (RG value) of 4-5

were used as SECM tips. A sample holder exposing a circular area of 0.67 cm2 to the solution was

used. During SECM scans the height of the tip over the sample was held constant at 5 µm using a

tool for automatic tilt correction implemented into the custom-made software. Area scans were

performed in a comb-like manner alongside a defined grid with a mesh width of 25 µm and a tip

velocity of 100 µm/s. The current value at each grid point was recorded after a waiting time of

2 s.

6.3.3 Surface modification with nitrophenyl groups

SECM patterning and imaging experiments were performed using a SECM equipped with a high-

resolution module (Sensolytics) and 25 µm diameter Pt microelectrodes as SECM tips. For

feedback mode experiments a PG100 (Jaissle Elektronik) was employed. Electrode patterning


114

was performed in 0.1 M acetic acid. Potentiostatic patterning in the direct mode of SECM was

performed by applying potential pulses of -0.7 V (base potential 0.3 V) to the glassy carbon plate

for 0.5 s using a PG310 potentiostat (HEKA). Galvanostatic patterning was achieved using a

µAutolab II (Metrohm Autolab) by applying current pulses with different magnitude for 0.5 s

with no passage of current before and after the pulse. The tip-to-sample separation during

patterning experiments was 4 µm. Feedback mode SECM imaging was carried out in 1 mM

K3[Fe(CN)6] in either 0.1 M HCl (pH1) or 0.1 M phosphate buffer (pH 7) with potentials of Etip =

0 V and Esample = 0.5 V (pH 1) or Etip = -0.1 V and Esample = 0.4 V (pH 7). For immobilization of the

alkaline phosphatase/avidin conjugate, the patterned glassy carbon electrode was wetted with

10 mM sulfo-NHS-biotin solution (Pierce) in 0.1 M phosphate buffer pH 7 for one hour and

rinsed with 0.1 M glycin, 0.1 M phosphate buffer pH 7. The biotinylated surface was incubated in

0.2 mg/ml alkaline phosphatase/avidin conjugate (Thermo Scientific) in 10 mM phosphate

buffer pH 8, 0.4 M NaCl and 0.5 mg/ml Tween 20 for 40 min. To remove unspecifically adsorbed

enzyme, the sample was extensively rinsed with phosphate buffer/NaCl/Tween solution. The

sample-generation/tip-collection mode measurement to detect locally bound alkaline phos-

phatase was carried out in 0.1 M carbonate buffer pH 9.4 with 2.5 mM p-aminophenylphosphate

at Etip = 0.3 V.

6.4 Scanning Droplet Cell Patterning

The scanning droplet cell was composed of a PTFE capillary, movable by stepper motors, which

was housing a miniaturized reference electrode and the Pt counter electrode. The force at which

the capillary was pressed onto the glassy carbon substrate was controlled using a KD45 2 N

force sensor (ME-Messsysteme). To prevent leakage, the capillary was approached to the

substrate until the force reached 350-450 mN. To determine the electrochemically active surface

area in the SDC, chronoamperometric measurements were performed at a globally deprotected

quinone-modified glassy carbon plate with 5 mM [Ru(NH3)]Cl3 and 0.1 M KCl as the electrolyte.

The potential was set to -0.19 V vs. SHE and the resulting cathodic current was plotted against

the inverse square root of the time. The data was fitted linearly and using the current-time

relation for systems with planar diffusion I = nr2FD1/2cπ1/2t-1/2, the diameter of the droplet was

calculated from the slope of the graph, with n the number of transferred electrons, r the radius of

the electroactive area, F the Faraday constant, D the diffusion coefficient (D = 9.1∙10-6 cm2 ∙ s-1 at

22 °C),[389] c the bulk concentration of [Ru(NH3)]Cl3 and t the time. For the fit, only current values

recorded between 2 s and 20 s after setting the working potential were taken into consideration

to assure complete decay of capacitive current as well as a stable diffusion layer within the

capillary ((D∙t)1/2 = 135 µm for t = 20 s). Coefficients of determination R2 were larger than 0.989


115

for all fits. The active surface area amounts to (8.6 ± 0.3) ∙ 10-5 cm2 (n = 3) corresponding to a

diameter of the droplet inside the capillary of (105 ± 2) µm.

6.5 Atomic Force Microscopy

All atomic force microscopy images were recorded on a NanoWizard®3 NanoScience AFM (JPK

Instruments AG) in intermittent contact mode using ACTA cantilevers (Applied NanoStructures,

Inc.). Polynomial line leveling was used to flatten images.

6.6 Fabrication and Handling of Carbon Nanoelectrodes

To fabricate single carbon nanoelectrodes, quartz glass capillaries (inner diameter 0.9 mm, outer

diameter 1.2 mm) (Sutter Instruments) were pulled to fine tips with a P-2000 laser puller (Sutter

Instruments). The instrument uses a CO2 laser to heat the capillaries while simultaneously

pulling the two ends apart with controlled force. The pulling process occurs in two steps: 1.

Initial heating while applying gentle force to test the softness of the glass. 2. Strong pulling when

the capillary has softened and the laser is switched off. The laser puller uses five different

parameters to control the pulling procedure. The parameter HEAT correlates to the power of the

laser and thus to the energy applied to heat the capillary. A higher value corresponds to more

power. The parameter FILAMENT controls the width of the heated area on the capillary. There is

no direct correlation between the parameter value and the width. More details are given in the

instrument manual. The parameter VELOCITY is a measure of the softness of the glass that has to

be reached before the second pulling step is initiated. A higher value corresponds to softer glass,

i.e. lower viscosity. DELAY positively correlates to a delay time between the first and the second

step, whereas the laser remains switched off. PULL controls the force applied to pull the two

ends of the capillary apart, whereas higher values correspond to larger force. For all

experiments presented in this work, the parameters where finely adjusted to the meet the

required specifications of the nanoelectrodes. The laser puller reacts sensitively to slight

variations in the dimensions of the glassware even for nominally identical capillaries and also is

sensitive to fluctuation in air temperature and humidity. These factors require constant small

adjustments to obtain nanoelectrodes with the anticipated dimensions. Also, different individual

instruments of the P-2000 model were used for the experiments presented. Sets of parameters

are generally not interchangeable between different individual instruments. However, on one

particular instrument a basic set of parameters ranged around HEAT 760, FILAMENT 4,

VELOCITY 45, DELAY 130 and PULL 120 and was slightly varied to tune the opening size of the

nanopipettes. Dual carbon nanoelectrodes were fabricated on another instrument with typical

parameters of HEAT 790, FILAMENT 3, VELOCITY 45, DELAY 130 and PULL 90. On a third


116

instrument HEAT 900, FILAMENT 3, VELOCITY 45, DELAY 130 and PULL 90 was most successful.

As it can be seen from these parameter sets, adjustments were mainly made by changing the

HEAT and PULL values. Higher values for these parameters result in smaller tip sizes and a

longer taper while reducing HEAT or PULL yields larger and shorter pipettes.

The nanopipettes were connected to a butane/propane (80:20) container (Campingaz) with

tygon tubing and the valve was completely opened and closed again. On a home built positioning

apparatus, the pulled nanopipette was inserted into an unpulled protective capillary of the same

specifications (Figure 60). This capillary was connected to an argon 4.6 cylinder. To control the

stream of Ar, the capillary was immersed in water and the Ar pressure was increased until small

and regular bubbles emerged from the opening. In the inert atmosphere, capillaries were heated

in the inner cone of a butane flame (ca. 1200 °C) of a jet torch lighter for typically 20 s. The

nanopipette was heated first at the very tip to clog the opening with deposited conductive

carbon. The properties of the carbon film deposited inside the pipette were investigated in detail

in reference [291].

Figure 60. Different stages of carbon nanoelectrode fabrication. Top left: Apparatus to position pulled nanopipettes inside protective capillary. Argon is applied from the tube on the left side, the butane/propane source is connected to the nanopipette. Top right: Carbon pyrolysis in the inner cone of a butane flame (about 1200 °C). Bottom left: Resulting carbon nanoelectrode inside protective capillary. Bottom right: Comparison of empty nanopipettes and carbon nanoelectrodes.

In the course of this work, it was observed that while working with nanoelectrodes, the quality

and size of the electrodes changed over time. In particular, touching the electrodes with the bare

hands and frequent immersion into any electrolyte solution deteriorated the reversibility of

electrochemical reactions at the nanoelectrodes and altered their size.[293] These observations

were also made by other authors, who attributed the damages to electrostatic discharges (ESD)


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occurring at the small electrodes.[138] Mirkin has demonstrated the utility of air plasma cleaning

to maintain the state of metal nanoelectrodes without changing the electrode geometry by

mechanical polishing.[390] In this work, to minimize the effects of electrostatic discharge,

experimenters wore ESD-protection straps in combination with a grounded ESD floor mat. In

addition, to minimize the effect of electrode fouling, solutions to be in contact with

nanoelectrodes were filtrated using Filtropur S 0.2 sterile filter (Sarstedt). These measures

improved the longevity of nanoelectrodes, even though not used consistently in all experiments

presented herein. The nanoelectrodes were used as fabricated without further treatment or

polishing. Electrodes were contacted by inserting Cu or Ag wires with diameters between

0.15 mm and 0.5 mm into the carbon-coated interior of the pipettes. Physical contact of the wire

to the carbon is sufficient to connect the electrodes.

6.7 Setups for Electrochemical Measurements and Positioning of Nanoelectrodes

All setups were housed in metal faraday cages. If possible, all electronically conductive parts

inside the shielding were grounded to the internal ground of the measuring amplifier. To avoid

ground loops, the amplifiers were operated in floating mode, i.e. the direct connection of the

internal ground to the protective earth was removed. In case of analog instruments

communicating via BNC connectors, the only connection to protective earth was indirectly via

the AD/DA converter which used the ground of the computer used for data acquisition. To

reduce electronic noise, cable connections were kept as short as possible. The shielding of BNC

cables connecting the electrochemical cell was grounded to the Faraday cage.

Setup 1

Electrochemical measurements were performed with an Axopatch 200B patch clamp amplifier,

an Axon Digidata 1322A, and a PC equipped with pClamp10 software (Axon Instruments). To

achieve cell penetration, an angle micromanipulator (Scientifica) was operated. The setup was

mounted on a damped table and shielded with removable electrical shields. Figure 61 shows an

image of the system. Position of the electrode and state of cells were optically followed by a

camera installed in the microscope (Watec WAT-902H Ultimate). For the analysis of the data,

recorded signals were digitally filtered with a 50 Hz lowpass filter. This setup was used for

experiments on brain slices and cell penetrations on DRG neurons.


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Figure 61. Setup used for experiments in relation to intracellular measurements at DRG neurons and brain slices. Image taken from [391].

Setup 2

Electrochemical measurements were performed with an Axopatch 700B two-channel patch

clamp amplifier, an Axon Digidata 1322A, and a PC equipped with pClamp10 software (Axon

Instruments). The electrodes and samples were housed in a Faraday cage built with conductive

glass windows. The petri dishes containing cultured cells or calibration solution were held by

the positioning stage built on top of an inverted optical microscope. The scan head of the SECM

instrument consisted of a PIHera P-621.2 XY nanopositioning Stage (Physik Instrumente) with

100 x 100 μm travel range that moved the sample and a LISA piezo actuator P-753.21C (Physik

Instrumente) with travel range 25 μm for electrode positioning along the Z-axis. Coarse

positioning was achieved with translation stages M-111.2DG (XY directions) and M-112.1DG (Z-

axis) (Physik Instrumente). Piezo actuators were powered by high voltage amplifiers E-503 and

E-505 (Physik Instrumente) and a servo module E-509 (Physik Instrumente) operating in closed

loop. The setup was controlled using software written in Delphi (Borland) and Code Composer

Studio (Texas Instruments) for a ScanIC controller (Ionscope). A 1 kHz low pass filter was

typically used. All Experiments involving dual carbon electrodes were performed at this setup.


119

Figure 62. Close-up image of the faraday cage housing the two headstages, piezo stage, z-motor and the cell dish in setup 2.

Setup 3

Electrochemical measurements were performed using a VA-10 voltammetric amplifier (npi) or a

L/M-EPC 7B whole cell/patch clamp amplifier (List-Medical), AD/DA converters (Measurement

Computing) and a home-built data acquisition system using custom-made software written in

Visual Basic 6. The electrochemical cell was located was in a metal faraday cage. The current

signals were filtered with in-built analog 100 Hz and 1 kHz lowpass filters, respectively, and

additionally with a digital 50 Hz lowpass filter. This setup was used for the detailed

characterization of PB-modified H2O2 sensors and investigation of Ni(OH)2 nanoparticle

electrodes.

Setup 4

Electrochemical measurements were performed using a VA-10 voltammetric amplifier (npi) or

an Axopatch 200B patch clamp amplifier (Axon Instruments). The electrochemical cell and a

micromanipulator P-853 (Physik Instrumente) were constructed on an Axiovert 25C inverted

optical microscope (Carl Zeiss AG) inside a metal Faraday cage. Controlled movements to

penetrate cells were performed with a D E-665 piezo amplifier/servo controller (Physik

Instrumente). To reduce electronic noise, the built-in light source of the microscope was

unplugged from the power supply and the microscope chassis was grounded to the Faraday

cage. As a light source light emitting diodes (LED MiniMatrix, Lumitronix) were used and

powered with a 24 V laboratory power source. An image of the setup is shown in Figure 63.


120

Figure 63. Setup used for cell penetrations at macrophage cells. Closeup image of the electrochemical cell in the Petri dish containing cells, the preamplifier and micromanipulator on an inverted optical microscope.

Setup 5

Electrochemical measurements were performed inside a metal Faraday cage using a Modulab

potentiostat (Solartron Analytical) equipped with a femtoammeter module. This setup was used

for characterization of Ni(OH)2 nanoparticle electrodes.

6.8 Protocols for the Modification and Characterization of Carbon Nanoelectrodes

6.8.1 General

All electrodes were characterized by means of cyclic voltammetry in either 5 mM [Ru(NH3)6]Cl3,

0.1 M KCl or 1 mM ferrocenemethanol, 0.1 M KCl. Only electrodes exhibiting a flat current

plateau for diffusion-limited oxidation/reduction of the mediator were further used. The

electrode radii were estimated according to equation (1) assuming diffusion coefficients of 9.1 ∙

10-6 cm2 s-1 or 6.1∙ 10-6 cm2 s-1 for the one-electron reduction of [Ru(NH3)6]3+ or oxidation of

ferrocenemethanol, respectively.

6.8.2 SEM imaging

SEM imaging of nanoelectrodes was conducted either using an EM Quanta 3 D FEG electron

microscope (FEI) without deposition of a metal film or after coating with 10 nm of chromium in

a sputter coater on all sides (Q150T S Quorum) in an FIB-SEM (Cross Beam Work Station Auriga,

Carl Zeiss). The FET tip was milled using a milling current of 200 pA at a working distance of

5 mm. Sections of 10 nm were ion-milled and imaged by SEM with a 5 keV acceleration voltage,


121

using a secondary electron in-lens detector. EDX spectra were recorded in the EM Quanta 3 D

FEG electron microscope (FEI).

6.8.3 Platinization

Platinum black was deposited on carbon nanoelectrodes by cyclic voltammetry in 2 mM H2PtCl6,

0.1 M HCl in a potential range from 0.4 V to -0.5 at 0.5 V/s for one or two cycles.

6.8.4 Fabrication and characterization of PB-based H2O2 nanosensors

Electrochemical etching was performed by means of CV from 0 V to 2 V in 0.1 M KOH, 10 mM KCl

for typically 15 cycles until the formation of a cavity occurred. Electrochemical deposition of PB

was achieved by potential cycling from 0.6 V to -0.4 V for 10 cycles in 1 mM FeCl3 and 1 mM

K3[Fe(CN)6] in 0.1 M HCl, 0.1 M KCl. After deposition the PB film was activated by cycling in the

same potential range in only 0.1 M HCl, 0.1 M KCl for at least 100 cycles. Alternatively, PB was

deposited by potential cycling from 0.6 V to 0.3 V with 0.1 V/s (50 cycles), followed by activation

for 20-40 cycles. Standard external K+ solution contained 150 mM NaCl, 6 mM KCl, 1 mM MgCl2,

1.5 mM CaCl2, 5 mM glucose and 10 mM HEPES pH 7.2. For the calibration curves, the

concentration of H2O2 stock solution was verified by titration with KMnO4. Standard internal

potassium solution contained 144 mM KCl, 2 mM MgCl2, 5 mM EGTA (ethylene glycol tetraacetic

acid) and 10 mM HEPES pH 7.2. Different potassium concentrations were achieved by addition

to the external solution of different volumes of 1 M KCl in water. In that case a Ag/AgCl counter-

reference electrode in a compartment filled with 3 M KCl and separated by a frit was used.

Potentiometric measurements were performed using a PG-100 bipotentiostat (Jaissle

Elektronik).

6.8.5 Fabrication and characterization of PPy FET nanosensors

Two wires were inserted into each barrel to make a connection with the carbon and thus

connect the drain and source electrode. A Ag/AgCl electrode was placed in solution acting as a

pseudoreference electrode. All electrochemical potentials and gate voltages are quoted against

this electrode in the corresponding solution. Pyrrole was used as received and stored under

argon atmosphere. Polypyrrole was deposited by sweeping the potential of both carbon

electrodes between -0.3 V and 0.6 V vs. Ag/AgCl/no Cl- in a deaerated solution of 0.5 M pyrrole

0.2 M LiClO4, and 0.1 M HClO4 in water. Electrochemical current was continuously measured

during the electrodeposition in order to monitor the growth of PPy on each electrode. In order to

detect the formation of a bridge between the two carbon electrodes a small drain-source voltage

was already applied and as soon as a significant drain-source current occurred the deposition

was stopped. Before usage all FET sensors were cycled between -0.3 V and 0.3 V in 0.1 M HCl

until a stable I-V curve was obtained. Every sensor was calibrated for pH in phosphate buffered


122

solutions. For all measurements on the FET, voltages of a few millivolts, typically 5 mV, were

applied between drain and source. For all measurements on cells and the associated calibrations

the gate voltage was swept between −0.3 and 0.3 V (0.4 V/s) and drain-source current was

continuously measured. To extract single drain-source current values the resulting I−V curves

were averaged and each averaged value represents one cycle. pH calibrations were made by

adding small amounts from a 0.1 M HCl stock to a solution of 120 mM NaCl, 5 mM KCl, 5 mM

MgCl2, and 12 mM phosphate buffer pH 7.4. The maximal concentration of added Cl− did not

exceed 20 mM, which has only a negligible effect on the behavior of the nano-PPy-FET. When

moving from 135 mM to 155 mM Cl−, the shift of the electrode potential of the Ag/AgCl

pseudoreference according to the Nernst equation is less than 4 mV and thus has no significant

effect on the gate voltage.

6.8.6 Immobilization of hexokinase on PPy-FET nanosensors

PPy-FETs were immersed into a solution of 25 % glutaraldehyde in water for at least 30 min

while continuously sweeping the gate voltage between -0.3 V and 0.3 V and monitoring drain-

source current. Afterwards, also monitoring transistor performance, the probe was dipped into a

solution of 500 U/ml hexokinase and glucose-6-phosphate dehydrogenase from S. cerevisiae

(Sigma Aldrich) for at least 30 min.

6.8.7 Deposition and characterization of Ni(OH)2 nanoparticles

For all experiments involving Ni(OH)2 nanoparticles a Ag/AgCl/3 M KCl counter-reference elec-

trode was used in a two-electrode system. Ni(OH)2 was deposited cathodically from 5 mM NiCl2

by applying -1 V vs. Ag/AgCl/3 M Cl- for varying electrolysis times. However, at the nanometric

electrodes only poor correlation between the electrolysis time and the amount of deposited

Ni(OH)2 was observed. CVs for the OER were performed in 0.1 M KOH at 0.01 V/s. Before the

analysis of peak currents as a function of the scan rate in 0.1 M KOH the electrodes were cycled

for at least 500 cycles in the same solution in a potential range between 0 and 0.8 V vs. Ag/AgCl/

3 M Cl-. The CVs for the analysis of Ni(OH)2 oxidation peaks are smoothed using an adjacent-

averaging filter with a width of 5 data points. However, the peak analysis was done before

smoothing by taking the average peak intensities from 3 adjacent cycles.

Particle sizes were estimated from the charge transferred during the anodic peak for the

oxidation of Ni(OH)2 according to Faraday’s law of electrolysis n = q/zF. The charge is

proportional to the amount of deposited catalyst material and thus the volume of individual

Ni(OH)2 particles assuming a density of 4.1 g cm-3.[365] From the particle volume, the particle

radii and surface areas were calculated assuming spherical particle geometry. Only those

electrodes where the calculated particle size was larger than the initial carbon electrode radius


123

were used for further analysis. Turnover frequencies were computed according to TOF =

iOER/4FnNi, with iOER the OER current extracted from the steady-state voltammograms at a

potential of 1.88 vs. RHE and nNi the amount of deposited Ni(OH)2.

6.9 Local Measurements at Cells

6.9.1 H2O2 detection in macrophage cells

The penetrations were carried out in PBS pH 7.4 on the stage of an inverted microscope inside of

a Faraday cage. Cells were taken freshly out of the incubator, the medium (Dulbecco's modified

Eagle's medium, DMEM) was removed and the cells were washed two times with PBS. To inhibit

catalase 20 mM 3-amino-1,2,4-triazole were added to the PBS shortly before starting the

measurements. The nanosensor was fixed in a 45° angle with respect to the cell dish. Under

optical control under the microscope, a micromanipulator was used to change the position of the

nanosensor until it touched the cell membrane. For cell penetration, a 2.5 to 5 μm movement of

the nanosensor along the 45° axis with respect to the cell dish was performed with the piezo

positioner. For LPS stimulation, the macrophages were incubated in DMEM containing 5 μg/ml

of LPS for 18 to 24 hours. Before the measurement, the medium was replaced with PBS buffer.

6.9.2 pH and ATP measurements using PPy-FET nanosensors

pH and ATP measurements to investigate cells were performed in unbuffered media containing

120 mM NaCl, 5 mM KCl, 5 mM MgCl2 and 20 mM glucose added in case of ATP measurements.

Prior to cell measurements, each ATP sensor was calibrated using a solution of adenosine

5′-triphosphate disodium salt hydrate (Sigma Aldrich) in the same medium used for ATP

measurements on cells. The pH of these stock solutions was carefully adjusted and matched with

the one of the media. Movements of the sensors with respect to cell specimens were performed

using stepper motors at setup 2. The measured distance from cells is the position of the motor

with respect to the point of closest approach shortly before touching the cell with the probe.

6.10 Cell Culture and Tissue Preparation

6.10.1 Brain slice preparation

Brain slice samples were prepared by Sergiy Tokar, Imperial College London.

Hippocampus slices were isolated from 28-days old rats using standard techniques.[392] After

decapitation of the rats was performed, the rat brain was isolated and placed into ice-cold ringer

solution, where the hippocampus isolation was performed. The hippocampus was sliced into

350-μm-thick transverse slices, using a Leica VT1200S blade microtome. After obtaining the cut


124

slices, they were stored in the same solution at 34 °C for 15 min. Then they were stored for at

least 1 h at room temperature. During and after isolation, the solution was constantly bubbled

with a 95% O2 and 5% CO2 gas mixture.

6.10.2 Dorsal root ganglia (DRG) neuronal culture

DRG neurons were prepared by Ainara López Córdoba, Imperial College London.

DRG from all spinal levels were obtained and pooled from freshly sacrificed postnatal day 0 to 2

(P0 to P2) Sprague Dawley rats in DMEM (Life Technologies). They were enzyme-digested in

0.2% collagenase (type I; Sigma-Aldrich) and 0.5% dispase II (Roche) in DMEM for 30 min at

37°C, and dissociated in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen),

penicillin (100 Units/mL; Sigma-Aldrich) and streptomycin (100 μg/ml; Sigma-Aldrich). Neurons

were plated on poly-L-lysine and laminin-coated (20 μg/ml; Sigma-Aldrich) and glass-bottomed

plastic petri dishes (MatTek Corporation) in DMEM supplemented with 10% FBS, penicillin (100

Units/ml), streptomycin (100 μg/ml) and 50 ng/ml Neuron Growth Factor (NGF) (all from

Sigma-Aldrich). The density was 600–1000 cells/dish and the media was renewed every two

days.[391]

6.10.3 Macrophage cell culture

Macrophages were cultured in collaboration with Melanie Mark, Ruhr-Universität Bochum and

Miriam Marquitan, Ruhr-Universität Bochum

Murine macrophages J774A.1 (ATCC LGC Standards) were cultured in DMEM (Sigma-Aldrich)

supplemented with 4500 mg/l glucose, L-glutamine, sodium bicarbonate, sodium pyruvate and

10 % FBS at 37 °C under a humidified atmosphere containing 5 % CO2. For splitting

macrophages, the medium was removed and the cells were washed with 2 - 3 mL of PBS pH 7.4.

Subsequently, they were incubated for three to five minutes in 1 ml of trypsin/EDTA until the

cells detached from the bottom of the dish. To inactivate the trypsin, 10 ml of DMEM/FBS were

added. The macrophages were removed from the Petri dish by mechanical trituration and plated

onto new dishes.

6.10.4 Melanoma and melanocyte culture

Melanoma and melanocytes were prepared by Yanjun Zhang, Imperial College.

The human malignant melanoma cell line A375M and human immortal melanocyte cell line

Hermes 3A were all obtained from the Wellcome Trust Functional Genomics Cell Bank (St.

George's, University of London, UK). Cell culture medium and reagents were purchased from

Sigma Aldrich. Melanoma cells were grown in DMEM with 10% fetal calf serum and


125

supplemented with L-glutamine (2 mM), penicillin (100 U per ml), and streptomycin (100 μg per

ml). Melanocytes were grown in RPMI 1640 medium supplemented with fetal calf serum (10%),

12-0-tetradecanoyl phorbol acetate (200 nM), cholera toxin (200 pM), human stem cell factor

(10 ng/ml) and endothelin 1 (10 nM). Cells were kept in a 5% CO2 incubator at 37°C and were

subcultured at 75% confluence.

6.10.5 Isolation of rat ventricular myocytes

Rat cardiac myocytes were prepared by Yanjun Zhang, Imperial College.

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published

by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Cardiac

myocytes from adult rats were isolated as previously described.[393] Briefly, male Sprague–

Dawley rats were heparinized, killed by cervical dislocation and the heart was rapidly excised

and placed in ice-cold Krebs–Henseleit (KH) solution of composition (mM): NaCl 119, KCl 4.7,

MgSO4 0.94, KH2PO4 1.2, NaHCO3 25, glucose 11.5, CaCl2 1 and equilibrated to pH 7.4 with 95%

O2/5% CO2. A Langendorff perfusion method was used and the interventricular septum with the

left ventricle was cut and shaken in 100% O2 enzyme-containing solution for 5 min. The

supernatant was centrifuged at 400 g for 1 min at room temperature. Cells were washed and

resuspended in the solution with 200 μmol/l calcium.

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8 Annex

146

8 Annex

8.1 Abbreviation List

3-AT –3-amino-1,2,4-triazole ATP – adenosine triphosphate AC – alternating current AFM – Atomic Force Microscopy BNC – Bayonet Neill–Concelman BQ – benzoquinone CE – counter electrode CNT – carbon nanotube CV – cyclic voltammogram/voltammetry CVD – Chemical Vapor Deposition DCFH-DA - 2′,7′-dichlorofluorescein diacetate DMEM – Dulbecco’s modified Eagle’s medium DNA – deoxyribonucleic acid DPN – dip-pen nanolithography DRG – dorsal root ganglia EDL – electrochemical double layer EDX – Energy-dispersive X-ray spectroscopy ESD – electrostatic discharge ESR – Electron Spin Resonance FB – feedback FBS – fetal bovine serum FET – field effect transistor FIB – Focused Ion Beam GC – glassy carbon GCE – glassy carbon electrode GOX – glucose oxidase HEPES - 4-(2-hydroxyethyl)-1-piperazine- ethanesulfonic acid HER – hydrogen evolution reaction HRP – horseradish peroxidase HOPG – highly oriented pyrolytic graphite HQ – hydroquinone LOD – limit of detection LPS – Lipopolysaccharides MOSFET – metal oxide semiconductor field effect transistor NADPH – nicotinamide adenine dinucleotide phosphate NGF – neuron growth factor NHS – N-hydroxysuccinimide NOS – nitric oxidase synthase NOX – NADPH Oxidase OCP – open circuit potential OER – oxygen evolution reaction ORR – oxygen reduction reaction pAP – p-aminophenol pAPP – p-aminophenolphosphate PB – Prussian Blue

PBS – phosphate-buffered saline PNA – peptide nucleic acid PW – Prussian White PPy – polypyrrole PTFE – polytetrafluoroethylene RDE – rotating disk electrode RE – reference electrode RHE – reversible hydrogen electrode ROS – reactive oxygen species RNS – reactive nitrogen species SAED – Selected-Area Electron Diffraction SAM – self-assembled monolayer SDC – Scanning Droplet Cell SECM – Scanning Electrochemical Microscopy SF – shear force SEM – Scanning Electron Microscopy SHE – standard hydrogen electrode SICM – Scanning Ion Conductance Microscopy SOD – superoxide dismutase STM – Scanning Tunneling Microscopy TBAHFP – tert-butylammonium hexafluoro-phosphate TBDMS – tert-butyldimethylsilyl UME – ultramicroelectrode TEM – Transmission Electron Microscopy TOF – turnover frequency WE – working electrode XPS – X-ray Photoelectron Spectroscopy XRD – X-ray Diffraction

8 Annex

147

8.2 Publications

[12] Zhang, Y.*; Clausmeyer , J.*; Babakinejad, B.*; López Córdoba, A.; Ali, T.; Shevchuk , A.;

Takahashi , Y.; Novak , P. Edward , C.; Lab, M.; Gopal, S.; Chiappini, C.; Anand, U.; Magnani, L.;

Coombes, C.; Gorelik, J.; Matsue, M.; Schuhmann, W.†; Klenerman, D.†; Sviderskaya, E.†; Korchev,

Y.† Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis. ACS Nano

2016, in press, DOI: 10.1021/acsnano.5b05211

[11] Clausmeyer, J.†; Schuhmann, W.† Nanoelectrodes: Applications in Electrocatalysis, Single-

Cell Analysis and High-Resolution Electrochemical Imaging. TrAC Trends Anal. Chem. 2016, in

press, DOI: 10.1016/j.trac.2016.01.018

[10] Clausmeyer, J.†; Masa, J.; Ventosa, E.; Öhl, D.; Schuhmann, W.† Nanoelectrodes Reveal

Electrochemistry of Single Nickelhydroxide Nanoparticles. Chem. Commun. 2016, 52, 2408–2411.

[9] Stratmann, L.*; Clausmeyer, J.*; Schuhmann, W.† Non-destructive Patterning of Carbon

Electrodes Using the Direct Mode of Scanning Electrochemical Microscopy. ChemPhysChem

2015, 16, 3477-3482.

[8] Švorc, Ĺ.; Jambrec, D.; Vojs, M.; Barwe, S.; Clausmeyer, J.; Michniak, P.; Marton, M.;

Schuhmann, W.† Doping Level of Boron-Doped Diamond Electrodes Controls the Grafting

Density of Functional Groups for DNA Assays. ACS Appl. Mater. Interfaces 2015, 7, 18949-18956.

[7] Matysiak, E.; Botz, A.; Clausmeyer, J.; Wagner, B.; Schuhmann, W.; Stojek, Z.; Nowicka, A.†

Assembling Paramagnetic Ceruloplasmin at Electrode Surfaces Covered with Ferromagnetic

Nanoparticles. Scanning Electrochemical Microscopy in Presence of Magnetic Fields. Langmuir

2015, 31, 8176-8183.

[6] Clausmeyer, J.; Schäfer, D.; Nebel, M.; Schuhmann, W.† Temperature-Induced Modulation of

the Sample Position in Scanning Electrochemical Microscopy. ChemElectroChem 2015, 2, 946–

948.

[5] Clausmeyer, J.; Actis, P.; López Córdoba, A.; Korchev, Y.; Schuhmann, W.† Nanosensors for

the detection of hydrogen peroxide. Electrochem. Commun. 2014, 40, 28–30.

[4] Actis, P.†; Tokar, S.; Clausmeyer, J.; Babakinejad, B.; Mikhaleva, S.; Cornut, R.; Takahashi, Y.;

López Córdoba, A.; Novak, P.; Shevchuck, A. I.; Dougan, J. A.; Kazarian, S. G.; Gorelkin, P. V.;

Erofeev, A. S.; Yaminsky, I. V.; Unwin, P. R.; Schuhmann, W.; Klenerman, D.; Rusakov, D. A.;

Sviderskaya, E. V.; Korchev, Y. E.† Electrochemical Nanoprobes for Single-Cell Analysis. ACS Nano

2014, 8, 875–884.

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[3] Clausmeyer, J.; Schuhmann, W.†; Plumeré, N. Electrochemical patterning as a tool for

fabricating biomolecule microarrays. TrAC Trends Anal. Chem. 2014, 58, 23–30.

[2] Clausmeyer, J.; Henig, J.; Schuhmann, W.†; Plumeré, N.† Scanning Droplet Cell for

Chemoselective Patterning through Local Electroactivation of Protected Quinone Monolayers.

ChemPhysChem 2014, 15, 151.

[1] Maleki, A.; Nematollahi, D.; Clausmeyer, J.; Henig, J.; Plumeré, N.†; Schuhmann, W.

Electrodeposition of Catechol on Glassy Carbon Electrode and Its Electrocatalytic Activity

Toward NADH Oxidation. Electroanalysis 2012, 24, 1932–1936.


† Corresponding authors

8.3 Conference Contributions

8.3.1 Oral presentations

Amperometric Nanosensors and Field-Effect Transistors for Extra- and Intracellular Chemical

Analysis

Clausmeyer, J.; Zhang, Y.; Marquitan, M.; López Córdoba, A.; Korchev, Y. Schuhmann, W.

66th Annual Meeting of the International Society of Electrochemistry, Taipei, Taiwan, 2015

Carbon Nanoelectrodes for Single-Cell Analysis: From Amperometric Sensing to Field-Effect

Transistors

Clausmeyer, J.; Zhang, Y.; Babakinejad, B.; Marquitan, M.; López Córdoba, A.; Actis, P.; Korchev,

Y.; Schuhmann, W.

17th Topical Meeting of the International Society of Electrochemistry, St. Malo, France, 2015

Destruction-Free Surface Patterning Using Electrochemical Scanning Probes and Functionalized

Nanoelectrodes for Single-Cell Analysis

Invited Lecture

Clausmeyer, J.

Aarhus Symposium on Surface Chemistry and Electrochemistry, Aarhus, Denmark, 2014

Functionalized Nanoelectrodes for the Detection of Oxygen Species Inside Single Living Cells

Clausmeyer, J.; Actis, P.; López Córdoba, A.; Korchev, Y.; Schuhmann, W.

Elecnano 6, Paris, France, 2014

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149

Electrochemical surface modification and sensing of pH and oxygen species

Invited Lecture

Clausmeyer, J.

Colloquium at the Imperial College London, London, UK, 2013

Scanning Droplet Cell for Chemoselective Patterning via Local Electroactivation of Protected

Quinone Monolayers

Clausmeyer, J.; Henig, J.; Schuhmann, W.; Plumeré, N.

Bioelectrochemistry 2013, Bochum, Germany, 2013

Direct mode SECM revisited: Increased chemoselectivity during local electrochemical cleavage of

a protecting group from electroactive quinone monolayers

Clausmeyer, J.; Plumeré, N.; Henig, J.; Stratmann, L.; Schuhmann, W.

7th Workshop on Scanning Electrochemical Microscopy (SECM) and Related Techniques, Ein Gedi,

Israel, 2013

Electrochemical Deprotection and Activation of Michael Acceptors for Electrode

Microstructuration

Clausmeyer, J.; Plumeré, N.; Henig, J.; Schuhmann, W.

62nd Annual Meeting of the International Society of Electrochemistry, Niigata, Japan, 2011

8.3.2 Poster presentations

Spearhead Field-Effect Transistor Nanosensors for High-Resolution Chemical Analysis

Won 2nd Poster Prize

Clausmeyer, J.; Zhang, Y.; Babakinejad, B.; López Córdoba, A.; Ali, T.; Shevchuk, A.; Edwards, C.;

Gopal, S.; Anand, U.; Korchev, Y.; Schuhmann, W.

8th International Workshop on Scanning Electrochemical Microscopy, Xiamen, China, 2015

Investigation of Single Ni(OH)2 Nanoparticles: Electrocatalysis and Energy Storage at Ultrafast

Mass Transport

Clausmeyer, J.; Masa, J.; Ventosa, E.; Schuhmann, W.

66th Annual Meeting of the International Society of Electrochemistry, Taipei, Taiwan, 2015

Electrocatalysis at the Nanoscale – Oxygen Evolution Reaction (OER) at Single Ni(OH)2

Nanoparticles

Clausmeyer, J.; Masa, J.; Ventosa, E.; Schuhmann, W.

17th Topical Meeting of the International Society of Electrochemistry, St. Malo, France, 2015

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150

Patterning of Electroactive Monolayers Using Scanning Droplet Cell

Clausmeyer, J.; Henig, J.; Schuhmann, W.; Plumeré, N.

12th International Fischer Symposium, Lübeck, Germany, 2012

Underscored name: Presenting author

Documents

Micro- and nanoelectrochemistry for surface patterning