Enhanced Electrochemical Nanoring Electrode for Analysis of Cytosolin Single CellsLihong Zhuang,† Huanzhen Zuo,‡ Zengqiang Wu,‡ Yu Wang,‡ Danjun Fang,*,† and Dechen Jiang*,‡

†School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu 210000, China‡Key State Laboratory of Analytical Chemistry for Life Science and School of Chemistry and Chemical Engineering, NanjingUniversity, Nanjing, Jiangsu 210093, China

*S Supporting Information

ABSTRACT: A microelectrode array has been applied for single cell analysis with relatively highthroughput; however, the cells were typically cultured on the microelectrodes under cell-sizemicrowell traps leading to the difficulty in the functionalization of an electrode surface for higherdetection sensitivity. Here, nanoring electrodes embedded under the microwell traps werefabricated to achieve the isolation of the electrode surface and the cell support, and thus, theelectrode surface can be modified to obtain enhanced electrochemical sensitivity for single cellanalysis. Moreover, the nanometer-sized electrode permitted a faster diffusion of analyte to thesurface for additional improvement in the sensitivity, which was evidenced by the electrochemicalcharacterization and the simulation. To demonstrate the concept of the functionalized nanoringelectrode for single cell analysis, the electrode surface was deposited with prussian blue to detectintracellular hydrogen peroxide at a single cell. Hundreds of picoamperes were observed on ourfunctionalized nanoring electrode exhibiting the enhanced electrochemical sensitivity. The successin the achievement of a functionalized nanoring electrode will benefit the development of highthroughput single cell electrochemical analysis.

Microelectrodes are powerful tools for single cell analysisbecause of their small size. The well-developed micro-

electrochemical strategy included the location of the micro-electrode near an individual cell to detect the cellular exocytosisin real time.1−5 The advantages of the microelectrode in thesingle cell analysis were unlabeled, quantitative, and in vivomeasurement; high sensitivity; and submillisecond timeresolution.6−8 Recently, the dimension of the microelectrodewas scaled down to the nanometer size so that the electrodewas inserted into the cell to record the intracellular information,which offered the opportunity to apply the electrode forintracellular analysis.9,10 The diameter of nanoelectrodes was farsmaller than the cell size resulting in the minor damage to thecells during the electrode insertion. The challenge of thisstrategy was the low throughput owing to the manual positionof the electrode near the cell and the low electrochemicalsensitivity owing to small electrode area.To achieve microelectrochemical single cell analysis with

relatively high throughput, the array of microelectrodes wasfabricated using photolithography. The individual cells werelocated automatically and cultured on the microelectrodes withthe aid of cell-size microwell traps.11−13 The parallel recordingfrom individual cells using a multichannel electrochemicalstation enabled the fast single cell analysis of cellular exocytosis.The throughput was not as high as that in flow cytometry orthe microfluidics; however, the assay was favorable for theanalysis of adherent cells because no removal of the cells fromthe support surface was needed during the analysis. Since thecells were cultured on the electrode surface, the surface

functionalization was absent to avoid the cytotoxicity of theelectrode surface on the cells, and thus, the electrochemicalsensitivity was limited. The insufficient sensitivity induced thedifficulty to apply this platform for the detection of themolecules in single cells with low concentration. Therefore, forbetter electrochemical sensitivity, the surface functionalizationwas required, which needed the spatial division of the electrodesurface and the cell support.The nanoring electrode is a particular class of nanoelectrodes

that is commonly used in generator-collector experiments.14,15

Typically, the diameter of the ring was at micrometer scale, andthe width or height of ring was at nanometer level. Ascompared with the microelectrode, the electrode dimensioninduced faster diffusion of analyte to the electrode surface andenhanced current sensitivity.16,17 Meanwhile, the apparentelectrode area of the nanoring electrode was much largerthan that of the traditional nanometer disk electrode, so thatthe nanoring electrode offered a larger current than that of thenanoelectrode. Taking in account that the center region of theelectrode was vacant and could be used for the cell culture, thenanoring structure should be perfect to achieve the isolation ofthe electrode surface and the cell support.In our work, a nanoring electrode embedded under a cell-

sized microwell trap was fabricated using one-step wet etchingto realize the surface functionalization and enhanced electro-

Received: July 3, 2014Accepted: November 3, 2014

Technical Note

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chemical sensitivity for single cell analysis. A cell sizedmicrowell trap was fabricated on the glass slide coated with180 nm of an indium tin oxide (ITO) layer. An etching solutionwas introduced to remove the ITO layer at the bottom surfaceof microwell leading to the exposure of the glass surface for cellculture. The vertical section of ITO layer at the bottom edge ofthe microwell, as shown in Figure 1A, behaved as a nanoringelectrode that can be functionalized for high sensitivity andselectivity. Despite that the nanoring electrode could be appliedfor the study of cellular exocytosis, it was believed to be morefavorable for the detection of intracellular molecules at singlecells. After the cell cultured in the microwell was lysed torelease the cytosol, the structure of the microwell slowed downthe diffusion of analyte away from the microwell. As a result,the analyte was not diluted dramatically near the electrodesurface in the initial period and could be detected by thesurrounding nanoring electrode. For the proof-of-conceptdemonstration of our platform, the electrode was modifiedwith prussian blue to detect the intracellular hydrogen peroxideat single cell level. The enhanced electrochemical property anddetection sensitivity of the nanoring electrode for hydrogenperoxide were discussed. The success of this novel design willprovide a new strategy for single cell analysis with enhanced

electrochemical sensitivity and, eventually, benefit the field ofhigh throughput single cell electrochemical analysis.

■ EXPERIMENTAL SECTION

Chemicals. Raw264.7 cells were obtained from the Instituteof Biochemistry and Cell Biology, Shanghai Institute forBiological Sciences of Chinese Academy of Science (Shanghai,China). SU-8-25 photoresist and the developer were obtainedfrom Microchem Corp (Newton, MA). All other chemicalswere from Sigma−Aldrich, unless indicated otherwise. Buffersolutions were sterilized.

Cell Culture. Raw264.7 cells were seeded in DMEM/highglucose medium supplemented with 10% fetal bovine serum(FBS) and 1% antibiotics (penicillin/streptomycin). Cultureswere maintained at 37 °C under a humidified atmospherecontaining 5% CO2.

Fabrication of Nanoring Electrodes. The glass slidecoated with 180 nm of ITO layer (Huayu United Inc.Shenzhen, China) was covered with 30 μm of SU-8 photoresist.After the soft baking at 65 °C for 3 min and 95 °C for 7 min,the photoresist was illuminated with the lithography system(JB-VIII, Pmled Optoelectronics, China) through an iron oxide

Figure 1. (A) The fabrication process for nanoring electrode. (B) (a) Bright-field image of the microwell; (b) luminescence image of the microwellbefore the wet-etching; (c) the overlap of the images in a and b; (d) luminescence image of the microwell after the wet-etching. (C) (a) SEM imageof the nanoring electrode at a 75° tilt; (b) the amplified image of the edge at the nanoring electrode.

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mask. Then, the photoresist was baked at 65 °C for 1 min and95 °C for 3 min. Unpolymerized photoresist was removedusing SU-8 developer yielding the microwell with 30 μmdiameter and the opening as the connection pad. The coverslipswith cell microwells were hard baked for 20 min at 120 °C. AnO-ring was attached on the ITO slide to include the microwell.For the etching of ITO layer under the microwell, a solution ofHCl/H2O/HNO3 (4:2:1) was introduced into the microwellfor 20 min. After washing, a nanoring electrode with the heightof 180 nm and the diameter of 30 μm was fabricated. The barenanoring electrode can be stored in the dark for more than onemonth.Modification of the Prussian Blue Layer on the

Electrode Surface. The deposition was initialed by cyclingthe potential from −0.2 to 0.6 V for 2 segments with a scan rateof 100 mV/s in 0.1 M KCl solution containing 2 mMK3[Fe(CN)6], 2 mM FeCl3, and 0.1 M HCl. Then, the layerwas activated in the solution with 0.1 M HCl and 0.1 M NaClby cycling the potential from −0.2 to 1 V at a scan rate of 100mV/s for 10 segments.Electrochemical Characterization. The nanoring elec-

trode was connected with the electrochemical station (CHI630E, CH Instruments) as the working electrode. Ag/AgCl andPt electrodes were the reference and counter electrodes,respectively. For the electrochemical characterization, theelectrolyte solution was 100 mM phosphate-buffered saline(PBS, pH 7.4) with 5 mM ferrocyanide. A cycle of voltageranged from 0 to 0.4 V.Imaging of Nanoring Electrode. The electrochemilumi-

nescence imaging was performed on the upright microscope(Mshot MF10, Microshot Tech., China) equipped with Evolve512 EM CCD (Photometrics, Tucson, AZ). The electrolytesolution was 100 mM PBS with 200 μM luminol and 1 mMhydrogen peroxide. A voltage of 1.0 V was applied on the ITOlayer before and after the wet-etching for the imaging, and theexposure time was 5 s. Scanning electron microscopy (SEM)was performed using a Hitachi S-4800 Instrument (Japan). Anaccelerating voltage of 10 kV with an Au coating of the samplewas used to image the nanoring electrode.Single Cell Analysis. For the trapping of a single cell in the

microwell, ∼100 cells in 10 μL of medium were added on theITO slide with the microwell. The ITO slide with the cells wasshaken gently for a few minutes until one cell was loaded intothe microwell. After the loading of a single cell into themicrowell, the extra cells outside the microwell were washedaway by a flow of medium. The succeeding rate was near 100%.After culturing the cell for 3 h, 200 ng/mL phorbol-myryslateacetate (PMA) was added into the medium to stimulate the cellfor 30 min at 37 °C. To break the cellular membrane, 3 mMNaOH was added into the solution and the cell membrane wasobserved to be broken in less than 1 s. After the analysis, due tothe contamination of the glass surface under the microwell bythe remaining cell lysate, the nanoring electrode was discarded.Estimation of Intracellular Hydrogen Peroxide Con-

centration. 105 stimulated cells were lysed using 3 mMNaOH, and the intracellular hydrogen peroxide in thesupernatant was measured using an amplex red assay kit. Theconcentration of hydrogen peroxide in each cell was estimatedfrom total hydrogen peroxide from the cell population. Theindividual cell volume was assumed to be 1 pL for calculation.Finite Element Simulation. The voltammetries of the

nanoring and recessed electrodes were simulated in 2D axial

symmetry using a combination of COMSOL 3.5a software(Comsol Inc.) and the diffusion equation.

■ RESULTS AND DISCUSSIONFormation of Nanoring Electrode. The process for the

formation of the nanoring electrode was exhibited in Figure 1A.As designed, after the fabrication of the cell-sized microwell onthe layer of ITO, the introduction of etching solution removedthe layer of ITO in the microwell. The exposed vertical sectionof the ITO layer at the bottom edge of the microwell formed aring with the diameter of 30 μm and vertical height of 180 nmas the nanoring electrode. To chase the removal of the ITOlayer, a simple luminol electrochemiluminescence (ECL)imaging technique was applied.18,19 In the presence ofhydrogen peroxide and luminol, the luminescence was emittedfrom the electrode surface under the potential.20,21 Thus, theluminescence image can be used to visualize the electrodesurface during the etching process. Before the wet-etching, thelayer of ITO in the microwell behaved as the recessed electrodeand was expected to emit the microwell-shaped luminescence.After the etching process, the removal of the ITO layer in themicrowell created a nanometer layer of electrode, and this areawas too small to generate the detectable luminescence. Figure 1B(a−c) showed the bright field, the luminescence, and theoverlaying images before the etching in the presence of luminoland hydrogen peroxide. A bright disk in luminescence wasobserved at the region of microwell, which revealed theexistence of the ITO layer in the microwell. The introduction ofwet-etching solution removed the ITO layer under themicrowell and the shrinkage of the luminescence area wasvisualized, as expected. When the etch time was over 20 min,the whole luminescence in the microwell disappeared, as shownin Figure 1B(d), exhibiting the complete removal of the ITOlayer under the microwell. As a result, 20 min was fixed as theetching time for the removal of ITO at the bottom layer of themicrowell.To confirm the formation of nanoring electrode after etching,

SEM imaging was applied to attempt the visualization of thenanoring electrode under the microwell. However, since thedepth of the nanoring (180 nm) was much smaller than that ofthe SU-8 microwell (30 μm), the observation of the nanoringunder the microwell was technically challenging. Therefore, forthe better imaging of the nanoring electrode, the SU-8 film waspeeled off by soaking in water for 2 days before SEM imaging.As shown in Figure 1C(a,b), a ring with the height of ∼180 nmwas observed, which was the same as the thickness of the ITOlayer. The diameter of the ring was measured to be 30 ± 1 μm,which was consistent with the diameter of the microwell in theSU-8 film. The agreement in the diameter and depth suggestedthat the etching removed the ITO layer in the SU-8 microwellto form a nanoring electrode.

Voltammetry of Nanoring Electrode. The formation ofnanoring electrodes was confirmed by investigating thevoltammetry of electrodes in the presence of ferrocyanide asan electrochemical probe. Before the etching, the ITO layerunder the microwell behaved as the recessed microelectrodewith 30 μm in diameter and 30 μm in depth. Thevoltammetries of the recessed electrode with different scanrates were recorded in Figure S1 (Supporting Information).Similar to the previous report,14 a transient response with aslow decay of the faradaic current was observed at a scan rate of100 mV/s, as shown in curve a. When the scan rate reached 20mV/s, a pseudosteady-state current was collected in curve b,

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which exhibited the radial diffusion of Fe2+ to the electrodesurface. The limiting current was measured to be 6.12 nA,which gave the current density of 0.91 mA/cm2. Following theequation of recessed electrode,22,23

π π= +i nFDcr H r4 /(4 )2(1)

where n was the number of electrons transferred, D was thediffusion coefficient (6.5 × 10−6 cm2/s), c was the bulkconcentration of Fe2+, H was the recessed depth (30 μm), and r(15 μm) was the apparent radius of the microelectrode. Thetheoretical current of 5.27 nA and the current density of 0.78mA/cm2 were calculated. The small discrepancy between theobserved and theoretical currents was reported to be associatedwith the edge effect, which created a larger actual electrode arearelative to the estimated geometric area.14 After wet-etching, asshown in Figure 2A, pseudosteady-state currents were observed

when the scan rates were 100 and 20 mV/s revealing a fasterdiffusion of probe to the nanoring electrode. The limitingcurrent was 4.44 nA at the scan rate of 20 mV/s, and thecurrent density was calculated to be 26.18 mA/cm2. Comparedwith the current density of 0.91 mA/cm2 on the recessedmicroelectrode, 28-fold enhancements in the current density onthe nanoring electrode supported fast diffusion of the probe onthe nanometer sized surface, which was favorable for thefollowing single cell analysis.To verify the enhanced current density on nanoring

electrode, the voltammetry was simulated using Comsolsoftware. The models of the recessed and nanoring electrodesare shown in Figure S2A,B (Supporting Information), and thesimulation procedure followed the protocol in the literature.24

As shown in Figure 2B, pseudosteady-state currents wereobtained from both the recessed and nanoring electrodes at ascan rate of 20 mV/s, and the limiting currents were 5.42 and3.24 nA, respectively. The similarity of the experimental andsimulated currents on the nanoring electrode confirmed the

formation of nanoring structure. As compared with the modelof recessed electrode, the discrepancy between the exper-imental and simulated currents on the nanoring electrode was alittle larger. The additional deviation might be caused by somesmall ITO regions remaining near the nanoring electrode afterthe etching, which was unavoidable in the wet-etching process.

Detection of Aqueous Hydrogen Peroxide. Todemonstrate functionalized nanoring electrodes for thedetection of biomolecules, hydrogen peroxide as a classiccellular messenger was chosen as the model. The nanoringsurface was modified with a layer of prussian blue, which waswell-known to increase detection sensitivity on hydrogenperoxide.25 In most of the literature, low or negative potentialis chosen to reduce hydrogen peroxide on prussian blue.However, the concentration of hydrogen peroxide releasedfrom a single cell in the microwell was typically a fewmicromolar, and thus, oxygen in aerated solution with aconcentration of ∼250 μM might be involved in the reductionprocess leading to the artifact. Therefore, a positive potential of600 mV on the electrode was chosen in our work to oxidizehydrogen peroxide for the detection, which had been reportedbefore.26,27 Since 3 mM NaOH was used to lyse the cell in thefollowing single cell analysis, 100 mM PBS with 3 mM NaOHwas chosen as the solution to correlate the current responses toaqueous and cellular hydrogen peroxide. After the collection ofthe background current in the solution, hydrogen peroxide witha concentration lower than 250 μM was injected into thesolution and the current was recorded continuously, as shownin Figure S3 (Supporting Information). After the extraction ofbackground current, the current increases were replotted withthe concentrations of aqueous hydrogen peroxide, as shown inFigure 3A. A gradual elevation of the oxidative current withmore aqueous hydrogen peroxide exhibited that the nanoringelectrode was responsible for hydrogen peroxide. The relativestandard deviation from triplicate measurement using oneelectrode was less than 9.5%, and the relative standard deviationfrom four electrodes was less than 11.2% for all concentrationsof hydrogen peroxide. The control experiment was performedby injecting the same amount of PBS into the solution, and nocurrent increase was observed, which confirmed that thecurrent increase in the presence of hydrogen peroxide wasascribed to the oxidation of hydrogen peroxide on the electrodesurface. The current density was calculated to be 22 A/cm2·M,which was beyond the highest current density (8.8 A/cm2·M)obtained on prussian blue modified macroporous micro-electrodes.28 It was noted that the current density of thenanoring electrode for hydrogen peroxide was not as high asthat on platinized nanoelectrodes (∼75 A/cm2·M in ref 10).However, the nanoring electrode offered the average current of818 pA for the detection of 250 μM hydrogen peroxide, whileplatinized nanoelectrode gave less than 20 pA for the sameconcentration of hydrogen peroxide. Therefore, the function-alized nanoring electrode with a relatively large current andcurrent density was advantageous to the following single cellanalysis.

Analysis of Hydrogen Peroxide in Cytosol at theSingle Cell. For single cell analysis, one cell was introducedinto the microwell and spread on the glass, which was thepreferred surface for cell culture. The spatial isolation of the cellfrom the nanoring electrode surface, as confirmed bymicroscopic observation, minimized the cytotoxicity of themodification layer on the cellular activity. To increase theintracellular hydrogen peroxide, the cell was stimulated by PMA

Figure 2. (A) Cyclic voltammograms of nanoring electrode in 0.1 MKCl with 5 mM ferrocyanide at scan rates of (a) 100 mV/s and (b) 20mV/s; (B) the simulated voltammograms of the (a) recessedmicroelectrode and (b) nanoring electrode at a scan rate of 20 mV/s.

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for half an hour to induce the production of hydrogen peroxideinside the cell.29 After applying a constant potential of 600 mVin 100 mM PBS (pH 7.4) to collect the background current,alkaline was introduced to break the cell membrane in less than1 s. As shown in Figure 3B, a transit current increase wasobserved in 0.7 ± 0.2 s from five independent single cellmeasurements, followed with the decay in the current. Sincesome other species inside the cells can be oxidized on theelectrode under the potential of 600 mV, the controlexperiment was performed on the nonstimulated cell and notransit current increase was observed, as shown in Figure S4(Supporting Information). These results confirmed that thecurrent observed on the stimulated cell was associated withintracellular hydrogen peroxide. The contribution of thecurrent response from intracellular hydrogen peroxide onlycould be explained by the relatively high concentration ofhydrogen peroxide after the specific cellular stimulation usingPMA, as compared with the concentration of other speciesinside the cells. Moreover, prussian blue as an excellent catalystfor the oxidation of hydrogen peroxide benefited the specificdetection of hydrogen peroxide. Compared with 1−2 pAcollected from a single cell on the nanoelectrode,10 the currentresponse using the nanoring electrode reached hundreds ofpicoamperes exhibiting the enhanced electrochemical sensitivity

for single cell analysis. Referring to the calibration curve inFigure 3A, the highest concentration of hydrogen peroxide inthe microwell was estimated to be 81 ± 34 μM from fivemeasurements.To support our experimental result, the concentration

change of hydrogen peroxide at the nanoring surface after themembrane broke was simulated using Comsol software. Themodel for the simulation is shown in Figure S5 (SupportingInformation), and 1 s was taken as the release time forintracellular hydrogen peroxide. The average amount ofhydrogen peroxide in each cell was determined to be 2.6fmole based on the analysis on the cell population using anamplex red kit. When 1 pL was taken into account as thecellular volume, the intracellular concentration of hydrogenperoxide was calculated to be 2.6 mM. This concentration wasconsistent with the literature value obtained using nano-electrodes.10 Figure 3C showed that the simulation resultexhibited a maximum concentration of 88 μM at 0.97 s and asubsequent decay of concentration associated with the diffusionof hydrogen peroxide away from the microwell. The match ofmaximum concentration between the simulated and exper-imental results confirmed the accuracy of our measurementusing a nanoring electrode. Moreover, since the maximumconcentration was correlated with the amount of intracellularhydrogen peroxide, the determination of the maximumconcentration in the experiment offered information aboutthe amount of intracellular hydrogen peroxide at single cells inthe biological study. The successful analysis of intracellularhydrogen peroxide exhibited that our nanoring electrode couldbe applied for single cell analysis with the enhanced sensitivity.However, it was noted that, since the electrode was not locatedunder the cells, our design had limitations for the localizedelectrochemical detections that needed the contact between theelectrode and the cells, such as the imaging of the moleculardistribution at the cell surface. As a result, the nanoringelectrode should be favorable for the analysis of chemicalrelease or cytosol from a single cell.

■ CONCLUSION

In this paper, nanoring electrodes embedded under themicrowell were fabricated to achieve the spatial isolation ofthe electrode surface and cell culture support, which permittedthe functionalization of the electrode surface with minorcytotoxicity. The electrode functionalization and the fasterdiffusion of analyte to the nanometer-scaled ring offeredenhanced electrochemical sensitivity in the analysis of cytosol atsingle cells. The future study aims to couple a highly sensitivesensing surface and nanoring electrode array for the analysis ofmore molecules at single cells. Connected to the multichannelelectrochemical station with low current amplifiers, thenanoring electrode array will be fabricated for high-throughputsingle cell analysis. Meanwhile, a new laser technique will beintroduced to lyse the cell in a microsecond so that thebioinformation in the cells will be maintained during the lysisfor more accurate measurement.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional information as noted in the text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

Figure 3. (A) The current responses of the prussian blue modifiednanoring electrode for hydrogen peroxide; the error bar represents thestandard deviation from four independent electrodes. (B) The typicalcurrent trace of the prussian blue modified nanoring electrode for theanalysis of hydrogen peroxide in cytosol from a single stimulated cell.(C) The simulated concentration of hydrogen peroxide at the surfaceof the nanoring electrode after breaking the cell membrane.

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■ AUTHOR INFORMATIONCorresponding Authors*Phone: 086-25-86868477. Fax: 086-25-86868477. E-mail:djf@njmu.edu.cn.*Phone: 086-25-83594846. Fax: 086-25-83594846. E-mail:dechenjiang@nju.edu.cn.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the 973 Program (2013CB933800), the National Natural Science Foundation ofChina (Nos. 21135003, 21105045, 21105049), the NationalScience Fund for Talent Training in Basic Science (No.J1103310), and Technology Foundation for Selected OverseasChinese Scholar, Ministry of Personnel of China.

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