Plasmonic Imaging of Electrochemical Impedance

AC10CH09-Wang ARI 3 May 2017 21:55

Plasmonic Imaging ofElectrochemical ImpedanceLiang Yuan,1 Nongjian Tao,1,2 and Wei Wang1

1State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry andChemical Engineering, Nanjing University, Nanjing 210093, China;email: [email protected] for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University,Tempe, Arizona 85287

Annu. Rev. Anal. Chem. 2017. 10:183–200

First published as a Review in Advance onMarch 6, 2017

The Annual Review of Analytical Chemistry is onlineat anchem.annualreviews.org

https://doi.org/10.1146/annurev-anchem-061516-045150

Copyright c© 2017 by Annual Reviews.All rights reserved

Keywords

electrochemical impedance microscopy, surface plasmon resonance,plasmonic imaging, electrochemical impedance spectroscopy, single-cellimaging

Abstract

Electrochemical impedance spectroscopy (EIS) measures the frequencyspectrum of an electrochemical interface to resist an alternating current.This method allows label-free and noninvasive studies on interfacial ad-sorption and molecular interactions and has applications in biosensing anddrug screening. Although powerful, traditional EIS lacks spatial resolutionor imaging capability, hindering the study of heterogeneous electrochem-ical processes on electrodes. We have recently developed a plasmonics-based electrochemical impedance technique to image local electrochemicalimpedance with a submicron spatial resolution and a submillisecond tem-poral resolution. In this review, we provide a systematic description of thetheory, instrumentation, and data analysis of this technique. To illustrate itspresent and future applications, we further describe several selected samplesanalyzed with this method, including protein microarrays, two-dimensionalmaterials, and single cells. We conclude by summarizing the technique’sunique features and discussing the remaining challenges and new directionsof its application.

183

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ANNUAL REVIEWS Further

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http://www.annualreviews.org/doi/full/10.1146/annurev-anchem-061516-045150


1. INTRODUCTION

Electrochemical impedance spectroscopy (EIS) has found numerous applications in analyticalchemistry, ranging from biosensing (1–3) to drug screening (4–6). EIS measures the frequencyresponse of an electrochemical interface to resist a small alternating current. By analyzing thefrequency spectrum of the voltage response in terms of a model electric circuit consisting ofresistors and capacitors, one can quantitatively investigate various processes at the interface,including molecular adsorption and interactions and electrochemical reactions. EIS is also apowerful technology for the label-free detection of molecular binding in real time (1, 3). Despitethese unique capabilities and features, traditional EIS measures the impedance averaged over theentire interface (or electrodes). As such, it lacks spatial resolution or imaging capability, which isessential for studying heterogeneous processes occurring at the interface and resolving individualnanoscaled materials on the electrode.

Two approaches were developed to achieve spatial resolution and imaging capability. One mea-sures the impedance of an array of microelectrodes (7–12), and the second approach measures localimpedance of a sample by scanning a microelectrode across the sample surface (13–17). The formerrequires fabrication of a microelectrode array and multiplexed recording of current from each mi-croelectrode in the array, which limits the total number of microelectrodes. Besides, the interelec-trode distance, which determines the spatial resolution, must be carefully designed and optimizedto minimize electronic and chemical cross talk between adjacent microelectrodes. In the latterapproach, the use of a scanning microelectrode provides high spatial resolution but at the expenseof temporal resolution. It often takes minutes to study fast processes or to obtain an impedance im-age, which is difficult for applications that require high throughput. A technique that can image theelectrochemical impedance with high spatial and temporal resolutions is highly desired for high-throughput readout of microarrays and for studying fast heterogeneous electrochemical processes.

Recently, we have developed plasmonics-based electrochemical impedance microscopy (P-EIM), which allows wide-field imaging of the interfacial distribution of electrochemical impedancewith submicron spatial resolution and submillisecond temporal resolution (18–20). Instead of mea-suring impedance electrically from local voltage (response to a modulating current), P-EIM obtainselectrochemical impedance images optically by converting the local surface charge associated withthe modulating current into a plasmonic signal. This method does not require the use of a scanningmicroelectrode, which leads to extremely fast imaging speed and avoids possible disturbance ofthe microelectrode to the sample in the scanning probe approach. In this review, we discuss indetail the basic principle of P-EIM and its experimental validation. We then describe the instru-mentation of P-EIM, including the optical microscope, electrochemical module, signal and imagesynchronization, and data processing algorithm. After presenting the principle and instrumenta-tion of P-EIM, we survey several of its applications, including (a) studying molecular interactionsand binding kinetics on microarrays, (b) visualizing the capacitance and defects of two-dimensional(2D) nanomaterials such as graphene, and (c) mapping the subcellular impedance distribution anddynamic evolution of single living cells. Finally, we discuss the major features and strengths ofP-EIM and the remaining challenges and potential research directions of this technique.

2. PRINCIPLE AND PROOF-OF-CONCEPT EXPERIMENTSOF PLASMONICS-BASED ELECTROCHEMICALIMPEDANCE MICROSCOPY

2.1. Surface Plasmon Resonance and Its Dependence on Surface Charge Density

The principle of P-EIM is based on the sensitive dependence of surface plasmon resonance (SPR)on the surface charge density of a metal film (electrode), which changes with the voltage applied

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to the electrode. Once the relation between SPR and surface charge density is determined, onecan then map the local surface charge density from SPR images, from which the local currentdensity and thus the impedance image is obtained.

Surface plasmons are collective oscillations of electrons near the metal film (electrode) sur-face, which can be excited optically under a proper optical configuration, leading to SPR (21,22). The most popular optical arrangement is the Kretschmann configuration (23), which useseither an optical prism (21–26) or a high numerical aperture objective (19, 27–39). As P-EIMis based on SPR microscopy (SPRM), the spatial resolution of P-EIM is the same as that ofSPRM. Prism-based SPRM has a wide-view window (from millimeters to centimeters), but itsspatial resolution is limited to a few microns to tens of microns, corresponding to a one- to ten-fold magnification (40). All of the commercial SPR instruments to date are based on the prismsetup, which either divides the sensing surface (metal film) into several parallel fluidic channelsfor binding kinetics measurement (21, 26) or prints a microarray of biomolecules on the sensingsurface for high-throughput studies (22, 24, 25). Objective-based SPRM provides higher spa-tial resolution (several microns along the direction of surface plasmon propagation and down to∼0.2 microns in the perpendicular direction) although its view window is decreased to a few hun-dred microns (27). The high spatial resolution, together with the improved signal-to-noise ratio,allows objective-based SPRM to image micro- to nanosized samples, including single cells (19, 28–30), bacteria (31, 32), virus particles (33), and various inorganic (34–38) and polymer (39) nanopar-ticles. For this reason, most P-EIM analyses to date were performed with the objective-basedsetup.

Figure 1a shows the schematic illustration of a P-EIM setup, which consists of an SPR mi-croscope, an electrochemistry module (including a cell and potential modulation electronics), anda signal synchronization and processing unit. SPRM is used to capture optical images duringelectrochemical modulation. The signal synchronization and processing unit helps convert thetime and potential-dependent SPR images into an electrochemical impedance image. For SPRMimaging, a collimated light beam (with a single wavelength between 630 and 700 nm) is focused onthe back focal plane of an oil-immersed objective (with a numerical aperture of 1.45 or above) toachieve parallel illumination onto a glass coated with 50-nm-thick gold film at a certain incidentangle. This incident angle can be adjusted by shifting the focal point on the back focal plane. Byscanning the incident angle (typically 60–75◦), the reflectivity can be obtained as a function ofincident angle, leading to an SPR intensity profile. This reveals a sharp dip known as the SPRangle (θSPR; Figure 1b) (21). At the SPR angle, a large fraction of the incident light is absorbedby surface plasmons and dissipated into heat, which causes the dip in the SPR profile. The SPRangle is determined by many factors, including the wavelength of incident light, refractive indicesof the prism (objective) and electrolyte, and the dielectric constant of metal film.

By measuring the change in the SPR angle, SPRM allows real-time and label-free detection ofmolecular interactions and electrochemical reactions on or near the gold film. The applicationsof traditional SPRM have been described in many reviews (21, 22, 26, 41), but here we focus onP-EIM. P-EIM is based on the dependence of θSPR on the dielectric constant of metal film, whichis a function of the electron density in the gold film. In P-EIM, the gold film serves not only asthe SPR sensing surface but also as a working electrode on which electrochemical processes takeplace. When changing the potential of the gold film, the surface charge density changes, whichaffects the electron density of the thin gold film and is detected from the associated change in theSPR angle, θSPR.

To examine the influence of potential on θSPR, SPR profiles at different potentials are plottedin Figure 1c, which shows that increasing potential leads to a positive shift in θSPR. This potential-dependent θSPR is described by the following equations. Based on a three-layer model, including

www.annualreviews.org • Plasmonic Imaging of Electrochemical Impedance 185

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a

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Figure 1(a) Schematic illustration of a P-EIM setup, consisting of an SPR microscope, an electrochemistry module, and a signalsynchronization/conditioning unit. Panel modified with permission from Reference 19. Copyright 2011, Nature Publishing Group.(b) A representative SPR reflectivity versus the incident angle profile showing a dip at the SPR angle. (c) The SPR reflectivity profile ofa gold film at different potentials. (d ) Electrochemical current (blue), SPR intensity (red ), and total transferred charge ( gold ) of the baregold surface during a potential modulation with a frequency of 20 Hz (black). Abbreviations: CCD, charge-coupled device; NA,numerical aperture; P-EIM, plasmonics-based electrochemical impedance microscopy; RE, reference electrode; SPR, surface plasmonresonance.

the prism (objective), metal film, and electrolyte, θSPR is given by (18, 42)

sin(θSPR) =√

ε1εm

(ε1 + εm) ε2, 1.

where ε1, ε2, and εm are the dielectric constants of the electrolyte, prism (objective), and metalfilm, respectively. According to the Drude model of metal, εm is a function of free electron density,ne, in the gold film (18):

εm( f0) = 1 − nee2

ε0me4π2 f02 , 2.

where e, ne, and me are the electron charge, density, and mass, respectively; f0 is the frequency ofincident light; and ε0 is the vacuum permittivity (8.85 × 10−12 F/m). When a positive potential isapplied on the gold film, electrons flow out of the gold film, resulting in a decrease in the electrondensity. This will increase εm according to Equation 2 and thus θSPR according to Equation 1 (43).

On the basis of the above analysis, a key task in P-EIM is to determine the potential-dependentθSPR. A convenient way to determine θSPR shift with potential is to measure reflected light (referred


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to as SPR intensity, ISPR) at an incident angle near θSPR. A shift in θSPR translates the SPR profileand thus the ISPR. For example, if the incident angle is fixed at 70.5◦ (dashed line in Figure 1c), anincrease in the metal film potential will lead to an increase in ISPR. This fixed-angle measurementof θSPR requires calibration, but it allows for the fast detection of θSPR shift.

In a typical P-EIM experiment, the potential is modulated by applying a harmonic (sine)waveform to the electrode, and corresponding ISPR is recorded versus potential (or time). Figure 1dshows such an example with a modulation frequency of 20 Hz. For a double-layer chargingprocess, the electrode–electrolyte interface is modeled with a capacitor, so a ∼90-degree phaseshift between potential and current is expected (top panel in Figure 1d ). However, SPR intensityfollows the potential without phase delay (middle panel in Figure 1d ). This is because ISPR (θSPR)measures charge density rather than current density, and the phase between charge and currentin a capacitor is 90◦. Figure 1d (bottom panel) compares ISPR with charge obtained by integratingcurrent over time, which shows good agreement between theory and experiments.

P-EIM provides quantitative local impedance and electrochemical current. In the absence ofFaradaic current, P-EIM measures the double layer charging current, which is given by

i = α · �θSPR · 2π f · A, 3.

where α, �θSPR, f, and A are conversion factor, fluctuation amplitude of the SPR angle, modulationfrequency, and electrode area, respectively. Note that α relates the optically measured θSPR to thedouble-layer charging current, which can be regarded as the sensitivity of P-EIM. Using the Drudefree electron gas model, α is calculated to be 28 C · m−2 · deg−1 for gold film on BK7 glass and withaqueous electrolyte (18). Conversion factor α was also calibrated experimentally, and the valuewas found to be 17 C · m−2 · deg−1, or in a more microscopic manner, 0.1 e− · nm−2 · mdeg−1 (20).Given the simplicity of the Drude model, the agreement between the theoretical and experimentalvalues can be considered reasonable.

So far, we have only considered double-layer charging current (non-Faradaic) in P-EIM, andits basic principle relies on the sensitive dependence of θSPR on the dielectric constant of the metalfilm, which is related to surface charge density. In practice, P-EIM measures the response of θSPR

to a potential modulation applied to the electrode (metal film), which includes both non-Faradaicand Faradaic currents (44, 45). The contribution of Faradaic process to P-EIM arises from acompletely different principle from that for the non-Faradaic charging process. In a Faradaicprocess, electrochemical reactions take place on or near the electrode, which are accompanied bythe conversion between oxidized and reduced species. This results in the change in the refractiveindex of the electrolyte near the electrode surface because oxidized and reduced species usuallyhave different refractive indices (34, 35, 44–46). The change in the electrolyte refractive index givesrise to the image contrast of a Faradaic process in P-EIM. Traditional EIS also measures bothnon-Faradic and Faradic processes. However, a quantitative comparison of P-EIM and traditionalEIS must consider the difference in the conversion factors of non-Faradic and Faradic processes(45). Although a slight change in the electrolyte refractive index occurred during non-Faradaiccharging due to the spatial reorganization of the double layer, it influenced a very thin layer of afew nanometers (an electrical double layer). Faradaic reactions, however, affected the electrolyterefractive index in a much thicker diffusion layer on the order of microns. Moreover, a differenttimescale was also utilized to distinguish non-Faradaic charging and Faradaic reactions, as thecharging process usually completed in a much shorter timescale.

We point out that P-EIM is different than simply combining SPR with EIS measurement, whereSPR is measured optically and impedance is determined electrically with the conventional EISmethod (47–49). In P-EIM, the electrochemical impedance or EIS is determined optically, whichprovides imaging capability. The current from the entire electrode can be recorded simultaneously


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in a P-EIM experiment, which is used as a reference to validate the local impedance obtained fromP-EIM.

2.2. Circuit Model

EIS measures the current response of an electrochemical interface to a periodic potential per-turbation at various frequencies. We have shown that P-EIM provides quantitative measurementof local impedance at a single frequency, and we now turn to spectral analysis, which performsP-EIM as a function of frequency. A simple example is the spectral analysis of a bare gold film in a0.5-M NaF solution. Figure 2a,b show the amplitude and phase spectra obtained with P-EIM andwith the traditional EIS, respectively. The impedance spectra may be analyzed with a simplifiedRandles circuit model (Figure 2c), which consists of a resistor Rs (solution resistance) in serieswith a capacitor Cdl (electric double layer) and another resistor Rp (Faradaic current) in parallel.

The traditional EIS is performed with a potentiostat that measures the current response to amodulating potential (Figure 2a). The current response based on the Randles model is expressedin terms of Rs, Rp, and Cdl:

I = V 0

Rs + (R−1p + jωCdl)

−1 , 4.

where ω = 2π f. At low frequencies, the circuit is dominated by Rs and Rp connected in series,which leads to a current amplitude of I = V0/(Rs + Rp) and a phase shift of 0. At high frequencies(e.g., >1,000 Hz under the experimental condition defined above), the circuit is dominated by asingle resistor, Rs, and the corresponding current amplitude and phase are V0/Rs and 0, respectively.In the middle frequency range, both the current amplitude and phase are strongly dependent onfrequency.

As previously discussed, P-EIM measures the response of SPR image intensity (or θSPR) topotential modulation, and the SPR response includes both double-layer charging and Faradaicprocesses. If we consider only the former, then P-EIM measures the electrode surface chargedensity, which is proportional to the potential drop across the double layer (Vdl = Q/Cdl, with Cdl

as a constant) (20) and given by

Vdl = V 0 − Rs

Rs + (R−1p + jωCdl)

−1 V 0. 5.

At low frequencies, most of the applied voltage falls across the electrode–electrolyte interface,leading to constant SPR amplitude and near-zero phase shift (Figure 2b). With the increasingfrequency, the fraction of Vdl gradually decreases toward zero due to the decreasing impedance ofthe double-layer capacitor. At high frequencies, there should be no change in the surface chargedensity and thus no P-EIM image contrast. However, in practice, the Faradaic process describedby Rp will change the concentrations of the reactants (e.g., dissolved oxygen) and products, stillleading to P-EIM image contrast because of the associated refractive index change. In the middlefrequency range, P-EIM measures both double-layer charging and Faradaic processes.

The impedance spectra obtained with both the traditional EIS and P-EIM can be quanti-tatively analyzed in terms of the Randles model, represented by Equations 4 and 5, respectively(Figure 2a,b). The corresponding fitting parameters from the traditional EIS and P-EIM are con-sistent with each other (20). For example, the double-layer capacitance of gold film was estimatedto be 9.68 ± 0.10 μF and 9.56 ± 0.13 μF from P-EIM and traditional EIS analysis, respectively.This level of agreement provides strong support for the basic principle of P-EIM. We note that thedouble-layer charging (non-Faradaic) process is expected to dominate the measured impedance


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Figure 2(Left column) Frequency-dependent amplitude and (right column) phase shift of the (a) current and (b) AC SPR signal of a bare gold filmin a 0.5-M NaF solution. (c) The Randles equivalent circuit used to model both the traditional and P-EIM impedance spectra. The redlines in panels a and b fit curves of the experimental data (black squares) with the Randles equivalent circuit model. Panels a and bmodified with permission from Reference 20. Copyright 2012, American Chemical Society. Abbreviations: P-EIM, plasmonics-basedelectrochemical impedance microscopy; SPR, surface plasmon resonance.


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in the absence of electrochemically active species. In this case, a Warburg impedance element,which is often used in EIS models to describe the resistance to mass transfer, does not play arole. However, adding electrochemically active species or redox probes, such as Fe(CN)6

3−/4−

and Ru(NH3)62+/3+, to the electrolyte will enhance the contribution of this Warburg impedance

component to the impedance, which is described by Rp in the Randles model. These redox probesare often used in traditional EIS-based biosensors to enhance the signal (47, 49). In the absenceof redox probes, Rp was found to be large compared to the impedance of the capacitor, given by1/ωCdl (20).

2.3. From Spectroscopy to Microscopy

The major benefit of P-EIM is its imaging capability, which is difficult to achieve in traditionalEIS. To illustrate the imaging capability (18), local impedance of an array of 1-dodecanethiol(DDT) spots printed on a gold film was imaged with P-EIM. Figure 3a shows the traditionalSPR image of a microarray, consisting of six DDT spots on the gold film. The SPR image contrastis poor because it depends on the refractive index of the DDT monolayer, which is relatively smallfor the small molecules. When applying a 50-Hz sine wave to the surface, the gold area exhibits alarge SPR response, whereas DDT regions show a much smaller SPR response (Figure 3c). Thisis because the area covered by the DDT monolayer has a much smaller interfacial capacitanceand thus a smaller change in the double layer charge density measured by SPR (Figure 3b). Infact, by analyzing the frequency-dependent SPR responses of the bare gold and DDT regions, thespecific capacitances were found to be 50.7 and 12.7 μF/cm2 for the two regions, respectively. Thisexperiment shows that P-EIM can provide better image contrast than SPR for small molecules.The reason is that the former measures interfacial impedance, whereas the latter is sensitive torefractive index change associated with molecular adsorption, which scales with the mass of themolecules. This difference suggests that P-EIM is more suitable for detecting small moleculesthan traditional SPR.

3. INSTRUMENTATION AND DATA ANALYSIS

P-EIM consists of three modules: an optical module for SPRM imaging, an electrochemicalmodule for potential modulation, and a data acquisition and processing unit for synchronizing theelectrochemical measurement and optical imaging. For SPRM, both prism- and objective-basedsetups have been used. The former setup offers a wide view mostly suitable for microarrays. Incontrast, the latter option provides high spatial resolution for imaging micro- to nanoscale objects,such as single cells, nanoparticles, nanowires, and 2D nanomaterials. Details of the prism- (18, 46,50, 51) and objective-based (27, 33, 36) SPRM setups are described elsewhere, and here we focuson the electrochemical module and signal synchronization and processing module.

The electrochemical module includes a liquid cell with reference and counter electrodes, apotentiostat to control the potential of the metal film (SPRM sensor chip), and a function gen-erator to create a sinusoidal potential applied to the electrode via the potentiostat. The signalsynchronization and processing module includes an analog/digital acquisition card to record thepotential, current (from the potentiostat), and a 5V-transistor–transistor logic signal indicatingthe time tags of each frame from the camera. Compact electrochemical workstations lacking ana-log input and output interfaces should be avoided. Once the synchronization signal and a seriesof time-lapsed SPRM images are collected, one can then extract the SPRM amplitude and phaseimages. Considering that a camera has a frame rate of k, and each frame has m × n pixels, thecollected dataset would be m × n × k × t over a time period of t. Fourier transform is applied to


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ΔθSP

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Time (s)

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Figure 3(a) SPR image and (b) P-EIM amplitude image of a microarray consisting of six DDT spots on a gold chip with a potential modulationof 50 Hz. Panels a and b modified with permission from Reference 18. Copyright 2008, American Chemical Society. (c) SPR intensitiesfrom the bare gold (red ) and DDT-modified (blue) areas showing the potential-dependent SPR angle. Abbreviations: DDT,1-dodecanethiol; P-EIM, plasmonics-based electrochemical impedance microscopy; SPR, surface plasmon resonance.

each pixel (pixel intensity versus time) to generate a spectrum in the frequency domain for thecorresponding pixel. The spectrum shows a peak at the frequency of potential modulation, and theamplitude and phase of the peak are then extracted for the pixel. This procedure is repeated for allpixels, which results in two images: the amplitude and phase images of P-EIM at a given frequencyof potential modulation. If we scan the modulation frequency, then P-EIM (amplitude and phaseimages) at different frequencies can also be obtained. We note that the Fourier transform spectrumalso contains zero frequency (DC) signals, corresponding to the time average of the SPR signal,which is the traditional SPRM image. Therefore, P-EIM provides not only impedance (amplitudeand phase) images but also the traditional SPRM image of the same sample.

The signal synchronization and processing module also records the electrode potential andcurrent over time during the entire experiment. By performing Fourier transform of the potential(versus time), the phase of the applied potential can be extracted, which is the reference phase. Bysubtracting this reference phase from the SPR phase, a P-EIM phase shift image is then obtained.The spatial resolution of P-EIM is the same as that of SPRM, but the temporal resolution ofP-EIM is determined by the modulation frequency and the maximal frame rate of the camera. Fora fast camera with a frame rate of 50,000 frames per second (FPS), applying 20 kHz of modulationresults in a temporal resolution of 0.2 ms. In practice, the temporal resolution is often compromisedby the signal-to-noise ratio, time constant of the electrochemical system, and memory size of thecamera.


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4. MAPPING HETEROGENEOUS INTERFACES

4.1. Microarray

One of the most popular applications of SPR is to detect and quantify the binding kinetics ofmolecules in solution with receptors immobilized on the SPR sensor (gold film). As discussedabove, the sensitivity of traditional SPR scales with the mass of the molecules. Low molecularmass molecules are thus difficult to study with SPR. P-EIM measures the interfacial capacitance,which does not scale with the mass of the molecules. For this reason, it is believed that P-EIMprovides superior sensitivity compared with traditional SPR for detecting small molecules.

To illustrate the capability of P-EIM for detecting small molecules, surfaces with differ-ent charge polarity were studied (52). To control the surface charge, ethylenediamine andethanolamine were chemically linked to a dextran-coated gold film. The surface charge was reg-ulated by adjusting the solution pH because ethanolamine, dextran, and ethylenediamine areterminated with hydroxyl (−OH), carboxyl (−COOH), and amine (−NH2) groups, respectively,and these terminal groups can be protonated/deprotonated at different solution pH values. Thecorresponding changes in the impedance of the surface were analyzed by both an electric circuit(Randles) model that described the potential distribution and a mechanical model that describedthe conformational response of three-dimensional dextran hydrogel. The mechanical model wasintroduced because the dextran hydrogel was mechanically flexible, and its response to the appliedpotential depended on the elastic properties of the hydrogel described with a spring constant.The mechanical flexibility of the dextran hydrogel led to the dependence on the charge of theterminal groups. For example, a repulsive electrical force between the charged terminal groupswill cause the dextran to expand, making the dextran more rigid and decreasing the amplitude ofSPR response to the applied potential modulation.

Charge-sensitive detection with P-EIM was used to investigate the posttranslational modifi-cations (38, 53), small-molecule binding events (54), and the conformational changes of DNAmolecules (55). For example, P-EIM was used to evaluate the binding kinetics of imatinib, asmall-molecule drug approved by the US Food and Drug Administration, with its target pro-tein (BCR-Abl), a protein kinase. This demonstrated significantly improved sensitivity comparedwith the traditional SPR measurements (54). In this work, protein molecules were deposited onthe polyethylene glycol–modified gold film surface by means of a noncontact piezoelectric inkjetprinter to generate a microarray of carboxyl- and hydroxyl-terminated protein spots. After col-lecting the P-EIM response upon the injection of imatinib onto a sensor chip spotted with Ablor other control proteins, SPR and impedance signals of each spot were extracted from the rawimages to obtain the kinetic constants (with the association and dissociation rates as constants)and binding affinity of small molecules with each spot in the protein microarray.

The charge-sensitive P-EIM method measured the binding kinetics in a protein microarrayformat for high-throughput detection of molecules, particularly small molecules. Additionally,the method was less sensitive to nonspecific adsorption, which was one of the most challengingissues in label-free molecular binding assays (56, 57). This feature was observed in protein–proteininteractions (20) and protein–small molecule interactions (54). In both cases, the traditional SPRsignal was easily affected by nonspecific binding and bulk effect, but P-EIM responses reflectedthe intrinsic kinetics of specific molecular interactions. One reason was that P-EIM was mostlysensitive to the capacitance of the electric double layer in the interface. A molecule nonspecificallyattached to the surface did not disturb the electric double layer as much as specific binding. Arecent comparative study also demonstrated the different sensing mechanism between SPR andP-EIM, in which SPR and P-EIM exhibited quite different responses to specific and nonspecificinteractions, respectively (58). This work showed that P-EIM was more sensitive to nonspecific


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adsorption. Clearly, further investigations are needed to fully identify factors that affect bothP-EIM and traditional SPR studies.

4.2. Two-Dimensional Nanomaterials

Using the objective-based setup, P-EIM is able to map the distribution of local electric fieldor capacitance with a submicron spatial resolution. A recent example of such research investi-gated the local electric field near a surface (metal film) generated by a micropipette (59). In thisstudy, the traditional SPR (DC signal) revealed the location and size of the micropipette tip, andP-EIM (AC signal) provided the local electric field and surface charge distribution. Both thelocal electric field and its distribution depended on the distance between the micropipette tipand gold electrode and the size of micropipette, which agreed with the numerical simulation re-sults by COMSOL software. This work represents an attempt to integrate P-EIM and scanningprobe-based electrochemical imaging techniques into a single system.

Graphene is a 2D material with many potential applications, including energy storage as asupercapacitor. In the energy storage application, the capacitance of graphene is critically impor-tant. Because of the low density of states near the Fermi level of graphene, quantum capacitancedominates the graphene capacitance. Graphene quantum capacitance has been studied with the tra-ditional electric method, which measured the charge or current responses to a voltage change (60).Such an approach lacks the spatial resolution needed to resolve local quantum capacitance of thegraphene (typically heterogeneous). Recently, P-EIM was used to map the local quantum capac-itance of graphene supported on a substrate (gold film) (37). The total interfacial capacitance ofgraphene in the experiment consisted of two contributions, electrical double-layer capacitance(CEDL) and quantum capacitance (CG) (Figure 4a). The two contributions were modeled as twocapacitors in a series, described by 1/CG + 1/CEDL = 1/C, where C is the interfacial capacitancemeasured by P-EIM. CEDL is large compared to the CG, so C is mainly due to the CG contribution.A more precise extraction of CG is also possible because CEDL can be obtained from the interfacial

b a

RS

CEDL

RS

RECE

WE

Au film

Electron puddles

Impurity polarity

Hole puddles

Electronpuddles

Graphene

CEDLCG

1

0

–1

Figure 4(a) Schematic illustration of the interfacial capacitance measurement of graphene with P-EIM. Illustrated arethe equivalent circuits of the (left) graphene area and (right) bare gold region. (b) P-EIM imaging of theelectron (blue) and hole (red ) puddles of graphene associated with the charged impurities at the average Diracpoint of graphene (−0.08 V). Figure adapted with permission from Reference 37. Copyright 2015, JohnWiley & Sons. Abbreviations: CE, counter electrode; P-EIM, plasmonics-based electrochemical impedancemicroscopy; RE, reference electrode; SPR, surface plasmon resonance; WE, working electrode.


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capacitance of the bare gold surface area. By knowing the CEDL, the graphene quantum capacitancecan be estimated according to the aforementioned equation.

The role of charged impurities was further investigated by controlling the surface charge withvarious molecules terminated with different charged groups, allowing the visualization of defects ingraphene, such as electron and hole puddles (37) (Figure 4b). The authors found that the graphenecapacitance was smaller than that of a bare gold surface but was larger than the theoretical valueof ideal quantum capacitance. This study demonstrates the important roles of charged impuritieson the heterogeneous distribution of quantum capacitance.

4.3. Imaging Single Cells and Intracellular Events

Traditional EIS has been widely used in cell-based assays to study various cellular events, includingcell-growth, wound-healing, and viability evaluations (4–6). Most of the assays either analyzethe frequency characteristics in the EIS spectrum or monitor the time evolution of impedanceduring particular cellular events. Traditional EIS lacks spatial resolution, so it measures only theaverage behavior of thousands of cells on the electrode (61–65). P-EIM allows for the imagingof single cells, which requires consideration of impedance of single cells. A single living cell isoften treated as a circuit involving capacitors (cell membrane) and resistors (cytoplasm) (66). Inaddition, many microscopic electrochemical reactions take place inside cells, including electrontransfer in respiration and photosynthesis as well as neurotransmitter release, which result in manyexciting findings in the development of single-cell electrochemistry (67). A capability to visualizethe subcellular distribution of electrochemical impedance in a single living cell provides detailedinformation to understand the microscopic mechanisms of the corresponding cellular processes.

P-EIM is used to study dynamic cellular processes, including apoptosis and electroporationof individual living cells (19). P-EIM reveals different stages of apoptosis in real time, includingcellular shrinking and condensation, followed by the fragmentation of nuclear material, formationof blebs, and eventual disintegration of the cells. It also allows study of the cell attachment processfrom the surface. For example, P-EIM images showed that the detachment started from thecell center immediately after the cell apoptosis began and then expanded toward the edge. Thisdetailed process could not be observed with conventional optical microscopy. Another importantcellular process is electroporation, which occurs by applying a voltage pulse to a cell on the surface.P-EIM revealed a large change in the image contrast, arising from the increase in the permeability(conductivity) of the cell plasma membrane (Figure 5). The simultaneously recorded brightfieldoptical image showed only subtle changes during and after the electroporation process. P-EIM

9 s 15 s 17 s 18 s 20 s 25 s 35 s 55 s

SPR

P-EIM

0

2.5 × 104RU

pS/pixel

2

800

Figure 5The dynamic electroporation process of a single cell monitored by time-lapsed snapshots of (top) SPR images and (bottom) P-EIMimages. Scale bar is 20 µm. Figure modified with permission from Reference 19. Copyright 2011, Nature Publishing Group.Abbreviations: P-EIM, plasmonics-based electrochemical impedance microscopy; RU, response unit; SPR, surface plasmon resonance.


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–0.2 s

+1.0 s

+2.2 s

+3.4 s

+0.2 s

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+0.6 s

+1.8 s

+3.0 s

pS/pixel–1

1

0

Figure 6Differential plasmonics-based electrochemical impedance microscopy images of individual cells afterhistamine stimulation. Adapted with permission from Reference 67. Copyright 2015, John Wiley & Sons.

provides a local conductivity map showing that electroporation started with a rapid and largeincrease in the center of the cell, followed by a slower recovery process lasting for tens of seconds.

In a recent work, P-EIM was used to study the endogenous G protein–coupled receptor(GPCR) stimulations of living HeLa cells with subcellular and millisecond spatial–temporal res-olutions (68). The study employed histamine to trigger GPCR activations of HeLa cells. P-EIMimages revealed the heterogeneous cellular responses to histamine stimulation in the initial pe-riod. The calcium flux after GPCR activation was clearly shown in the P-EIM images. Based onsuch observations, the heterogeneity of local distributions and transient activities of ion channelsduring agonist-activated calcium flux in single HeLa cells were investigated (Figure 6). Interest-ingly, the results showed that a subpopulation of cells was more active toward agonist stimulation,underscoring the value of single-cell imaging.

The optical setup of P-EIM is compatible with traditional SPR and optical imaging, allowingone to combine different imaging capabilities into a single system. SPR has long been recognizedas a label-free detection technique for studying molecular interactions of biologically relevantsamples, such as protein, DNA, and living cells (41, 69, 70). Operating P-EIM in the DC mode(traditional SPR) allows mapping of the local binding kinetics of molecules with single cells.For example, when lectin proteins flowed over the surface of a single living adherent cell, lectinmolecules bound to the glycoproteins in the cell membrane, which was observed in the SPRimage contrast. The association and dissociation processes were monitored in real time, leadingto detailed mapping of binding kinetics between ligand proteins and membrane proteins on singleliving cells (29). This approach does not require isolation, purification, and immobilization ofmembrane proteins used in traditional binding kinetics assays. These procedures are not only


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time consuming, but they also possibly change the native functions and conformations of themembrane proteins.

The binding kinetics of a monoclonal antibody drug, Herceptin, with its receptor, human epi-dermal growth factor receptor 2 (HER2) was determined at a single-cell level (71, 72). The resultsrevealed significantly weakened binding kinetics in Herceptin-resistant cells compared with thosein drug-sensitive cells (71). These findings help clarify the molecular mechanism for Herceptinresistance, which is currently a challenge for improving the clinical performance of the drug. Thisapproach also demonstrates P-EIM’s capability to quantify the expression level of membraneproteins on single cells [e.g., HER2 (71) and epidermal growth factor receptor (73)]. In additionto mammalian cells, the binding kinetics of molecules with an individual bacterium was demon-strated, from which the heterogeneity in a microbial population was analyzed (32). Furthermore,micromotion of a single bacterium was quantified and used to determine the metabolic activityof bacteria. The study observed significantly slower micromotion when exposing the bacteria toantibiotics (31).

5. REMARKS AND FUTURE PERSPECTIVES

Although optical microscopy has been a routine tool to map the optical and spectroscopic prop-erty in heterogeneous and microscopic interfaces, mapping the distribution of the electrochemicalproperty in such interfaces remains challenging. Now SPRM provides a bridge to connect opticalproperty and electrochemical behaviors with a quantitative optical-to-electrochemical conversionmodel. By analyzing fluctuations in the SPRM images accompanying electrochemical modulations,P-EIM achieves the wide-field imaging of electrochemical impedance at a submicron spatial reso-lution and a submillisecond temporal resolution. It has been utilized to resolve the binding kineticsof small molecules with protein microarray, and it exhibits superior sensitivity for small molecules,as it is sensitive to the surface charge instead of mass. Its high spatial resolution further allowsthe imaging of quantum capacitance in a layer of graphene and the study of multiple biologicalprocesses of single living cells.

Despite the capabilities of P-EIM, many questions remain to be addressed. Traditional EISinterpreted data in terms of the electrical circuit, which consists of capacitors and resistors. Thisconcept is hard to apply to heterogeneous chemical and biological systems. In addition, the meth-ods of expressing the P-EIM image contrast in terms of molecular-scale processes require furtherresearch. This issue is particularly important for imaging complex biological samples, such ascells. Combining P-EIM with fluorescence microscopy and other imaging techniques will help todevelop a molecular-scale understanding of P-EIM imaging of cells. This is because fluorescencemicroscopy has proven to be a powerful tool to study cellular processes with high chemical speci-ficity. The P-EIM setup in the authors’ labs is built on an inverted optical microscope, which iscompatible with epifluorescence microscopy and total internal reflection fluorescence microscopyto achieve simultaneous SPR, EIS, and fluorescence imaging of the same cell (29, 32).

The temporal resolution of P-EIM is largely limited by the frame rate of the camera. Nyquistsampling theory requires that the camera frame rate be at least twice as fast as the modulationfrequency. Traditional EIS studies of cells are often operated at tens of kilohertz (63), because atsuch frequencies, the impedance response of a monolayer of adherent cells is mostly sensitive toenvironmental or cellular events. Cameras with a frame rate up to one million FPS are commer-cially available and may be applied to study cellular processes at high frequencies. High-frequencyP-EIM requires optimization of the light source to achieve a sufficient number of photons perpixel per frame to minimize shot noise. To enable high-frequency P-EIM, it may be useful to


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develop an efficient imaging algorithm to process the large amount of images captured with thefast camera.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We are grateful for financial support from the National Natural Science Foundation of China(grants 21522503, 21327008, 21327902, 21527807) and the Natural Science Foundation of JiangsuProvince (grants BK20150013, BK20140592).

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New From Annual Reviews:Annual Review of Cancer Biologycancerbio.annualreviews.org • Volume 1 • March 2017

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http://www.annualreviews.org/r/nevs

AC10-TOC ARI 8 April 2017 7:25

Annual Review ofAnalytical Chemistry

Volume 10, 2017Contents

Chemical and Biological Dynamics Using Droplet-Based MicrofluidicsOliver J. Dressler, Xavier Casadevall i Solvas, and Andrew J. deMello � � � � � � � � � � � � � � � � � � � 1

Sizing Up Protein–Ligand Complexes: The Rise of Structural MassSpectrometry Approaches in the Pharmaceutical SciencesJoseph D. Eschweiler, Richard Kerr, Jessica Rabuck-Gibbons,

and Brandon T. Ruotolo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �25

Applications of the New Family of Coherent MultidimensionalSpectroscopies for Analytical ChemistryJohn C. Wright � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Coupling Front-End Separations, Ion Mobility Spectrometry, andMass Spectrometry for Enhanced Multidimensional Biological andEnvironmental AnalysesXueyun Zheng, Roza Wojcik, Xing Zhang, Yehia M. Ibrahim,

Kristin E. Burnum-Johnson, Daniel J. Orton, Matthew E. Monroe,Ronald J. Moore, Richard D. Smith, and Erin S. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Multianalyte Physiological Microanalytical DevicesAnna Nix Davis, Adam R. Travis, Dusty R. Miller, and David E. Cliffel � � � � � � � � � � � � � � �93

Nanosensor Technology Applied to Living Plant SystemsSeon-Yeong Kwak, Min Hao Wong, Tedrick Thomas Salim Lew, Gili Bisker,

Michael A. Lee, Amir Kaplan, Juyao Dong, Albert Tianxiang Liu,Volodymyr B. Koman, Rosalie Sinclair, Catherine Hamann,and Michael S. Strano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Coded Apertures in Mass SpectrometryJason J. Amsden, Michael E. Gehm, Zachary E. Russell, Evan X. Chen,

Shane T. Di Dona, Scott D. Wolter, Ryan M. Danell, Gottfried Kibelka,Charles B. Parker, Brian R. Stoner, David J. Brady, and Jeffrey T. Glass � � � � � � � � � � 141

Magnetic Resonance Spectroscopy as a Tool for AssessingMacromolecular Structure and Function in Living CellsConggangg Li, Jiajing Zhao, Kai Cheng, Yuwei Ge, Qiong Wu, Yansheng Ye,

Guohua Xu, Zeting Zhang, Wenwen Zheng, Xu Zhang, Xin Zhou,Gary Pielak, and Maili Liu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Plasmonic Imaging of Electrochemical ImpedanceLiang Yuan, Nongjian Tao, and Wei Wang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 183

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AC10-TOC ARI 8 April 2017 7:25

Tailored Surfaces/Assemblies for Molecular Plasmonics and PlasmonicMolecular ElectronicsJean-Christophe Lacroix, Pascal Martin, and Pierre-Camille Lacaze � � � � � � � � � � � � � � � � � � � 201

Light-Addressable Potentiometric Sensors for Quantitative SpatialImaging of Chemical SpeciesTatsuo Yoshinobu, Ko-ichiro Miyamoto, Carl Frederik Werner,

Arshak Poghossian, Torsten Wagner, and Michael J. Schoning � � � � � � � � � � � � � � � � � � � � � � 225

Analyzing the Heterogeneous Hierarchy of Cultural HeritageMaterials: Analytical ImagingKaren Trentelman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Raman Imaging in Cell Membranes, Lipid-Rich Organelles,and Lipid BilayersAleem Syed and Emily A. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

Beyond Antibodies as Binding Partners: The Role of AntibodyMimetics in BioanalysisXiaowen Yu, Yu-Ping Yang, Emre Dikici, Sapna K. Deo, and Sylvia Daunert � � � � � � � 293

Identification and Quantitation of Circulating Tumor CellsSiddarth Rawal, Yu-Ping Yang, Richard Cote, and Ashutosh Agarwal � � � � � � � � � � � � � � � � � 321

Single-Molecule Arrays for Protein and Nucleic Acid AnalysisLimor Cohen and David R. Walt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 345

The Solution Assembly of Biological Molecules Using Ion MobilityMethods: From Amino Acids to Amyloid β-ProteinChristian Bleiholder and Michael T. Bowers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 365

Applications of Surface Second Harmonic Generationin Biological SensingRenee J. Tran, Krystal L. Sly, and John C. Conboy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

Bioanalytical Measurements Enabled by Surface-Enhanced RamanScattering (SERS) ProbesLauren E. Jamieson, Steven M. Asiala, Kirsten Gracie, Karen Faulds,

and Duncan Graham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 415

Single-Cell Transcriptional AnalysisAngela R. Wu, Jianbin Wang, Aaron M. Streets, and Yanyi Huang � � � � � � � � � � � � � � � � � � � 439

Errata

An online log of corrections to Annual Review of Analytical Chemistry articles may befound at http://www.annualreviews.org/errata/anchem

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Documents

Plasmonic Imaging of Electrochemical Impedance