Robust Microelectrodes Developed for Improved

Stability in Electrochemical Characterization of

Biomolecular Layers

Yuksel Temiz, Anna Ferretti, Enrico Accastelli, Yusuf Leblebici, Carlotta Guiducci

Institute of Bioengineering and Institute of Electrical Engineering

Ecole Polytechnique Federale de Lausanne (EPFL)

CH-1015, Lausanne, Switzerland

e–mail: yuksel.temiz@epfl.ch

Abstract— This paper presents a robust electrochemical detec-tion system composed of microfabricated electrodes and a poten-tiostat circuit developed for quantitative detection of biomoleculesby Cyclic Voltammetry (CV) measurements. Compared to elec-trochemical cells employing external counter electrodes (CE) andpolymer passivation layers, this system offers more reliable andstable operation owing to on-chip Pt CE and very stable oxidepassivation layer, withstanding aggressive cleaning techniquesand chemicals involved. The adhesion of oxide to Au and Ptis significantly enhanced by adding slots to the metals andoptimizing the metal lift-off process. Different electrode configu-rations and sizes are tested by CV of redox species before andafter self-assembled organic molecular layer formation, and it isconcluded that the proposed system offers a low-cost and reliablemicroelectrode array solution for real time and high sensitivebiomolecular detection.

I. INTRODUCTION

Characterization of electrochemical interfaces is a power-

ful and assessed mean for investigating molecular layers. A

number of quantitative techniques are available to determine

the layer formation, as well as its compactness and passivation

properties, the composition of mixed layers and the presence of

electroactive or enzymatic labels led by target recognition [1],

[2]. The development of such techniques on microfabricated

arrays is driven by one or more of the following requirements:

(i) reagents and time reduction, (ii) parallel measurements, (iii)

on-chip CMOS integration, enabling multiplexing and very

high parallelism.

Electrochemical sensor arrays are usually fabricated either

on passive substrates (such as silicon [3] and glass [4]),

enabling low-cost and disposable chips, or directly on top

of an active CMOS chip by the integration of electronics

with the sensing sites through post-CMOS processing, offering

higher array densities and performances [5]–[7]. Although

both approaches propose promising solutions for biosensing

applications, the reliability of the electrodes in such electro-

chemical sensor arrays is still a matter of discussion. For

instance, the response of the sensors is noticeably affected

by the process variations due to very small sizes of electrodes

, and the materials used for the electrodes, as well as for the

passivation, are not always resistant to typical surface cleaning

techniques and to the chemicals involved [8]. Therefore, the

system reported in this paper aims to provide more robust

microelectrodes suitable for medium-density biosensor arrays.

II. ELECTROCHEMICAL MEASUREMENT SYSTEM

This section describes the operation of the potentiostat

circuit and the fabrication of the microelectrodes designed for

the characterization of the biomolecular layers. Two versions

of microelectrodes are developed: the first version involves

basic microfabrication process steps, while the layout of the

electrodes and the process flow are modified in the second

version in order to improve their reliability and stability.

A. Potentiostat Circuit and Microelectrode Design

Fig. 1 shows the simplified diagram of the electrochemical

measurement system used for the detection of redox species

through the CV technique [1]. The system comprises a three-

electrode electrochemical cell, a potentiostat circuit, and a

data acquisition (DAQ) board controlled by the LabVIEW

software. Among different potentiostat topologies [9], the

Ag/AgCl reference electrode

Pt counter

electrode

t

Vin

-

+

IWE

IWE

Vin vs VRE

PDMS

Reservoir

-

+

-

+

t

IWE

Vin

Au

working

electrode

LabVIEW

Fig. 1. Simplified schematic of the three-electrode cyclic voltammetrymeasurement system.

978-1-4244-8168-2/10/$26.00 ©2010 IEEE 1051 IEEE SENSORS 2010 Conference

standard grounded-WE configuration is preferred since it is

relatively simple to implement, it enables bi-directional current

measurement, and it allows both to apply the input potential

and to measure the output current with respect to ground.

In the control part of the potentiostat circuit, a feedback

loop ensures that the potential difference between the on-

chip WE and the external RE is kept at the applied level,

while preventing any current to flow through the RE [10].

In the current measurement part, a transimpedance amplifier

keeps the WE potential at the ground (virtual) potential and

converts the current flowing between the CE and the WE to

a voltage through a feedback resistor. The output voltage is

then digitized and processed to be displayed in the CV plots.

B. Basic Design and Fabrication for Microelectrodes

The first version of the electrochemical sensor chip is fab-

ricated through a series of standard microfabrication steps, as

illustrated in Fig. 2. Au and Pt microelectrodes are fabricated

on a 100 mm diameter silicon wafer with 500 nm of top

silicon dioxide (SiO2, hereafter shortened as “oxide”) film for

isolation. First, a standard double-layer lift-off process is used

to realize Au WE electrodes. In this step, spin-coated lift-off

resist (LOR) and positive-tone photoresist are patterned by

photolithography, then 20 nm of Ti and 200 nm of Au are

deposited by e-beam evaporation. Metals on the unexposed

regions are removed by stripping the resists, forming the

electrodes and the interconnections. The same lift-off process

steps are repeated for the CE, which consists of 20 nm of Ti

and 200 nm of Pt. The whole surface is then covered with 1 µm

of sputtered oxide to passivate interconnections and the Ti

adhesion layer. Oxide is chosen as the passivation layer since it

is more resistant to surface cleaning methods (such as oxygen

plasma) and to the chemicals used in the electrochemical tests

compared to polymeric materials (such as polyimide, parylene,

SU8, etc.). Moreover, oxide is a microelectronic compatible

material, which can be deposited and etched with the standard

3. Oxide sputtering (1µm)

for the passivation

2. Ti/Pt (20nm/200nm) lift-off

for the counter electrode (CE)

4. Oxide Etching (Wet)

SiO2

Si

1. Ti/Au (20nm/200nm) lift-off

for the working electrode (WE)

Fig. 2. Simplified process steps used in the fabrication of microelectrodescomposed of Au WE and Pt CE.

17.4mm

17.4mm

Fig. 3. Camera photo of the fabricated chips after SiO2 etching and pho-toresist stripping. Each chip measures 17.4 mm by 17.4 mm, and comprises10 WEs with diameters ranging from 50 µm to 1 mm.

200µm

dia.

100µm

dia.

50µm

dia.

Au

PtSiO2

SiO2

delamination

SiO2

delamination

Cracks and

defects

500µm

dia.

Fig. 4. Colorized SEM photos of the microelectrodes showing severe oxidedelamination problem.

microfabrication processes. Finally, the oxide on the sensing

sites and the socket contacts is etched with the buffered

hydrofluoric acid (BHF 7:1) by using patterned photoresist

as the masking layer.

Fig. 3 shows the camera photo of the fabricated chips

after oxide etching and photoresist stripping. Although these

process steps enable the realization of electrodes with no

visible defect prior to measurements, scanning electron mi-

croscope (SEM) images taken after surface cleaning steps

and electrochemical measurements show that the oxide layer

tends to delaminate due to poor adhesion of sputtered oxide

to Au and Pt layers. This phenomenon is shown in Fig. 4.

To achieve a reliable and robust electrochemical measurement

system, such delamination cannot be tolerated since it gives

misleading measurement results by affecting the total electrode

area. Additionally, it is also observed that the layer delamina-

tion propagates during the electrochemical tests, resulting in

random drift in the output signal.

C. Improved Design and Fabrication for Microelectrodes

In the improved version of the microelectrodes, the lift-off

process and the electrode design are modified to solve the

oxide delamination problem. Fig. 5 illustrates the difference

1052

Si

SiO2

LOR

Photoresist

Au

Notches at

the edges

Standard Lift-off Process

Final view

Modified Lift-off Process

Fig. 5. Illustrative 3D picture of the standard and modified lift-off processes.

Au

(After SiO2

etching)

Sputtered

SiO2

SiSiO2

Notch

Fig. 6. Colorized SEM photo of the cross-section of the improved versionof electrodes with the notches at the metal edges.

between the lift-off processes employed in the two versions of

microelectrodes. In the modified version, the LOR undercut

is reduced to have notches at the metal edges in order to

enhance the oxide adhesion. However, reducing the undercut

too much may result in deposition of almost or completely

conformal metal film, which would lengthen the lift-off pro-

cess considerably and possibly require ultrasonic agitation to

break the metal. Thus, the resist development time has to be

adjusted to optimize the LOR undercut, providing notches at

the edges, while maintaining easy and precise metal patterning.

The cross-section of the improved version of the electrodes is

shown in Fig. 6. Besides, it is observed that the delamination is

more significant on the larger metal regions. Therefore, the lay-

out of the electrodes is modified by introducing slot openings

to the critical points, especially to the wider interconnections.

Fig. 7 shows the SEM photos of the improved electrodes with

notches and slots, taken after a series of cleaning steps and

electrochemical tests, demonstrating that the electrodes stay

stable throughout the experiments.

III. MEASUREMENT RESULTS

This section provides the results of the electrochemical

measurements involving the CV of potassium ferricyanide, fer-

rocenium hexafluorophosphate, and self-assembled monolayer

(SAM) obtained with ferrocene functionalized alkanethiols.

The chips featuring different sizes of microelectrodes are

Au

Pt

SiO2

Slots

Notches at the

edges of the

metal, improving

the SiO2

adhesion

Fig. 7. Colorized SEM photos of the improved electrodes, demonstrating novisible defect even after a series of cleaning steps and electrochemical tests.

tested with a very compact potentiostat circuit implemented

with a small number of high-performance components, en-

abling experimental flexibility and reliability. The system al-

lows to address each WE and CE independently and to perform

differential measurements by simultaneously acquiring the

signal coming from two on-chip WEs. The triangular signal

required for the CV is generated by the LabView software

and applied to the electrolyte through the control part of

the potentiostat circuit connected to the external Ag/AgCl

pellet RE (Phymep, France) and on-chip Pt CE. The generated

current in response to Faradaic and non-Faradaic processes

is converted to voltage by a transimpedance amplifier and

recorded with a DAQ board (National Instruments).

Prior to the experiments, chips are cleaned with oxygen

plasma to remove the possible organic contaminants from

the surface. A polydimethylsiloxane (PDMS) chamber is used

as the reservoir for the solutions during the CV tests and

the washing steps. All chemicals mentioned hereafter are

purchased from Sigma (Buchs, Switzerland).

A. Cyclic Voltammetry of Potassium Ferricyanide

Potassium ferricyanide (K3[Fe(CN)6]) is a reversible redox

species commonly used for the electrode characterization [7],

[11]. Recently, it has been also employed for the electrochemi-

cal detection in bioaffinity sensors [12], [13]. Fig. 8 shows the

CV results of 50 µm, 100 µm, and 200 µm diameter electrodes

in response to different concentrations of (K3[Fe(CN)6]) in

200 mM KCl solution, and the dependency of the output

current on the WE area. The linear relation between the

current level and the electrode area demonstrates that the

oxide passivation layer, which defines the total electrode area,

is stable without any delamination. These CV measurements

also verify that the system gives reliable and quantitative

response, with a forward peak current of 143 µA/cm2 for

1 mM concentration of K3[Fe(CN)6].

B. Cyclic Voltammetry of Ferrocenium

Ferrocene cation, called Ferrocenium (Fc+), is an electroac-

tive DNA label which interacts electrostatically with the major

groove. It has been employed in electrochemical sensors for

DNA hybridization detection [14], [15]. A solution of 0.4 mM

ferrocenium hexafluorophosphate in TE buffer at pH 8 is

employed to test the response of the system to this label.

1053

-12

-10

-8

-6

-4

-2

0

2

4

-0.1 0 0.1 0.2 0.3 0.4 0.5

Cu

rre

nt (n

A)

Potential (Vin vs Ag/AgCl)

50µm dia. Electrode

200mM KCl

10µM K3Fe(CN)6

100µM K3Fe(CN)6

1mM K3Fe(CN)6

Scan Rate: 100mV/s

-30

-25

-20

-15

-10

-5

0

5

10

-0.1 0 0.1 0.2 0.3 0.4 0.5

Cu

rre

nt (n

A)

Potential (Vin vs Ag/AgCl)

100µm dia. Electrode

200mM KCl

10µM K3Fe(CN)6

100µM K3Fe(CN)6

1mM K3Fe(CN)6

Scan Rate: 100mV/s

-100

-80

-60

-40

-20

0

20

40

-0.1 0 0.1 0.2 0.3 0.4 0.5

Cu

rre

nt (n

A)

Potential (Vin vs Ag/AgCl)

200µm dia. Electrode

200mM KCl

10µM K3Fe(CN)6

100µM K3Fe(CN)6

1mM K3Fe(CN)6

Scan Rate: 100mV/s

-100

-80

-60

-40

-20

0

20

40

-0.1 0 0.1 0.2 0.3 0.4 0.5

Cu

rre

nt

(nA

)

Potential (Vin vs Ag/AgCl)

1mM K3Fe(CN)6 in 200mM KCl

Scan Rate: 100mV/s

y = 0.1431x - 1.1324R² = 0.9986

0

10

20

30

40

50

0 100 200 300 400

Fo

rward

Peak C

urr

en

t (n

A)

Electrode Area (x10-6 cm2)

200µm dia.

100µm dia.

50µm dia.

200µmdia.

100µm dia.

50µm dia.

(a) (b)

(c) (d)

50µm diameter Electrode 100µm diameter Electrode

200µm diameter Electrode

Fig. 8. CV measurements of potassium ferricyanide for 50 µm (a),100 µm (b), 200 µm (c) diameter electrodes, and the dependency of theoutput current on the surface area of WE (d).

Two identical electrodes placed 12 mm far away from each

other on the same chip are measured simultaneously by the

differential electronics in order to investigate the mismatch due

to process variations. The measurement results show that the

error due to mismatch in the output current is less than 10%

also including the mismatch introduced by the electronics, as

plotted in Fig. 9.

C. Cyclic Voltammetry on SAM of Ferrocene Functionalized

Alkanethiols

Ferrocene is employed as a covalent label for electro-

chemical aptamer based sensors (aptasensors). Aptamers are

single-stranded nucleic acid –either DNA or RNA– able to

specifically bind to the target against which they are selected.

Nucleic acid can be attached to Au surfaces through a thiol

group at one of their ends and they can be labeled, for

instance, with ferrocene. Upon recognition of the target, the

conformational change of the probe brings ferrocene in close

proximity to the surface, resulting in a higher voltammetric

response [16]–[18].

CV measurement on a ferrocene-labeled alkanethiol SAM

is performed in order to investigate the possibility to detect

the ferrocene covalently linked to the surface. Microelec-

trodes are incubated in a 2 mM ethanolic solution of 6–

(Mercaptohexyl)ferrocene for 1 h, rinsed with ethanol and

measured in a 0.1 M HClO4, 10 mM HCl solution. Fig. 10

shows the result of a 50 µm diameter electrode. The oxidation

and reduction peaks of 0.9 nA are clearly observable over the

background signal recorded before the incubation.

IV. CONCLUSION

In this paper, the development of robust microelectrodes

designed to enhance the stability and the reliability in elec-

trochemical sensors is presented. Microelectrodes comprising

Au WE and Pt CE are fabricated on a silicon substrate and

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

-0.1 0.1 0.3 0.5

Cu

rre

nt (n

A)

Potential (Vin vs Ag/AgCl)

Identical 50µm dia. Electrodes

TE Buffer 1

0.4mM FC 1

TE Buffer 2

0.4mM FC 2

Scan Rate: 200mV/s

Cell 1Cell 2

Identical 50µm diameter Electrodes

(a)

-8

-6

-4

-2

0

2

4

6

8

-0.1 0.1 0.3 0.5

Cu

rre

nt (n

A)

Potential (Vin vs Ag/AgCl)

Identical 100µm dia. Electrodes

TE Buffer 1

0.4mM FC 1

TE Buffer 2

0.4mM FC 2

Scan Rate: 200mV/s

Cell 1Cell 2

Identical 100µm diameter Electrodes

(b)

Fig. 9. CV of TE buffer and 0.4mM Ferrocenium (Fc+) in TE buffer for50 µm (a), and 100 µm (b) diameter electrodes, recorded simultaneously fromtwo identical cells placed 12 mm far away from each other.

passivated with sputtered silicon dioxide, withstanding the

chemicals and the surface cleaning techniques involved. The

oxide delamination problem observed in the microelectrodes

fabricated with the standard processes is solved by modifying

the design and the lift-off process. Additionally, a compact

potentiostat circuit, which enables to select each electrode

independently and to read two WEs differentially, is imple-

mented to perform cyclic voltammetry measurements.

The microelectrodes are characterized by the CV of various

redox species which are commonly employed in the electro-

chemical sensors. The results demonstrate that the developed

1054

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

-0.2 0 0.2 0.4

Cu

rre

nt

(nA

)

Potential (Vin vs. Ag/AgCl)

50µm diameter Electrode

Before

Incubation

After 1hr of

Incubation

Scan Rate:

100mV/sec

Fig. 10. CV of the 50µm diameter electrode before and after incubationwith 6-(Mercaptohexyl)ferrocene.

microelectrodes provide stable and repeatable signals, suggest-

ing that they are good candidates for reliable biomolecular

detection.

ACKNOWLEDGMENT

This work is supported by the Nano-Tera.CH program of

the Swiss Confederation and by the Integrated Systems Centre

(Centre SI) of EPFL. Authors would like to thank Jean-

Baptiste Bureau and Cyrille Hibert for the discussions on the

microfabrication processes, and Giampaolo Zuccheri for his

precious support and feedback on the electrochemical tests.

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