Electrochemistry at nanoelectrodes

Andrew J. WainDOI: 10.1039/9781849737333-00044

2.1 Introduction

The advent of nanotechnology has had a colossal impact on the modernworld and scientific discoveries continue to be made in a variety of discip-lines as a result of our ability to measure and understand processes at thetiniest of scales. Electrochemistry is no exception to this trend. Indeed, it isdifficult to overstate how this field is thriving through the development ofdevices with nanoscale dimensions and the fabrication of new and excitingnanostructured materials with unique behaviours. Applications of electro-chemistry at the nanoscale vary widely and include sensing, catalysis, cor-rosion science, energy conversion technologies and cellular biology to nameonly a few. In fact, given that electrochemists are primarily concerned withcharge transfer across interfaces, it would be easy to argue that all elec-trochemistry is nanoelectrochemistry. Such a broad topic would be difficultto review, and so in this chapter we will focus specifically on nanoelectrodesand their arrays.

Formally the term nanoelectrode conventionally refers to electrodes witha critical dimension falling in the 1–100 nm range, although some of theexamples discussed in this chapter do strictly fall outside of this upper limit.Nanoelectrodes exhibit vastly different properties to their macroscopicequivalents, and although several parallels can be drawn with microelec-trode behaviour, electrodes with nanometre dimensions display many pe-culiarities that further set them apart. Much of the distinctive behaviour ofnanoscopic electrodes can be linked to the fact that, unlike microelectrodes,the electrode dimensions are comparable to the thickness of the electricaldouble layer, and are approaching the molecular scale. Moreover, theirsmall size imparts various beneficial properties, often rendering them su-perior electrodes for both fundamental studies and sensing applications. Ahighly touted advantage is the significantly enhanced mass transport asso-ciated with vanishingly thin diffusion fields, which not only yields highcurrent densities but lends unrivalled access to kinetic information per-taining to rapid electron transfer processes. Similarly, their inherent efficacyin confining local electrochemical measurements to increasingly smallspaces is one attribute that continues to find novel applications, particularlyin scanning electrochemical microscopy (SECM). Rapid response times, lowcapacitive currents and the scope to undertake measurements in more re-sistive media are further intrinsic benefits of nanoelectrodes that are fre-quently exploited.

It is not the purpose of this chapter to provide an extensive account of theentire field of nanoelectrodes, but to present the some of the major con-tributions of the last five years, during which remarkable advances in both

National Physical Laboratory, Hampton Road, Teddington, TW11 0LW, UK.E-mail: andy.wain@npl.co.uk

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�c The Royal Society of Chemistry 2014

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theoretical and applied elements have been achieved. The interested readeris also referred to a number of review papers that have emerged over thepast decade on this topic.1–5 We will begin this chapter with a brief overviewof the critical theory underpinning electrochemical measurements atnanoelectrodes and present key developments in the computational simu-lation of these systems. After this we turn our attention to the latest ex-perimental advances, first by reviewing the state-of-the-art in nanoelectrodefabrication and subsequently focusing on the latest and most innovativeapplications to the areas of fundamental electrochemistry, electroanalysisand electrochemical imaging.

2.2 Theory and modelling of nanoelectrodes

Whilst robust theoretical models have been developed for microelectrodesand their arrays, the field of nanoelectrochemistry is less mature and acomprehensive theory is currently lacking.6 However, great steps have beenmade in the past decade towards the understanding of charge and masstransport at nanoscopic interfaces and these advances will be discussed inthis section.

2.2.1 Diffusion-only considerations

Mass transport in a convection-free system is described by the Nernst-Planck equation, in which the flux Ji, of a species i, is governed by aombination of Fickian diffusion and charge migration, i.e.:

Ji ¼ �Di rci þziF

RTcirj

� �ð2:1Þ

where Di, ci and zi are the diffusion coefficient, concentration and charge ofthe species i respectively, j is the potential and F, R and T take their usualmeanings. We first consider the diffusion-only case, which applies when theaddition of an inert supporting electrolyte is sufficient to compress anypotential gradient to within a short distance of the electrode-solutioninterface, such that the rj term in equation (2.1) can be neglected. We willreturn to discussing the validity of this assumption in the context of theelectrical double layer in section 2.3.

As with microelectrodes, diffusive transport to nanoelectrodes on con-ventional voltammetric timescales is dominated by convergent, as opposedto planar, diffusion. Therefore, for a simple electron transfer process, thevoltammetric response at steady state is characterised by a sigmoidal shape.Simulation of such voltammetry requires solution of the diffusion equationtypically with a Nernstian or Butler-Volmer boundary condition for the rateof electron transfer at the electrode surface, depending on its reversibility.For simple, uniformly accessible, electrode geometries analytical solutionsof these equations are available, and so for a disk electrode we obtain thefamiliar equation for the current (ilim) in the limit of diffusion control:

ilim ¼ 4nFrDc� ð2:2Þ

where n is the number of electrons transferred, r is the disk radius and c* isthe bulk concentration of the redox active species. For hemispherical

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electrodes the above limiting current can simply be multiplied by a factor ofp/2.7 For simulation of non-uniformly accessible nanoelectrodes, numericalmethods are necessary and so in many cases it is helpful to draw analogieswith the equivalent micrometre scale electrode. An example of this is micro/nanoband electrodes, which have been shown to reach a condition of quasi-steady-state where the plateau current is a function of the critical electrodedimension (e.g. the band width), the total electrode area and to a smalldegree the voltammetric scan rate.8–10

More recent works have demonstrated the application of numericalsimulation to exploring non-uniformly accessible 3D nanoelectrode geom-etries. For example, Streeter and Compton employed the finite differenceapproach to examine diffusion limited currents at isolated spheroidal andhemispheroidal nanoparticle electrodes immobilized on inert substrates.11

Building on this, Ward et al. used numerical methods to simulate isolatedspherical nanoparticle voltammetry in the limit of irreversible electrontransfer kinetics and derived a simple expression describing the voltam-metric wave-shape:12

i ¼ 4p ln 2ð ÞFDCr2k0

k0rþDexpaFRT

� �E � E0

f

� �� 0:5

� � ð2:3Þ

where r is the nanoparticle radius, k0 is the heterogeneous electron transfer

rate constant, a is the transfer coefficient, E is the electrode potential and E0f is

the formal electrode potential for the reaction. Furthermore, it was high-lighted that the voltammetric response for a single unsupported (uniformlyaccessible) spherical nanoparticle could be conveniently mapped onto that forthe same particle placed on a surface using a simple transformation. Theprocedure established involves simply introducing a current scaling factor of0.693 and translating the potential scale in the positive direction by a value of

RT/2aF mV by adjusting E0f , or the dimensionless rate constant.

Whilst modelling the diffusion characteristics of individual nanoelec-trodes may be relatively straightforward, a significant complication ariseswhen multiple nanoelectrodes are arranged in an array or ensemble anddiffusive ‘cross-talk’ becomes a problem. For microelectrode arrays thediffusion domain approach to modelling mass transport, in which a diffu-sional space or cell is defined for each electrode, has proven useful.13,14

From this and experimental observations it is well-established that amicroelectrode array will transition from entirely independent microelec-trode behaviour at sufficient electrode separations (signified by sigmoidalvoltammetry at conventional scan rates) through to fully overlapping dif-fusion fields at shorter separations leading to overall planar diffusion acrossthe array (manifested as peak-shaped voltammetry analogous to that of amillimetre scale electrode).

The situation becomes more complicated for nanoelectrodes arrays sincethey typically have a total footprint in the order of microns, and hence evenwhen adjacent diffusion fields fully overlap, behaviour akin to that at asingle microelectrode is still observed. The extension of the diffusion domainapproximation to nanoelectrode arrays was explored by Godino et al., whocompared simulated voltammetry generated by 2D and 3D modelling with

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experimental data.15 Importantly, these authors highlighted the limitationsof applying the diffusion domain approximation to such arrays since devi-ations arise from the non-equivalence of electrodes at different locations.The additional importance of radial diffusion at the array peripheries isoverlooked by this model, which becomes more significant as the array sizedecreases. The different diffusion regimes are illustrated in Fig. 2.1, whichdepicts simulated 2D concentration maps close to micro- and nanoelec-trodes arrays with overlapping diffusion fields. In the case of a microelec-trode array (Fig. 2.1a) the diffusional overlap leads to a predominantlyplanar profile across the array, whilst for the nanoelectrodes array(Fig. 2.1b), the profile is clearly hemispherical.

The importance of timescale on diffusional independence was also high-lighted in an earlier paper by these authors, in which the transient potentialstep behaviour of electrode arrays were compared by theory and experi-ment.16 It is noteworthy that the electrode separation required for truediffusional independence under conventional experimental timescales (vol-tammetric scan rates in the tens of mV s�1) is typically of the order ofhundreds of microns, a fact that can quite easily be verified by consideringEinstein’s relation for diffusion lengths, d = OpDt. Hence, it is clear thatelectrode spacing and experimental timescale are critical considerationswhen exploiting the mass transport advantages inherent to nanoelectrodes,a fact that will be addressed further in Section 4.2.

A final point to note in the context of diffusional transport at nanoelec-trodes is that as the electrode size begins to approach the molecular level,the continuum approximation that underpins Fick’s laws of diffusion maybreak down as statistical fluctuations due to single molecular events becomesignificant.17 Stochastic phenomena such as this were previously reported atnanoelectrodes with dimensions in the 10 nm region.18,19 More recently, theissue of current fluctuations at very small nanoelectrodes (o2 nm radius)has been discussed, which may also point towards individual surfacebinding events.20,21 These works emphasize the danger of overinterpretingvoltammetric data generated by the smallest of nanoelectrodes, since thecontinuum approach may indeed be compromised. Similarly, the studiesalso highlight that experimental results appearing to be defective should notnecessarily be discarded as erroneous! The stochastic nature of

Fig. 1 Graphical representation of 2D simulations of a plane perpendicular to a row of diskelectrodes within a 10� 10 array. (a) Disk radius 5 mm, 100 mm intercenter distance, (b) diskradius 50 nm, 1 mm intercenter distance. Concentration = 1 mM and D= 6.5� 10�6 cm2 s�1.Reproduced from ref. 15 with permission from the & American Chemical Society.

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nanoelectrochemistry is not limited to mass transport but has also beenlinked to fluctuations in electrode potential. This concept was explored in arecent paper by Garcıa-Morales and Krischer, who concluded that theobserved discreteness of electron transfer at this scale leads to more rapidelectrochemical kinetics than would be predicted by macroscopic models.22

2.2.2 Double layer effects

The influence of double layer effects on nanoelectrochemistry has beenalluded to for many years but until recently the influence of these phe-nomena on mass transport and electron transfer has not been well under-stood.23–25 In particular, deviations from classical behaviour emerge whendiffusion field thicknesses approach the Debye length (the distance overwhich the electrostatic effect of a charge in solution persist), as a result ofthe high rates of mass transport at nanoelectrodes. Under these conditions,the influence of migration on mass transport can no longer be neglected.Hence a significant challenge lies in finding a rigorous solution to equation(2.1), by imposing the Poisson condition:

r2jþ F

e0es

Xi

zici ¼ 0 ð2:4Þ

where e0 and es are the permittivity of free space and the relative permittivityof the solvent respectively. The simplest approach is to apply the electro-neutrality approximation, in which the summation term in equation (2.4) isassumed to be zero. However, it has been demonstrated that this may not beapplicable at nanometre length scales when the electrode size and the Debyelength are comparable.26

Full evaluation of equation (2.4) thus requires knowledge of the chargedistribution at the electrode – electrolyte interface, a problem that has beenexplored in various works.25,27–29 For example, Dickinson and Comptonrecently used numerical modelling to solve the Poisson – Boltzmannequation, which describes the electric field in an electrolyte solution underthermodynamic equilibrium, for hemispherical electrodes.28 The simu-lations revealed a transition between two classical limits; a planar doublelayer as predicted by the Gouy – Chapman model and the spherical doublelayer associated with a point charge (Coulomb’s Law). This is illustrated inFig. 2.2, in which the dimensionless charge density, �Q (=Frq/RTe0es)is plotted as a function of the dimensionless hemispherical electrode radius,Re (= rO(F2c/RTe0es)).

In practical terms, the simulation indicated that under conventional ex-perimental conditions (i.e. ionic strengths of the order of 100 mM),curvature of the diffuse double layer can no longer be neglected fornanoelectrodes with radii smaller than 50 nm. Whilst the Gouy – Chapmanmodel for describing the diffuse double layer has been successfully imple-mented for large electrodes, the deviations from this classical behaviour inthe case of small electrodes have in practice been largely ignored. Notably,the deviation from planar behaviour also becomes more marked with de-creasing electrolyte concentration, such that the capacitive properties ofnanoelectrodes under weakly supported conditions in particular are likelyto deviate significantly from the traditional Gouy – Chapman prediction.

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Returning to the issue of migrational transport at nanoelectrodes, amajor problem is that in the limit of a vanishingly small electrode size,charged species within the depletion zone are largely unscreened, resultingin profound effects on the predicted voltammetry. For example, Liu et al.demonstrated that significant edge effects in the dielectric field can be ob-served at such nanoscale interfaces, due to the inequivalence of solventdipoles at the interface.29 This leads to a non-uniform potential distributionin the double layer, which itself can impact electron transfer kinetics due toposition-dependent electron tunnelling rates at the electrode. Dickinson andCompton recently investigated the various effects of the diffuse double layeron voltammetry by simulating the one electron oxidation of species carryingcharges of � 1, 0 and þ 1 under steady-state conditions.30 For an electroderadius of the order of 10 nm and at low supporting electrolyte concen-trations, the three cases produced very different results. For the negativelycharged electroactive species the voltammetry is distorted and mass trans-port dominated currents greatly exceed the diffusion-only prediction due tothe unscreened Coulombic attraction between the anion and a positivelycharged surface. For the neutral species sigmoidal voltammetry is generallyobserved, although the predicted currents are reduced by retarded apparentelectron transfer kinetics within the double layer (so-called Frumkin effects).Finally, for the cationic species, a trade-off is observed between attractiveand repulsive forces that can, in some cases, leads to peak-shaped voltam-metry. These stark differences highlight the practical considerations neces-sary when undertaking nanoelectrochemistry and the care required ininterpreting nanoelectrode voltammetry.

2.2.3 Electron transfer kinetics

We have already touched upon how nanoelectrodes might exhibit differentelectrode kinetics to their larger counterparts, and although a thorough

Fig. 2 Simulated dimensionless charge density, �Q, as a function of the dimensionless hemi-spherical electrode radius, Re, plotted alongside ideal values predicted by Coulombs law and theGouy-Chapman model. Reproduced from ref. 28 with permission from the & AmericanChemical Society.

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discussion of this is beyond the scope of this text it is noteworthy to touchbriefly upon the validity of different models of heterogeneous electrontransfer kinetics at nanometre sized electrodes. Chen’s group have arguedthat intrinsic electron transfer kinetics can be affected when the electrodesize approaches nanometre dimensions on the basis that the distance de-pendence of the electron tunnelling probability becomes significant.29 Morerecently this group also questioned the appropriateness of Butler-Volmer orMarcus formalisms in the context of electron transfer at nanoelectrodes,emphasizing that at high electrode overpotentials (i.e. those departing sig-nificantly from the formal potential of the redox active species) departuresfrom these theories may occur.31 The Butler-Volmer equation dictates thatthe heterogeneous rate constant increases as an exponential function of theelectrode potential without limit, whilst Marcus theory instead describes aninversion region, and so deviations at high overpotentials might be ex-pected. In this study, the authors compared these two models with the morecomputationally demanding, but arguably more rigorous Chidsey modeland concluded that their viability varies with electrode size, heterogeneousrate constant and reorganization energy. For example, measurable devi-ations were observed for nanoelectrodes with radii ofo50 nm when the rateconstant was close to 0.1 cm s�1. Similarly, Amemiya et al. developed aMarcus-Hush-Chidsey model for kinetic measurements using nanoelec-trodes at macroscopic substrates in an SECM configuration, and againnoted deviations from the Butler-Volmer theory.32 However, it has beenpointed out recently that the Chidsey based formalism is also not without itsown limitations for slow electrochemical processes.33

Whilst it is clear from the above that a generalizable model that can fullydescribe electrochemical processes at nanoelectrodes has not yet beenrealised, it is evident that our theoretical understanding of the associatedconcepts underpinning their unique behaviour is beginning to mature.

2.3 Nanoelectrode fabrication

Recent progress in electrochemical applications of nanoelectrodes has to alarge extent been driven by significant advances in electrode and arrayfabrication technologies. Early approaches to nanoelectrode fabricationinvolved simply depositing a thin layer of metal onto an insulating sub-strate, followed by application of an insulating film and polishing the crosssection to reveal a nanoband electrode.34 Since this seminal work, a revo-lution in nano-engineering, deposition and processing methods has pavedthe way for a plethora of new and more intricate approaches to nanoelec-trode construction. In this section we consider some of the latest methodsthat have arisen in this field.

2.3.1 Ion and electron beam methods

Focused ion and electron beam methods have emerged as invaluable toolsfor producing nanoelectrodes and are particularly advantageous in thefabrication of ordered nanoelectrode arrays and assemblies where precisionand scale-up are critical.10,35–39 In focussed ion-beam (FIB) milling, a finelyfocused beam of ions, typically gallium (Gaþ), is used for the site-specific

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removal of surface material and can machine features as small as 10 nm. Theapplication of this method to nanoelectrode fabrication was first demon-strated by Lanyon and coworkers, who produced recessed nanoband10 andnanopore36 platinum electrodes and arrays by first insulating a flat platinumsurface with a 500 nm thick layer of silicon nitride and subsequently millingaway nanobands or spots of the passivation layer directly using FIB. Bandwidths as narrow as 80 nm and pore radii in the range 75–200 nm could bemachined in this way and although the 500 nm recess has significant im-plications on mass transport the convenience of this approach was clearlydemonstrated. Fig. 2.3a depicts a scanning electron microscope (SEM)image of a typical nanopore array and example voltammetry for the singleelectron oxidation of ferrocenemonocarboxylic acid at three different sizearrays is presented in Fig. 2.3b.

An alternative top-down approach to nanoelectrode fabrication is viaelectron beam, or ‘‘e-beam’’, lithography (EBL). Whilst more intensive thanthe direct-write approach highlighted above, this method is widely used innanofabrication and can be applied to the production of well-resolvednanoelectrode structures with extremely high precision.40 The EBL processinvolves using an electron beam to write a nano-array pattern directly intoan electron-sensitive resist layer that is coated onto the substrate, in amethod akin to photolithography. For lift-off approaches, the resist layeritself is used as a shadow mask for sputtering or evaporating a thin(o100 nm) layer of metal such as gold or platinum, typically with the aid ofa B5 nm thick adhesion layer of titanium or aluminium.38 Removal of themask then reveals a gold/platinum nano-patterned electrode substrate.Alternatively, the electrode material can be deposited prior to the appli-cation of the resist, and the electron beam is then used to expose regions ofthe resist and/or metal to be subsequently removed by chemical etching. Insome cases the remaining resist material is sufficiently insulating andchemically stable to negate the requirement for its removal post develop-ment. For example, Moretto et al. reported a novel polycarbonate-basedresist for high resolution EBL which was used to fabricate recessed nano-disk electrodes with radii as small as 75 nm.41 The recess depth depended on

Fig. 3 (a) SEM image of a 3� 3 array of platinum nanopores. (b) Cyclic voltammetry atdifferent size arrays for the single electron oxidation of 1 mM ferrocenemonocarboxylic acid in0.01 M phosphate buffered saline solution at pH 7.4 (scan rate 5 mV s�1, nanopore radius225 nm). Reproduced from ref. 36 with permission from the & American Chemical Society.

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the thickness of the resist layer deposited, but could also be controlled byfilling the nano-cavities with metal by electrodeposition, thus circumventingissues associated with mass transfer resistance. Kleijn and co-workers used asequence of steps including EBL, metal evaporation, lift-off, and chemicalvapour deposition of a passivating silicon nitride layer to yield individualgold nanoband electrodes, with widths down to 160 nm, on silicon wafers.42

The EBL approach is not limited to the fabrication of metallic nanoelec-trodes, but has also been applied to the fabrication of nanocrystallineboron-doped diamond (BDD) arrays.43 A conducting BDD substrate wasselectively masked with SiO2 and then coated with insulating diamond.Subsequent removal of the SiO2 yielded an ordered array of recessed BDDdisks, with electrode radii in the 150–250 nm range.

A related technique for the fabrication of nanoelectrode arrays isnanoimprint lithography which, although does not involve ion or electronbeams directly, is worthy of a mention in light of growing attention. San-dison and Cooper were the first to use nanoimprint lithography fornanoelectrode array fabrication which simply involves the application ofcompressive moulding, using a nanostructured stamp (typically fabricatedby EBL) to imprint a pattern into a polymer-coated electrode.44 Subsequentetching of the material at the base of the compressed regions yields ananopatterned recessed electrode surface in much the same way as the EBLapproach, only with the added advantages that large areas are imprinted ina single step and the stamp itself can be used multiple times. Such nano-technologies are beginning to find electrochemical applications in fuel cell,45

battery46 and biosensing47 research.The myriad of approaches discussed has been used to produce bands,

disks/pores, interdigitated arrays, and various other electrode architectureswith critical dimensions approaching 100 nm.48–50 However, only recentlyhave such techniques enabled this limit to be truly surpassed. Martinez-Rivas and co-workers implemented a combination of EBL and photolitho-graphy to produce interdigitated arrays comprising nanoband electrodeswith a width of 45 nm at wafer scale with high repeatability, but theirapplication to electrochemical measurement was not realised.51 In a moreelaborate approach using a sequence of photolithographic and etching stepsfollowed by FIB, Rauf et al. produced arrays of 100nm deep nanoholes withgold electrodes at their base and surface radii of approximately 25 nm.52

Steady state voltammetry for the oxidation of ferrocenedicarboxylic acid wasobserved at scan rates below 100 mV s�1 and deviations above this wereattributed to a large radial component to the mass transport at small dif-fusion field thicknesses. Dawson and co-workers presented an alternativehybrid approach to discreet nanowire electrode fabrication using EBL andphotolithography.53 The process consists of a number of steps, shown inFig. 2.4. First, an e-beam is used to write the nanowire pattern onto a resist-coated silicon substrate, which is then coated by evaporation of gold.Subsequent lift-off of the surrounding mask yields a gold nanowire with awidth in the order of 100 nm and a length of B45 mm. Next, a sequence ofphotolithographic steps enables robust electrical connection to the goldnanowire through patterning, exposure and metal deposition to generatemacroscopic contact pads. Finally, these electrical contacts are passivated

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using an insulating photoresist, leaving an exposed window containingmultiple, individually addressable, gold nanowire electrodes. The nanowireswere characterised electrochemically and exhibited good quality steady statevoltammetry, with low background capacitance, for the oxidation of fer-rocenecarboxylic acid, the oxidation of ferricyanide and the reduction ofruthenium hexamine. Moreover the voltammetry showed excellent agree-ment with simulated voltammetry based on Butler-Volmer kinetics.

2.3.2 Wet and dry etching methods

The group of White and later Zhang have pioneered the field of nanoelec-trochemistry and have presented a range of intricate approaches to singlenanoelectrode fabrication. In a recent paper, Zhang’s group reported theproduction of nanotrench electrodes in which a nanoband electrode sand-wiched between two insulators could be situated at various recesseddepths.54 Their fabrication method builds upon the very early approach tonanoband construction described by Wehmeyer and co-workers,34 with anadditional electrochemical etching step to remove the gold electrode tocontrollable depths. Nanotrenches with widths of 12.5 nm and 40 nm werefabricated with depths ranging from B10 nm to 4 mm and the associatedsteady state and transient voltammetry was compared with theory up toscan rates as high as 1000 V s�1. A schematic depiction of the electrode ispresented in Fig. 2.5 which includes example voltammetry for the singleelectron oxidation of ferrocene in acetonitrile before and after the etchingstep. Whilst some evidence of planar diffusion was observed at faster scanrates, indicating electrode imperfections such as cracks and gold delami-nation, these contributions were greatly minimised in the deeper nanotrenchelectrodes, demonstrating their potential future applications in sensing andfundamental studies.

A related electrochemical approach to nanoelectrode fabrication waspreviously demonstrated by Penner’s group.55,56 In this case a nanotrenchelectrode was instead used as a template for the electrodeposition of metalnanowire electrodes in a method referred to as lithographically patternednanowire electrodeposition (LPNE). Simple photolithography was used todefine a sacrificial nickel or silver nanoband electrode sandwiched between

Fig. 4 Schematic depiction of hybrid EBL/photolithography sequence for fabrication ofdiscrete nanowire electrodes. Reproduced from ref. 53 with permission from the & AmericanChemical Society.

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the base substrate and a photoresist layer. Electrochemical stripping of theexposed sacraficial metal produces an undercut horizontal nanotrench ofdefined height which can then be filled with the target electrode material(e.g. gold, platinum and palladium). Subsequent removal of the resist andthe remaining sacrificial metal then exposes addressable nanowire electrodeswith controllable widths as narrow as 40 nm. Elaborate nanoelectrodegeometries have been deposited in this fashion, although the voltammetricresponse of the final nanowire electrodes were not featured in these works.

Nanoskiving has emerged a convenient means produce metal nanowires,the roots of which can again be considered to stem from the originalnanoband approach by Wightman. Here a thin film of the target electrodemetal is deposited and cured between two layers of epoxy and thin crosssections are sliced off using a microtome, producing nanomembranes con-taining a metal nanowire sandwiched in epoxy.57 Removal of the sur-rounding epoxy using dry etching (e.g. oxygen plasma), subsequently yieldsa free standing nanowire with dimensions defined by the thickness of themetal layer and that of the nanomembrane slice. The electrochemicalcharacterisation of such nanowires has since been investigated and, whenmounted on a silicon chip support, the nanowire electrodes exhibit goodsteady state voltammetry at scan rates below 1 V s�1 and fast electrontransfer kinetics.58 The potential application of these devices to the elec-trocatalytic detection of hydrogen peroxide was also demonstrated.

2.3.3 Encapsulated wire nanoelectrodes

The fabrication of individual disk nanoelectrodes by sealing a metal wire orcarbon fibre within an insulating glass or other insulating sheath is a methodthat has long found considerable attention. This has at least in part beendriven by the potential application of high aspect ratio nanotips for highresolution electrochemical imaging (vide infra) but the same sharp electrodegeometry also finds use in a host of biological applications, for example forin-vitro and in-vivo cellular studies.59–61 The simple planar disk geometry

Fig. 5 Example voltammetry for the single electron oxidation of 5 mM ferrocene in acet-onitrile (supported with 0.1 M tetra-n-butylammonium hexafluorophosphate, TBAPF6) at aflat nanoband and etched nanotrentch electrode (width=12.5 nm, length=0.96mm, nano-trench depth 1260 nm, scan rate 50 mV s�1). Shown also is a graphical representation of thenanoband electrode before and after etching. Reproduced from ref. 54 with permission fromthe & American Chemical Society.

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combined with controlled insulator dimensions and minimized electrolyteor medium perturbation allows for the intricate measurement and model-ling of electron transfer processes on a highly localised scale. Disk electrodescan be produced routinely on the micron scale, and indeed are readilyavailable commercially, but at the nanoscale challenges related to imperfectelectrode geometry, poor electrode sealing, protrusions and consequentlyexperimental repeatability become increasingly significant. Hence at thisscale there is an even greater requirement for novel approaches to nanodiskfabrication with tighter control over size, geometry and above all quality,and this has been the focus of a number of recent investigations.62

Traditionally, glass-encapsulated disk microelectrodes were fabricated byheat-sealing the electrode wire or fibre in a borosilicate glass capillary undervacuum using a basic pipette puller or heated resistor coil.63,64 Since then,the automated laser-heated pipette puller has revolutionised the sealingprocess, although issues of pulling repeatability are a known result of theirsensitivity to various conditions such as laser alignment and capillarycleanliness. The many practical considerations for the use of laser pullers forthis purpose have been reported and reviewed.65,66 The earliest demon-stration of a nanodisk electrode to be produced in this way was reported byShao and co-workers, who varied the parameters of a laser pulling programto seal a 50 mm platinum wire into borosilicate and pull it into a fine point,and used a chemical or mechanical etch to subsequently expose a platinumdisk at the apex.67 Disk electrodes with effective radii in the 50 nm rangewere produced routinely but the report highlighted the pitfalls and limi-tations of using sub-10 nm electrodes produced in this way. Building on this,Katemann and Schuhmann later presented experimental details for a re-fined fabrication procedure, and reported a similar lower limit in the 10 nmregion.68 More recently, through further modification of the laser-assistedprocedure, Li et al. took a major step towards the reproducible productionof sub-10 nm nanodisk electrodes.69 Their procedure, which is depictedschematically in Fig. 2.6a, involves feeding a platinum microwire into anarrow silica sleeve (internal diameter B80 mm, outer diameter B350 mm),and the resulting assembly is inserted into a wider silica tube (internaldiameter B400 mm, outer diameter B1.2mm). A high temperature lasersealing step, followed by a laser pulling programme generates a platinumnanodisk electrode at the apex of a sharp capillary. In a second sealing stepthe resulting pulled capillary can be inserted into an even wider (2mm outerdiameter) borosilicate tube which can be melted around the narrow capil-lary using a hydrogen flame. The improved robustness of the electrode aidsthe process of platinum disk exposure, by enabling conventional mechanicalpolishing. Using a 25 mm diameter platinum wire the authors reported anaverage effective nanodisk radius of 3.3 nm (see Fig. 2.6b), as determined byvoltammetry. This could be reduced to 2 nm by chemically etching theplatinum microwire prior to the initial sealing step but attempts to decreasethis further were met with poor repeatability. More impressive was thequality of the voltammetry presented, an example of which is shown in Fig.2.6c, which exhibits text-book sigmoidal behaviour for the single electronoxidation of ferrocene in acetonitrile. Despite the high signal to noise ratiopresented by these authors, the electrical detection of miniscule (sub-pA)

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currents also becomes an issue for electrode dimensions approaching 1 nm,at which point instrumental factors may become limiting.70

The use of platinum wire as an electrode material for the above laserbased pulling approaches has proved advantageous as a result of its hard-ness and high melting point. However, the use of these techniques becomesmore challenging when alternative electrode materials are sought. Somesuccess of the direct laser-assisted method has been reported for silvernanodisk electrodes using a modified pulling procedure, although the pro-cess resulted in larger electrodes than those discussed above (B50 nm radius)due to the low melting point of the target metal.71 Gold nano-disk elec-trodes with effective radii as small as 7 nm have also been prepared usingthermal stripping of polystyrene-coated etched gold microwires.72 In analternative approach, a number of examples have appeared in the literaturewhich circumvent the difficulties associated with low melting point metalsby using electrodeposition. A recessed nanodisk or nanopore electrode canbe produced by chemical or electrochemical etching of a planar platinumnanodisk (as produced by one of the methods discussed above) and thecavity can be filled with the target metal by electroplating. Metals such asgold73 and mercury74 have been deposited and, since the resulting electrodedimensions are at least in part controlled by the pore size, nanodisks in thesub-10 nm range are possible. Deposition of other metals could allow formore unusual nanodisk electrodes to be fabricated, such as those composed

Fig. 6 (a) Schematic representation of the laser-assisted pulling process, (b) transmissionelectron microscope (TEM) image of 3 nm radius platinum nanodisk electrode and (c) vol-tammetry observed at various sized nanodisk electrodes for the single electron oxidation of5 mM ferrocene in acetonitrile (0.2 M TBAPF6). Reproduced from ref. 69 with permission fromthe & American Chemical Society.

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of palladium or iridium, or even mixed or alloyed materials that may be ofinterest for catalytic studies. The pore-filling approach was explored furtherin the context of potentiometric measurements for ion selective determin-ation although for larger electrodes (100–500 nm).75 In one example, irid-ium oxide, which is known to exhibit a super-Nernstian potentialdependence on pH, was electrodeposited into the cavity of a platinumnanopore electrode to yield a pH nanoprobe suitable for highly localisedmeasurements and imaging (vide infra). Similarly, deposition of silver intothe a nanopore, followed by a layer of silver chloride, allows the nanoe-lectrode to serve as a potentiometric chloride sensor.

The fabrication of carbon nanodisk electrodes has also been achieved bymaking use of shear force detection to control the process of electrode in-sulation.76 Encapsulation of a carbon fiber into a tapered glass capillary wasachieved using the standard pipette pulling approach, however allowing thesealed fiber to protrude beyond the end of the glass. The tip of the carbonfiber was then sharpened via electrochemically etching and was subsequentlyinsulated in an anodic electrophoretic paint. However, in order to prevent thesharpened apex of the carbon fiber itself being coated, shear force detectionwas used to touch the tip to a soft silicon rubber during the electrodepositionstep. This yielded conical electrodes with effective radii as small as 46 nm, andsince they are inherently suitable for shear force positional control, they havepotential application in electrochemical imaging (vide infra).

Individually addressable dual disk electrodes have also been successfullyfabricated using a laser-assisted approach, albeit with larger dimensionsthan those presented above.77 These were prepared using a borosilicate y-capillary and a combination of laser pulling and sealing steps to encase twoetched platinum microwires. Platinum disk radii in the range 75–200 nmwere produced, with a separation of 1–2 mm. Both electrodes produced well-behaved voltammetry and the generator – collector properties of the dualassembly were characterised, paving the way for some interesting analyticalapplications in addition to their potential use for electrochemical imaging.A dual disk electrode was also produced by Gao et al., who used a phenol-allylphenol co-polymer to coat two 10 mm gold wires, one of which wasetched to an ultrafine point, and the two wires were sealed into a pulled y-capillary using ethyl a-cyanoacrylate.78 The result was an asymmetric dualgold electrode consisting of a 10 mm disk electrode situated alongside a20 nm disk electrode.

While glass-encapsulation has been employed as the predominant meansto insulate nanowire-based electrodes, there are examples of similar highaspect ratio needle nanoelectrodes being produced by a variety of othermethods. For instance, Yum and co-workers used mounted boron nitridenanotubes (BNNT) as a template for gold sputtering followed by insulationusing electrophoretic paint and cross sectioning using FIB milling to yield anano-ring electrode (Fig. 2.7).79 This attractive approach, which was re-ported to yield nanoelectrode radii as small as 40 nm, resembles earlierfabrication of carbon nanotube-templated electrodes used for electro-chemical imaging applications.80

Other approaches to encapsulating nanoelectrodes with an insulating ma-terial include: chemical vapour deposition of silicon nitride80 or Parylene C;81

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atomic layer deposition of HfO2;82 electrodeposition of co-polymers.80

Whatever the nature of the coating, the most critical element is to produce aconformal, pin-hole free, non-porous layer and this requirement becomesincreasingly significant the smaller the electrode.

2.4 Applications of nanoelectrodes

The practical applications of nanoelectrodes continue to diversify, but it isonly in recent years that our ability to fabricate well-characterised elec-trodes has enabled confident interpretation of the associated electro-chemical measurements. In this section we divide the latest developmentsinto three themes: (i) fundamental studies relating to heterogeneous electrontransfer at nanoelectrodes; (ii) sensing applications of nanoelectrodes andtheir arrays and; (iii) electrochemical imaging using nanoelectrodes.

2.4.1 Fundamental studies of electron transfer and electrocatalysis

The high rates of mass transport associated with nanoelectrodes rendersthem particularly useful for the determination of heterogeneous electrontransfer kinetics, an application that was reviewed recently by Mirkin’sgroup.83 For a range of electrode geometries the mass transfer coefficient,which essentially defines the limit as to the fastest accessible kinetic par-ameters, is of the order of D/r. Thus, heterogeneous rate constants of theorder of 100 cm s�1 may be discernible at nanometre sized electrodes fortypical diffusion coefficients of 10�5 cm2 s�1. However, this advantagecomes at a price; the uncertainty in electrode geometry associated with

Fig. 7 Schematic representation of the BNNT-templated nanoelectrode fabrication process:(a) sharpened tungsten wire, (b) attachment of BNNT, (c) gold sputter coating, (d) insulation,(e) FIB milling of apex, (f) optical image of final nano wire electrode. Reproduced from ref. 79with permission from the American Chemical Society.

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nanoelectrodes can cast doubt on the accuracy of kinetic parameters de-termined in this way.

In many of the examples discussed in section 2, the ‘‘effective’’ size of thefabricated nanoelectrode was quoted. In other words, a certain electrodegeometry was assumed (e.g. planar disk, hemispherical, conical) and thecritical dimension was calculated on the basis of the mass-transport limitingcurrent for a model Faradaic process by comparison with theory. There is ofcourse an innate danger in taking this approach because of the many geo-metric uncertainties inherent to experimental systems, and hence there isalways a need for claims on the basis of voltammetry to be substantiatedand geometric models validated with surface area measurements and highresolution microscopy. Whilst such uncertainties are commonly overlookedin the context of calculating electrode size, to do so in the determination ofrapid electrode kinetics is ill-advised. A number of authors have reportedthat in supported electrolytes electrode size has no substantial effect onmeasured k0 values for many outer sphere electron transfer processes.69,83–85

Conversely, uncertainties in electron transfer kinetics are commonly at-tributed to imperfections in electrode geometry.85 Hence, whilst producingsmaller and smaller electrodes has advantages, without knowledge of thetrue electrode geometry and the presence of defects, these advantages areimmaterial if one wishes to confidently ascertain kinetic parameters for aparticular Faradaic process. This is especially true of non-planar electrodesand the smaller the electrode is, the bigger the relative impact these un-certainties will have. The requirement for multiple experiments in thesecircumstances then becomes imperative. A more detailed discussion on thevarious pitfalls and problems associated with determination of electrontransfer kinetics from nanoelectrochemistry, and their potential solutions,can be found in Mirkin’s review.83

One approach to address some issues of imperfect geometry is to measurethe electrochemical response of the nanoelectrode in close proximity toanother (macro)electrode or insulating surface in a thin layer configuration.In practice this can most easily be achieved using a scanning electrochemicalmicroscope in which a positioning device is employed to control the sep-aration of the two electrodes.83,84,86 The advantage of this method is thatthe measured current vs. separation response (i.e. the approach curve) ishighly sensitive to protrusions and defects in the nanoelectrode geometry,and in some cases the extent of these imperfections may be determined byfitting experimental approach curves to theory. This sensitivity is particu-larly pronounced for the feedback effect, occurring when products elec-trogenerated at the nanoelectrode are rapidly turned over by the secondelectrode, regenerating the original electroactive species and replenishing itsconcentration locally (see Fig. 2.8a). As an example, Sun and Mirkinsimulated approach curves for recessed disk electrodes and highlighted theprofound effect of recess depth on feedback current (Fig. 2.8b).86 Using afitting procedure, these authors were able to characterise the geometry ofrecessed nanodisk electrodes with good agreement between experimentand theory.

Notwithstanding the practical issues associated with confident electrodecharacterisation discussed above, nanoelectrodes have provided a wealth of

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information about heterogeneous electron transfer. An interesting examplewas presented by Velmurugun et al., who noted a small, but statisticallysignificant difference between the k0 measured at gold (13.5� 2 cm s�1) andplatinum (17.0� 0.9 cm s�1) nanodisk electrodes for the single-electron re-duction of ruthenium hexamine in 0.5 M KCl.87 The authors attributed thisdependence of outer sphere kinetics on the electrode material to an elementof non-adiabaticity in the electron transfer process. Conversely, hetero-geneous rate constants determined for other electron transfer processes suchas the single electron oxidation of ferrocenemethanol (aqueous) and ferro-cene (in acetonitrile solution) were almost identical for the two differentelectrode materials, a fact that points towards fundamental differences inthe nature of the electron transfer process for ruthenium hexamine.Moreover, the observation that heterogeneous rate constants measured atnanoelectrodes were notably higher than those determined previously forlarger electrodes suggests that non-classical influences may be at work; thefact that these differences appear more pronounced for the triply chargedRu(NH3)6

3þ ion than for neutral electroactive species is not inconsistentwith a double layer or Frumkin effect.25 It is noteworthy at this point tomention the work of Guo and co-workers, who studied outer sphere elec-tron transfer, albeit more qualitatively, at chemically modified nanodiskelectrodes.88 A monolayer of 4-aminothiophenol (ATP) was immobilized,by self-assembly, onto a gold nanodisk electrode (85 nm radius) and cyclicvoltammetry was undertaken in aqueous solutions of ruthenium hexamineand ferrocenemethanol. In both cases the mass-transport limited currentwas significantly reduced compared to the unmodified gold nanoelectrode,but more interestingly the half wave potential for ruthenium hexaminereduction was unchanged whereas that for the oxidation of ferrocene-methanol was shifted more positive by close to 100 mV. There are, ofcourse, many possible explanations for this apparent change in electrodekinetics, but one could speculate that the difference in behaviour of thesetwo electroactive species is connected their different charges (and the rela-tive change in charge upon reduction/oxidation at the electrode). Indeed,

Fig. 8 (a) Schematic representation of positive feedback effect for a recessed disk electrodeapproaching a conducting substrate. (b) Simulated approach curves demonstrating the effectof recess depth on the electrode current (normalised with respect to the current in bulksolution) for a model positive feedback system. L = displacement normalised with respectto electrode radius and approach curves corresponding to normalised recess depths between0 and 2 are presented. Adapted from ref. 86 with permission from the & American ChemicalSociety.

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this could be ascribed to solvation effects, a perturbation of the electricaldouble layer by the assembled ATP molecules or simply mass transportconsiderations, and so more work is required to understand these funda-mental processes at the nanoscale.

Nanodisk electrodes have not only been utilised in the quantification ofelectron transfer kinetics, but have also featured in more mechanistic studiesof adsorption/desorption and nucleation/growth processes.89,90 For ex-ample, Zhan et al. presented compelling evidence for hydrogen spilloverphenomena in glass-sealed platinum nanodisk electrodes.90 The adsorptionand rapid surface diffusion of hydrogen adatoms across the platinum/glassinterfaces is believed to result in the accumulation of hydrogen within theglass phase. Such effects become more pronounced with small disk elec-trodes, wherein the size of the platinum/glass interface is significantly largerthan the exposed metal surface. In this work, the spillover was manifested inthe form of unusual observations in the hydrogen adsorption/desorptionvoltammetry of such nanodisk electrodes, such as the appearance of anunexplained peak in the double layer region, and unfeasibly large roughnessfactors calculated by integration of the hydrogen adsorption charge. Notonly does this work offer a new perspective on adsorptive processes at glass-sealed nanoelectrodes, and indeed highlights a potential pitfall in the use ofsuch adsorption/desorption voltammetry to characterise electrode surfacearea, but moreover has possible implications for the development ofhydrogen storage materials.

Building on their work on nanometre scale disk electrodes, Zhang’s grouphave undertaken electrochemical investigations at the individual nano-particle level.91 The measurement and understanding of electron transportkinetics at single isolated nanoparticles is a challenge that has thwarted thedevelopment of nanomaterials for a range of energy applications such asfuel cells, solar cells and related energy conversion technologies. Singlenanoparticle electrodes (SNPEs) were prepared by chemically assemblinggold nanoparticles from a colloidal suspension to an oxidised platinumnanodisk electrode, using (3-aminopropyl)trimethoxysilane (APTMS) as alinker molecule (Fig. 2.9a and b). In this case, estimation of the goldnanoparticle surface area was achieved using copper underpotential de-position (UPD), wherein a monolayer of copper adatoms is deposited andthe associated charge is determined. Larger than expected geometric areaswere reported, suggesting that the underlying platinum nanodisk, despite itsoxide layer, may also contribute to the copper UPD process. This is also inkeeping with the above discussion on adatom spillover,90 and again high-lights a possible danger in using the UPD approach alone for characterisingelectrode surface area, especially in the case of mixed systems in whichdifferent metals might have similar UPD properties. Cyclic voltammetrywas found to be a more reliable means to characterise the size of the an-chored gold nanoparticles and for particle radii in the range of 7–12 nmthe calculated radii were within 20% of those determined by TEM. Sig-moidal voltammetry was presented for model electron transfer reactions(ferricyanide and ruthenium hexamine, Fig. 2.9c) at the SNPE, but also foroxygen reduction in alkaline media (Fig. 2.9d), where interestingly thelarger (24 nm) gold nanoparticles exhibited the lowest overpotential. This is

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in contrast to observations based on gold nanoparticle ensembles and mayrelate to the specific surface faceting of the immobilized particle.92 In anycase, the characterisation of the oxygen reduction response at the singlenanoparticle level represents a significant step forward in the context ofunderstanding electrocatalytic processes, particularly those relating to en-ergy conversion technologies such as fuel cells, and paves the way tounderstanding a variety of inner sphere reactions with a wide range ofpotential applications.

More recently, Sun’s group has also made progress in the area of singlenanoparticle-modified electrodes using alternative fabrication methods.93,94

One approach was to use electrochemically-assisted attachment of a singleor multiple gold nanoparticles to a platinum nanodisk electrode from astabilised colloidal suspension.93 Repeated scanning of the platinum elec-trode potential between � 0.1 and 1.2 V was found to result in the emer-gence of a gold oxide stripping peak attributed to the electrostatically drivencapture of a gold nanoparticle with a radius in the range 3–10 nm from thecolloid. The authors reported an interested phenomenon in which the goldoxide stripping peak potential became more negative and the gold oxidationpeak potential became more positive with decreasing nanoparticle radius.Although these results were preliminary, the observations may be related to

Fig. 9 (a) Graphical representation and (b) TEM image of SNPE. (c) Voltammetric responsefor the single electron reduction of 5.0 mMK3Fe(CN)6 in a 0.2 M KCl solution using a 9 nm Ptelectrode: bare Pt electrode (black), APTMS-modified Pt electrode (red), and Au SNPE (green).(d) Voltammetry in O2-saturated 0.1 M KOH solution using bare 7 nm diameter Pt nano-electrode (black), a 14 nm Au SNPE (red), an 18 nm Au SNPE (green), and a 24 nm Au SNPE(blue) (all scan rates 10 mV s�1). Adapted from ref. 91 with permission from the & AmericanChemical Society.

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the various surface facets exposed at different nanoparticle sizes and theirrelative stability with respect to oxidation. Of course, since the underlyingplatinum electrode was potentially exposed in this work, the study waslimited to investigating gold surface/oxide processes, as distinct fromFaradaic electron transfer with solution phase species.

Nanoparticle electrodes produced from colloidal suspensions in themethods discussed above have the added complication of a stabilisingmonolayer (e.g. of citrate) adsorbed on the surface of the nanoparticlewhich unless removed will likely effect molecular adsorption and innersphere electron transfer processes. In a later paper by Sun’s group the be-haviour of ‘‘naked’’ individual gold nanoparticles attached to platinumnanodisk electrodes was investigated.94 This was achieved simply by thespontaneous electroless deposition of a gold nanoparticle directly onto theplatinum by rapid exposure (<0.5 s) to a solution of 1% HAuCl4. Goldnanoparticle radii in the range 3–15 nm were studied by cyclic voltammetryin sulphuric acid solution and the position of the gold oxide stripping peakwas again observed to shift to more positive potentials with decreasingnanoparticle radius. It was noted that the nanoparticle growth mechanismpresents the possibility of alloying between the gold and the underlyingplatinum electrode, which potentially adds to the stability of the grownparticle as compared to assembly-based methods.

The application of nanoelectrodes to fundamental investigations is notlimited to encapsulated nanodisk electrodes, but extends to other electrodegeometries. In particular, nanowire electrodes are finding increased atten-tion, which has again been driven, by and large, by advancements in fab-rication methodologies. For example, Li et al. produced single platinumnanowire electrodes by chemically etching away the silica surround of ananodisk electrode (see Fig. 2.10a), and made some interesting observationsregarding the effect of nanowire length on outer sphere (ferrocene oxi-dation) and inner sphere (oxygen reduction) voltammetry.95 For a 4 nmradius nanowire, the half wave potential, for the electrochemical processwas found to shift to more positive potentials for ferrocene oxidation and to

Fig. 10 (a) TEM image of 40 nm long platinum nanowire with radius of B6 nm. (b) Oxygenreduction voltammetry in an oxygen saturated 0.1 M KOH solution observed at 4 nm radiusplatinum disk and different length platinum nanowires (scan rate 10 mV s�1). Current scale hasbeen normalised with respect to the limiting current for each nanowire length. Reproducedfrom ref. 95 with permission from the & American Chemical Society.

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more negative potentials for oxygen reduction with increasing nanowirelength in the range 0–950 nm (Fig. 2.10b). By itself this observation might beattributed to mass transport effects, but the fact that the magnitude of theoverpotential shift was notably larger for oxygen reduction than for fer-rocene oxidation, coupled with subtle differences in the normalised limitingcurrent densities, suggests that this was in fact more likely an electro-catalytic phenomenon. The authors highlighted that these observations areconsistent with the apex of the platinum nanowire electrode having a higherelectrocatalytic activity towards oxygen reduction than the sidewalls, whichitself may arise from the influence of variable surface stress or atomic ar-rangements at the different nanowire positions.

A unique approach to investigating heterogeneous kinetics at nanowireelectrodes was presented by Unwin’s group, who used the mircocapillaryelectrochemical method (MCEM) to undertake voltammetry at individualmetal nanowires with diameters as narrow as 32 nm.96 The nanowires werefabricated using templated electrochemical deposition of gold, platinumand palladium onto addressable single walled carbon nanotubes grown onsilicon wafers. In the case of polycrystalline gold nanowires, a micro-capillary containing a reference/counter electrode (chlorinated silver) waspositioned such that the solution meniscus contacted only a single nanowireon an otherwise dry substrate, enabling the formation of a confined elec-trochemical cell within which voltammetric measurements could be under-taken. Aqueous redox systems investigated included outer sphere(ferrocyanide and the (trimethylammonium)methylferrocene cation,FcTMAþ) and inner sphere (hydrazine) oxidations. Simulated voltammetrybased on Butler-Volmer kinetics was used to extract a heterogeneous rateconstant of 0.10� 0.03 cm s�1 for FcTMAþ oxidation. Interestingly, thesame analysis was undertaken for a bare SWNT and yielded a k0 of 2�1 cm s�1, consistent with other results determined using SECM,97 althoughsome deviation between model and experimental voltammetry were notedand attributed to double layer effects. Building on the MCEM technique,the same group have developed a more advanced method for undertakingelectrochemical measurements at the local scale called scanning electro-chemical cell microscopy (SECCM).98 In a recent study, the electrocatalyticbehaviour of platinum nanoparticles with diameters of the order of 100 nm,immobilized as an ensemble, was investigated.99 Electrocatalytic activitywith respect to oxygen reduction and hydrogen evolution was probed at thesingle particle level and revealed that in some cases similar sized particlesexhibited dramatically different activities. This observation, although notentirely unexpected, stresses the importance of factors other than particlesize such as surface faceting and substrate interactions. Moreover, the workhighlights how the ability to study electrochemical response at individualnanoparticles can offer new insights into nanoelectrochemical phenomena.

The study of fundamental electron transfer processes at nanoelectrodeshas also been extended to the field of bioelectrochemistry, notably in theelucidation of enzyme electron transfer kinetics and mechanism via proteinfilm voltammetry. This typically involves immobilizing a film of redox activeenzymes onto an electrode such that electronic contact is achieved betweenthe enzyme active site and the underlying surface, enabling voltammetry to

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be undertaken. Until recently, protein film experiments have been confinedto relatively large ensembles of enzyme molecules grafted to an electrode.However, as with nanoparticle electrocatalysis, there is a significant thrusttowards single enzyme measurements,100 not least because of the complexityof conformational variations that typically accompany redox processes inproteins, and become averaged in the case of ensemble experiments. Hoebenet al., took a significant step towards scaling down such enzyme measure-ments by employing individual nanoelectrodes as the substrate for proteinfilm voltammetry.101 Gold nanoelectrodes, which had dimensions as smallas 70� 70 nm2, were fabricated on silicon substrates by EBL and werepretreated with an electrode modifier, polymyxin, before immobilization ofa submonolayer of the redox active enzyme. The enzyme selected for in-vestigation was an [NiFe] hydrogenase, which can reversibly oxidize mo-lecular hydrogen and reduce protons, with a catalytic activity comparable toa platinum metal catalyst. Although significant challenges were en-countered, proton reduction/hydrogen oxidation voltammetry at the im-mobilized protein was successfully achieved at this scale. Based on theturnover currents measured, it was estimated that in the region of 8–46active enzyme molecules contributed to the voltammetric response, which isbelieved to be the smallest number detected thus far by this technique.

2.4.2 Nanoelectrodes for electroanalysis

The discussion thus far on the fundamental applications of nanoelectrodeshas focused mainly on individual nanoelectrodes. In contrast, the appli-cation of nanoelectrodes to the detection and determination of trace ana-lytes typically requires multiple electrodes distributed in an array orensemble format. In theory, this allows the high mass transport and lowcapacitive charging associated with nanoelectrodes to be exploited withoutthe need for low (pA) current measurement. The many practical advantagesof nanoelectrode arrays were demonstrated in a recent paper by Freemanand co-workers, who undertook a comparison between a 50 mm radiusplatinum disk and a square array of 50 nm nanobands of comparable totalelectrode area (Fig. 2.11a).102 The authors highlighted that not only cannanoelectrode arrays offer a two order of magnitude enhancement in masstransport limited currents, itself leading to improved access to rapid elec-trokinetic parameters, but also a three orders of magnitude lowering in thelimit of detection and a reduced susceptibility to convective effects. Thiswork also emphasized the danger of diffusional overlap, the theoreticalbasis of which was introduced in Section 2. As with microelectrode arrays,in order to realise the high mass transport advantages of nanoelectrodearrays in practice, the issue of overlapping diffusion fields between adjacentelectrodes and the impact of inter-electrode spacing and experimentaltimescales are critical considerations.

Examples of this are presented in Fig 2.11 for the square nanoband array,which shows cyclic voltammetry for the oxidation of ferrocenecarboxylicacid at scan rates of 10 mV s�1 (Fig. 2.11b) and 10 V s�1 (Fig. 2.11c).Notably, even at moderately fast scan rates, capacitive charging is minimal,but more importantly there is subtle evidence of peak-shaped voltammetryat the slowest scan rate, indicating diffusional overlap between

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neighbouring nanoband apertures (in this case separated by 90 mm) on thistimescale. A simple calculation based on Einstein’s relation for the diffusionlength (OpDt) indicates that for this system diffusional overlap can occur inless than 5 s, in keeping with this observation. A more thorough and sys-tematic experimental study of the effect of array dimensions on electro-analytical performance was also presented more recently by theseauthors.103

Having discussed the many practical issues related to electroanalysis atnanoelectrode arrays in general, we now turn our attention to some specificexamples that have emerged recently and demonstrate this application.

2.4.2.1 Detection of non-biological analytes. The advantages of usingnanostructured electrode surfaces for electroanalytical detection strategieshave long been recognised, and have commonly been exploited via thecombination of metal nanoparticle-modified electrodes with stripping an-alysis. Although such nanoparticle decorated surfaces do essentially con-stitute nanoelectrode arrays or ensembles, extensive research has beenundertaken in this particular area and so these will not be discussed in anydepth in this chapter. For a more detailed account the interested reader isdirected to several reviews of this field.104–107 Here, we will instead focus onordered and templated nanoelectrode architectures and highlight somespecific examples of their use in trace the detection of inorganic and non-biological analytes.

The determination of arsenic (III) in water is of great importance andelectroanalytical approaches have received significant attention.108 In thiscontext, a number of works have demonstrated the utility of gold

Fig. 11 (a) Schematic depiction of nanoband array, comprising 30 mm� 30 mm apertures, eachcontaining a 50 nm Pt nanoband electrode around its internal perimeter. Cyclic voltammetryfor the oxidation of 1.6 mM ferrocenecarboxylic acid at a 42� 42 array of nanobands at scanrates of (b) 10 mV s�1 and (c) 10 V s�1. Reproduced from ref. 102 with permission from theRoyal Society of Chemistry.

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nanoparticle ensembles, for example deposited on ITO109 and carbon110,111

electrodes, as a sensing platform. Building on this, Ugo and co-workersused templated nanoelectrode ensembles for arsenic detection.112 Ensemblesof gold disks were prepared by electroless plating onto track-etched poly-carbonate membranes and were characterised using cyclic and square waveanodic stripping voltammetry. As in earlier reports, As(III) was determinedvia direct reduction and stripping, whilst As(V) was first chemically reducedto As(III) using cysteine. The limit of detection (0.005 mg/L) and sensitivity(65.6 mA/mg L�1) of this approached compared well with other nanos-tructured gold electrodes, with the advantage that the templated growthapproach allows for a more controllable and reproducible fabrication ofnanoelectrode ensembles. A similar method was also adopted for the se-lective determination of lead, achieved via the modification of the goldnanoelectrode ensemble with bismuth.113

Trace analysis using nanoelectrodes is not limited to heavy metals but hasalso been applied to the determination of inorganic anions such as iodide114

and nitrite.115 More recently, the application of discrete nanowire arrays hasalso been extended to the sensitive detection of nitroaromatic explosives.116

Gold nanowire arrays were fabricated on silicon chips using the procedureoutlined in Section 3.1 (Fig. 2.4) and square wave voltammetry wasundertaken in solutions of trinitrotoluene (TNT) and various analoguesincluding nitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene and 1,3-dini-trobenzene. Well-defined cathodic peaks were observed in the square wavevoltammetry corresponding to the nitro-group reduction and the peakpotential was found to be sensitive to the nitroaromatic compound,allowing for selective determination of TNT with sub-150 ng mL�1 limits ofdetection. Moreover, the repeatability of the response was found to be highacross numerous nanowire chips.

2.4.2.2 Biosensing and bioelectrochemistry. The overwhelming majorityof electroanalytical applications of nanoelectrodes fall under the umbrellaof biosensing. As with the detection of trace inorganic analytes, the use ofimmobilized metal nanoparticles is ubiquitous in the world of bioelec-trochemistry and many reviews have emerged that summarise the variousdevelopments.117–121 Therefore in this section we will limit our discussion tothe major recent advances in biomolecule determination, again focusing onmore structured nanoelectrode assemblies.

Of the many bioanalytical fields impacted by nanoelectrochemistry, glu-cose detection continues to receive by far the most attention, predominantlydue to its relevance to the management of diabetes, and the associateddemand for miniaturised point-of-care diagnostics. A multitude of elec-trochemical glucose detection strategies have been proposed, many of whichare based on the enzymatic aerobic oxidation of glucose, catalysed byglucose oxidase, GOx:122

GlucoseþO2 �!GOx

Gluconic AcidþH2O2

Amperometric measurement of the hydrogen peroxide generated orthe oxygen consumed gives a direct means of glucose quantification,

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but more preferable is the use of a redox active mediator species to rapidlyshuttle electrons between the enzyme and the electrode, thus subvertingthe need for high overpotentials and avoiding interference problems asso-ciated with the direct approach. Generically this detection scheme can bewritten as:

GlucoseþGOxox þO2 ! Gluconic AcidþGOxred

GOxred þMox ! GOxox þMred

Mred !Mox þ e� electrodeð Þ

The GOx and mediator, M, may be anchored to the surface or free insolution. Since this is a catalytic cycle, significant enhancement factors areobserved in the current measurement at the electrode which, combined withthe advantageous properties of nanoelectrodes and their arrays (high masstransport, fast electron transfer, low capacitance) leads to a highly sensitivedetection technique.

O’Riordan’s group made use of single, 100 nm wide, gold nanowireelectrodes for this purpose.123 Using ferrocenecarboxylic acid as a mediator,the nanowire electrodes exhibited excellent voltammetry for mediated glu-cose oxidation across the concentration range 10 mM–100 mM (Fig. 2.12aand b). A particularly high sensitivity of 7.2 mA mM�1 cm�2 was observedin the lower concentration range and a limit of detection of 3 mM was de-termined. Furthermore, the nanowire sensor was demonstrated to be highlyselective, performing well in the presence of interfering oxidizable speciessuch as uric acid, ascorbic acid and other sugars.

One drawback of the above single nanowire biosensing is in the com-plexity of the electrode fabrication procedure. Simpler enzymatic ap-proaches have also been examined recently, such as the directelectrochemical detection of hydrogen peroxide generated by glucose oxi-dation, using templated metal nanoelectrode arrays. Claussen andco-workers used porous anodic alumina supported on a silicon chip for the

Fig. 12 (a) Cyclic voltammetry (scan rate 10 mV s�1) at a single nanowire electrode in a so-lution containing 10 mM ferrocenecarboxylic acid, 1mg mL�1 glucose oxidase and 10 mMphosphate buffered saline (pH 7.4), with various concentrations of glucose in the range1–100 mM. (b) Calibration plot showing background corrected limiting current densitiesas a function of glucose concentration. Reproduced from ref. 123 with permission from theRoyal Society of Chemistry.

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controlled growth of gold nanorods, only the tips of which were exposed,124

in a similar fashion to the track-etched polycarbonate template methoddiscussed above in the context of arsenic detection.112 Polycarbonate can beunsuitable for biosensing applications because of their reactivity with someproteins, and hence porous anodic alumina was used as a biocompatiblealternative. Rather than having GOx dissolved in the solution phase, theenzyme was instead immobilized onto the gold nanoelectrodes by means ofa monolayer of linker molecule, self-assembled onto the gold surface. Bothshort and long-chain alkanethiol molecules were tested as conjugates in anattempt to control the biosensor performance. The long chain linker mol-ecules were found to significantly impede the heterogeneous electrontransfer between the hydrogen peroxide generated and the gold nanoelec-trode surface. The best results were obtained with the shortest alkanethiolmonolayer (3-mercaptopropionic acid), and although these assembliesyielded higher limits of detection than the nanowire example highlightedabove (100 mM), the ease of fabrication gives this approach an advantage.The significant difference in performance associated with the two methodscan most likely be attributed to the large signal enhancement conferred bythe catalytic redox cycle of the mediated approach.

Another simple approach to glucose detection was recently proposed byTsai and co-workers, who employed atomic force microscopy (AFM) lith-ography to score nanochannels in a poly(methyl methacrylate)(PMMA) filmcoated onto a platinum substrate.125 Subsequent electrodeposition of theGOx enzyme into the nanochannels resulted in the growth of horizontal GOxnanorods, with heights as small as 80 nm. The analytical figures of merit forthis approach, which was tested for 25� 8 mm long channels, did not competewith the above examples, but the sensor is yet to be optimised and the sim-plicity of the approach makes it accessible to any research laboratory.

Hydrogen peroxide sensing itself finds applications not only in glucosedetection but also more broadly in oxidase based biosensing, and so someattention has been given to more generic electrochemical means of hydrogenperoxide quantification. In a recent example, Lupu et al. developed ananoelectrode sensor array for hydrogen peroxide determination usingnanosphere lithography.126 The electrode fabrication process begins with apolycrystalline gold surface which is coated with a self-assembled layer ofpolystyrene nanobeads distributed in a hexagonal arrangement. The beadsare etched using oxygen plasma and the resulting smaller beads act a maskfor silicon oxide chemical vapour deposition. Subsequent removal of thebeads yields an ordered gold recessed nanodisk array (Fig. 2.13a) which inthis case was modified by electrodepositing polypyrrole and Prussian Blue.Prussian Blue is known to effectively catalyse the electrochemical reductionof hydrogen peroxide,127 and so in this work was co-deposited with theconducting polymer, polypyrrole, in the form of nanopillars, thus facili-tating hydrogen peroxide detection at low overpotentials. The voltammo-gram in Fig. 2.13b demonstrates the response of the immobilized PrussianBlue nanoarray in the absence and presence of hydrogen peroxide, in whicha typical electrocatalytic response is observed.

A detection limit of 10�9 M was achieved using this nanoelectrode sensor,with a linear range of up to 10�4 M. Although this range is not as large as

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that demonstrated in earlier work using Prussian Blue-modified nano-structured electrode surfaces,128 an order of magnitude improvement in thelimit of detection may stem from the more reproducibly controlled nano-electrode assembly offered by the nanosphere lithography approach.

Moving on from glucose and hydrogen peroxide sensing, nanoelectrodeshave also been used for immunosensing and DNA recognition applications.Rauf and co-workers recently adopted a ‘‘drill and fill’’ approach to label-free immunosensing, exploiting FIB-milled recessed nanodisk arrays inwhich the pores were filled by electrodepositing platinum (see Section3.1).39,52 The electrodes were rendered immunosensitive to the HER-2(human epidermal growth factor receptor-2) antigen by first assembling amonolayer of mercaptohexanol/mercaptohexanoic acid followed by thecovalent attachment of the appropriate antibody, anti-HER-2. Specificbinding of the target antigen was monitored electrochemically using im-pedance spectroscopy and ferricyanide as a probe for interfacial electrontransfer resistance. Upon exposure of the antibody-modified electrode tothe antigen, the charge transfer resistance was found to measurably increasedue to the blocking effect caused by the binding of the antigen to the sur-face. A small difference in the Nyquist plot was observed in the presence of100 pg mL�1 antigen and, although some issues relating to non-selectivitywere observed, the nanoelectrode platform provides a promising directionfor immunosensing. A similar approach was also adopted by Viswanathanand co-workers for the detection the epithelial ovarian cancer antigen-125, aprotein biomarker linked to ovarian cancer.129

Other examples of immunosensing using nanoelectrodes have made useof a two-antibody assay format. In this approach, the recognition element(i.e. the antibody) is immobilised on the electrode or surrounding sensorsurface and binds the target antigen as above, but then a secondary anti-body containing a label moiety is introduced and binds also to the capturedantigen. Release of a redox active molecule from the label can then be in-duced, which is detected electrochemically at the electrode surface. Mucelliand co-workers,130 and later Kelly et al.,131 demonstrated the application ofthis approach using similar gold nanoelectrode array platforms. Mucelli

Fig. 13 (a) AFM image of recessed nanodisk array fabricated by nanosphere lithography.(b) Cyclic voltammetry of Prussian Blue/polypyrrole modified nanodisk array in 0.1 M PBS(pH 7) in the absence and presence of 1 mM hydrogen peroxide. Adapted from ref. 126 withpermission from Elsevier.

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used horseradish peroxidase as a label for the detection of trace HER-2antigen, whilst Kelly employed a biotin label in conjunction with silvermetal stripping for the analysis of prostate specific antigen, both studieshighlighting the remarkable efficacy of the nanoarray methodology.

Some of the themes discussed in the context of immunosensing also applywidely to the use of nanoelectrodes for DNA sensing. Ramulu and co-workers fabricated arrays of free standing, vertically aligned gold nanowiresfor the detection of DNA binding events.132 Single stranded DNA probeswere grafted to the gold nanowires and incubated with various target DNAstrands, (complementary, single-mismatched and non-complementary) andmethylene blue was used as a redox active intercalator to probe the level ofhybridisation, measured by differential pulse voltammetry. A low concen-tration of target DNA could be detected (6.78 nM), which was a factor oftwo improvement on that for an equivalent macroscopic gold electrode. Asin the immunosensing application, these authors also used electrochemicalimpedance spectroscopy to measure the increase in electron transfer re-sistance between the electrode and solution phase ferricyanide resultingfrom electrode blocking due to monolayer assembly and target binding. Analternative use of electrochemical impedance spectroscopy to study DNAbinding events was explored by Bonanni et al., who used an interdigitatednanoband electrode array to measure the double layer capacitance of aprobe DNA monolayer under exposure to various target strands.133 Goldnanoparticle labelling was employed for signal amplification, and statistic-ally significant differences in capacitance were observed between the mutanttarget DNA strand and non-complementary and wild-type samples.

2.4.3 Nanoelectrodes for electrochemical imaging

A unique advantage of small electrodes that has not been alluded to in anydepth so far in this chapter is their suitability for electrochemical imagingapplications. The combination of electrochemical measurement with vari-ous forms of scanning probe microscopy (SPM) via the precise positioningof an electrode probe is well established as a means to undertake highlylocalised analysis of solid-liquid interfaces and the associated heterogeneouselectron transfer processes. The lateral imaging and electrochemical reso-lution of such techniques are controlled largely by the probe dimensions,and so a substantial focus in this area continues to be in the developmentnanoscale electrodes and innovative approaches to controlling their pos-ition with respect to the surface. In this section we review the latest en-deavours to push the frontiers of electrochemical microscopy via theexploitation of novel nanoelectrode probes.

2.4.3.1 SECM. In conventional amperometric SECM an electro-chemical probe, typically a glass-encapsulated microdisk electrode, isbrought into close proximity to the interface of interest, and electrochemicalreactions are driven in the small gap between the tip and the surface.Current can be measured at the tip and/or at the substrate and the responseis highly sensitive to both the nature of the surface and the tip-surfaceseparation. There are various modes of SECM, such as feedback mode,generation-collection mode and redox competition mode, each of which can

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glean different levels of information about the surface. What lends SECMits remarkable efficacy is its unique ability to yield insights into local surfacechemistry, and various applications have emerged.134–136

As we have seen in Section 3.3, the fabrication of glass-encapsulated wirenanoelectrodes with exposed disk radii in the tens to hundreds of nano-metres is now considered routine in some laboratories. However, SECMtypically requires the tip-surface separation to be of the order of the elec-trochemical probe dimensions, and hence the application of such nano-electrodes to SECM requires a means to approach the surface within a fewhundreds of nanometres. For imaging applications, this challenge can beaddressed by introducing a mechanism for the probe to feedback off thesurface topography in order to perform a constant distance measurement(vide infra), but constant height SECM imaging is also possible withnanoscale resolution provided the sample is sufficiently flat and levelledacross the imaging range. Alternatively, for inert substrates, the applicationof constant-current mode facilitates the tracking of surface topography.Laforge et al. presented some examples of nanoscale SECM using a plat-inum tip with a radius of 140 nm, including compact disks and silicon wafermicrocircuits.137 Surface features as small as 200 nm were easily resolvedand in the case of purely insulating substrates, SECM maps across a rangeof several square microns could simply be transformed into topographicalimages by comparison with theory. In the case of electrochemically activesubstrates, variations in the surface activity could to some extent be de-lineated by comparison with AFM imaging, but constant height SECMimaging by itself suffers the limitation of a convoluted topographical-activityresponse. However, for substrates with negligible topography, constantheight SECM can provide a means to map variations in activity caused bylocal electrochemical stimuli, as demonstrated by Matysik’s group formonolayers of rat kidney epithelial cells immobilised on a flat silicon dioxidesubstrate, using platinum tips with radii in the 100–600 mm range.138

Mirkin’s group also exploited nanoelectrodes for constant height SECMimaging of single mammalian cells and expanded this application to probingintracellular electrochemistry.139 By employing ultra-sharp nanoelectrodes,the membrane of human breast epithelial cells could be pierced with min-imal damage, allowing redox processes within the cell to be examined. Forexample, using ferrocenemethanol as a redox probe, the potential dropacross the cell membrane was determined and the effect of cell depolar-ization upon exposure to valinomycin observed. Furthermore, mass andcharge transport rates across the cell membrane were investigated bymeasuring approach curves using menadione as a mediator, and permea-bility coefficients of the order of 0.15 cm s�1 were determined. More re-cently, this work has been extended to the intracellular detection of harmfulreactive oxygen and nitrogen species within macrophages, cells essential forthe performance of the immune system.60

Returning to the imaging application of SECM, for substrates with asignificant degree of topographical or (electrro)chemical non-uniformity,the use of nanoelectrodes for high resolution electrochemical mappingcomes with the requirement for constant-distance scanning in order todeconvolute variations in activity from topographical undulations. Various

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approaches have been proposed to introduce positional feedback, which fallbroadly into two categories: (i) the integration of a surface detectionmechanism into conventional or modified glass-encapsulated nanoelec-trode, such as shear force or voltage switching feedback and (ii) the inte-gration of a nanoelectrode onto probes for an existing topographicallysensitive SPM technique, such as AFM or scanning ion conductance mi-croscopy (SICM). Recent advances in each of these methods will now beconsidered.

2.4.3.2 Shear force SECM. In the shear force feedback approach, aglass-encapsulated electrode probe is oscillated laterally close to the sub-strate of interest ({1 mm), and the physical attenuation of the oscillation asit approaches the surface is monitored and used to define a set point forpositional control. This can be achieved by various means, such as tuningforks or optical methods, but the first application of shear force detection toSECM probes with nanoscopic dimensions was demonstrated by Schuh-mann’s group,140 who used a piezoelectric sensor to gauge the vibrationaldampening. The approach typically requires the fabrication of fine needle-like disk nanoelectrodes, but more recently this group has also exploited theshear force response itself to assist in the electrode fabrication process (seeSection 3.3). A number of recent applications making use of shear force-controlled nanoelectrodes for SECM have emerged, in particular in theimaging of single living cells.141 An excellent example of this is in the workof Matsue’s group, who devised an elegant means to combine nanoelec-trochemical mapping with fluorescence imaging using a novel shear force-regulated probe.142 The probes consisted of a pulled optical fiber, with anend radius of the order of 40 nm, coated with a B75 nm film of platinummetal followed by a B200 nm insulating layer of Parylene. Using FIBmilling, exposure of the apex yielded a platinum nanoring electrode with anoptical fiber at its core (Fig. 2.14a). The probes exhibited sensible steadystate voltammetry for ferrocenemethanol oxidation (Fig. 2.14b), butmoreover provided a unique means to simultaneously monitor differentprocesses taking place inside and outside of individual living HeLa cells (atype of human cell line commonly employed in laboratories) with topo-graphical imaging provided by shear force detection.

Two types of reporter proteins were used as a measure of activity, greenfluorescent protein (GFP), which responds within the cell to local illumin-ation via the optical fiber, and secreted alkaline phosphatase (SEAP), whichcatalyses the hydrolysis of p-aminophenyl phosphate to generate p-amino-phenol outside of the cell, which can be oxidized electrochemically at thenanoring electrode, in a substrate generation tip collection experiment.Single cell images are shown in Fig. 2.14, which depicts the topographicalresponse determined from the shear force feedback (Fig. 2.14c), the intra-cellular fluorescence response excited by the optical fiber (Fig. 2.14d) andthe extracellular response measured by the nanoring electrode (Fig. 2.14e).By making this triple measurement, the authors were able to confidentlylocate the optical fiber electrode close to the cells but moreover verify thecellular activity with two very different, but in many ways complementarydetection strategies.

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Another use of shear force controlled nanoelectrodes was presented byMezour and co-workers, who measured the hydrogen peroxide producedduring oxygen reduction reaction at immobilized cobalt-porphyrin elec-trocatalysts.143 Porphyrin monolayers were formed via thiol self-assemblyonto gold electrodes and a substrate generation tip collection experimentwas performed using a 230 nm radius platinum disk electrode positioned byshear force feedback. By comparison with theory, a heterogeneous rateconstant for the 2 electron reduction of oxygen at the porphyrin catalystwas estimated. The authors noted that one advantage of using nanometredimension SECM probes is that, unlike larger probes, they may not ne-cessarily require numerical simulation for quantitative interpretation sincephysical perturbation of the solution due to the presence of the probe itselfis minimised. In a more recent study, Dincer et al. explored the applicationof phase-operated shear force detection to image the electrochemical ac-tivity of BDD nanodisk arrays.144 This positional feedback approach en-abled SECM at a miniscule working distance of 45 nm, facilitatingelectrochemical imaging of a single 160 nm radius BDD disk, with a lateralresolution of the order of 100 nm.

2.4.3.3 SECM-AFM. The combination of SECM with AFM is one ofthe most attractive methods of achieving electrochemical imaging withnanoscale resolution. The integration of a nanoelectrode probe onto an AFM

Fig. 14 SEM image of optical fibre nanoring electrode (a) and cyclic voltammogram for theoxidation of 0.5 M ferrocenemethanol. Simultaneous topographic (c), fluorescence (d) andelectrochemical (e) images of a single HeLa cell measured in shear force-regulated constantdistance mode. Reproduced from ref. 142 with permission from the & American ChemicalSociety.

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cantilever promises a route to electrochemical mapping alongside highresolution topographical imaging. New approaches to SECM-AFM probedesign continue to appear in the academic literature, although a reliablecommercial probe is yet to emerge. A major challenge is improving the re-liability and durability of the probe insulation, in order to ensure that allcurrent contributions are confined specifically to the electrode region of theprobe, without compromising the nanoscale probe dimensions required forhigh resolution topographical imaging. Recent approaches to SECM-AFMprobe construction have involved either bottom-up production using modernmicrofabrication technologies145,146 or modification of existing AFM probesusing semiconductor processing techniques.147–150 In particular, probes basedon a pyramidal tip geometry have been developed and exploited by a numberof groups, typically via insulation of metal or carbon coated silicon AFMprobes, followed by exposure of a conical or disk-shaped nanoelectrode theapex using FIB milling, chemical etching or mechanical abrasion.145,147,148

High-aspect ratio SECM-AFM probes have also been developed recently, inwhich an addressable metallic needle is attached to the end of the AFM tipbefore insulation and FIB exposure to yield nanodisk or hemisphere elec-trodes with radii in the 50nm–200nm range.81,149,151

A unique approach to SECM-AFM measurements was proposed byAnne et al., who tethered the SECM redox mediator, in this case ferrocene,to the electrode using flexible polyethylene glycol chains in order to addressthe diffusional limitations associated with solution phase mediators inconventional SECM (Fig. 2.15a).152,153 This method, termed tip-attachedredox mediator (TARM) SECM-AFM, was shown to be suitable as ameans to probe the local electrochemical heterogeneity in planar substrateswhilst providing nanometre resolution topographical imaging using tappingmode. Example images are presented in Fig. 2.15, which depicts the topo-graphical response (Fig. 2.15b) at a highly ordered pyrolytic graphite

Fig. 15 (a) Schematic depiction of TARM probe. (b) Simultaneous topographical and(c) electrochemical images, including line profiles, of HOPG substrate. Tip biased at 0.3 V vs.SCE, substrate biased at 0.2 V. Reproduced from ref. 152 with permission from the &AmericanChemical Society.

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(HOPG) substrate alongside the electrochemical response recorded simul-taneously (Fig. 2.15c).

Step edges in the basal carbon surface are observed in the topographicalscan, which in some cases (but interestingly not all), exhibit enhanced ac-tivity with respect to the reduction of the oxidized ferrocene moiety tetheredto the tip, in keeping with current views on the relative activity of the edgevs. basal planes. Although the physical size of the tip itself was rather large(spherical radius B500 nm) the area probed by the tethered mediatormolecules was estimated to be confined to a disk of radius of the order of50 nm. Moreover, by removing the need for a solution phase redox medi-ator, the necessity for perfect, defect free insulation over the surroundingareas of the SECM-AFM probe is circumvented.

In many of the above examples, the topographical imaging point and theelectrode are either located at the same position, or in very close proximityto each other, which often necessitates the use of the dual pass Lift Modetscanning approach in order to undertake the electrochemical measurementat a controlled working distance. An alternative approach that continues tobe pioneered by Kranz’s group is to separate the topographical and elec-trochemical imaging locations on the probe by employing a recessed ringelectrode geometry, where a sharp contact point defines a fixed electrode-substrate separation, circumventing the need for the dual pass ap-proach.146,150 This method does come with the disadvantage that theelectrode itself has dimensions closer to the micron scale, such that elec-trochemical resolution is not as high as in some other approaches. However,as discussed in earlier sections, smaller is not always better when it comes atthe cost of less well defined geometry, and particularly in the case of SECM-AFM, less robust and reliable electrode insulation. It is noteworthy that inmany of the examples highlighted above, little and in some cases no elec-trochemical imaging data is presented. This is a testament to the challengingnature of the SECM-AFM approach. Nevertheless, the fact that new workscontinue to appear in the literature provides an indication as to how highly-sought this electrochemical imaging technique is.

2.4.3.4 SECM-SICM. SICM itself is an electrochemical imaging tech-nique that is, in theory, insensitive to variations in surface activity, and isused for topographical imaging of soft samples such as living cells. Thismethod employs a nanopipette filled with electrolyte solution and referenceelectrode, pulled to a fine aperture (>50 nm), through which an ionic cur-rent is driven. The ionic current varies as a function of the tip-substrateseparation, and hence can be used to define a set-point for positioningcontrol. A number of approaches have emerged in which a second Faradaicelectrode is integrated into this pipette assembly in order to simultaneouslyundertake SECM measurements with simultaneous SICM topographicalfeedback. One method, developed by Matsue’s group, is to coat a normalSICM barrel with an electrode material (e.g. gold), add a layer of insulation,and then use FIB to expose a ring electrode at the tip apex (Fig 2.16a).154

Pulled capillaries with aperture radii of 220 nm were typically fabricated andring electrodes with an inner radius of 330 nm and band thickness of 220 nmwere exposed (Fig 2.16b). In addition to preliminary image characterisation

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using a standard platinum microband array, the probes were used to mapthe activity of immobilized spots of the enzyme glucose oxidase in thepresence and absence of glucose, using ferrocenemethanol as a redox me-diator. Furthermore, the permeability of living cardiac myocyte cells wasinvestigated using a hopping mode scanning approach; whilst the cells werepermeable to ferrocenemethanol, ferrocyanide was observed to not cross thecell membrane, understandably due to its high charge density and hydro-philicity. Also, an increase in oxygen reduction current was observed inclose proximity to the cells, resulting from oxygen permeation from theintracellular space. Other examples of high resolution electrochemical im-aging of living cells can be found in a recent review.155

Hersam’s group adopted a similar approach to SECM-SICM probefabrication, but instead gold deposition was confined to one side of thepipette, generating a crescent electrode geometry upon FIB exposure(Fig. 2.16c and d).156 An advantage of this method is the smaller nanoe-lectrode dimensions (effective radius of 294 nm) allow for high resolutionelectrochemical imaging, and features as small as 180 nm could be dis-cerned. Morris and co-workers also developed half gold coated pipettes forSECM-SICM that, despite having significantly larger exposed electrodedimensions, were capable of imaging the permeation of redox probesthrough a nanoporous membrane substrate.157

An alternative route to fabricating SECM-SICM probes with smallerelectrode dimensions has been explored by Takahashi and co-workers, whodeveloped a simple procedure for making dual-barrelled probes from thetacapillaries.158 In this process, one of the barrels of a pulled capillary is filledwith carbon via the pyrolytic decomposition of a hydrocarbon gas feed,

Fig. 16 Schematic depictions and SEM images of nanoelectrode probes for SECM-SICM.(a) and (b) refer to the ring electrode geometry employed by Matsue’s group and (c) and(d) relate to the crescent electrode geometry used by Hersam’s group. Reproduced from refs.154 and 156 with permission from the & American Chemical Society.

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whilst the second barrel is left clear. By controlling the amount of carbondeposition, the result is a flat or hemispherical carbon nanoelectrode, withan effective radius as small as 50 nm, that can be employed for FaradaicSECM measurements, situated adjacent to an SICM aperture (also with a50 nm radius) to be employed for topographical feedback. The very smallprobe dimensions (total apex radius of the order of 100 nm) enables ex-ceptionally high resolution imaging, and this was demonstrated again byway of a number of different test samples, including platinum bands,nanoporous polyethylene terephthalate membranes and individual livingsensory neuron cells. The latter example is presented in Fig. 2.17, whichdepicts the topographical (Fig. 2.17a) and electrochemical (Fig. 2.17b) re-sponse of neuron cells in ferrocenemethanol solution, where the tall cellbodies and extending dendritic features are clearly resolved. The enhancedelectrochemical response over the cells is again attributed to the permea-bility of the cellular membrane to ferrocenemethanol. The authors were alsoable to use these probes to measure the local release of neurotransmittersfrom PC12 neuron cells. Cell depolarization was stimulated either via in-jection of a high potassium ion concentration using a second micropipette,or more elegantly, by electrochemically driving the flux of potassium ionsthrough the SICM barrel by applying a high bias. This second chemicaldelivery approach is a unique advantage of the SICM-coupled electro-chemical imaging techniques and has significant potential for the study oflocal cellular and related stimulated events.

2.4.3.5 Other constant distance imaging. Not all approaches to constantdistance SECM require the fabrication of specialist probes, but insteadmake use of electronic and position modulation. For example, Takahashiet al. introduced voltage-switching mode SECM in which the potential of asimple glass encapsulated nanodisk electrode is switched between two ex-tremes to perform two different electrochemical processes.159 At one ex-treme, the diffusion limited current due to oxidation or reduction of a redoxmediator (e.g. ruthenium hexamine) in solution is measured and the asso-ciated negative feedback is used to ascertain the surface topography. At thesecond voltage, the nanoelectrode probes the surface activity by collecting

Fig. 17 Simultaneous topographical (a) and electrochemical (b) imaging response of neuronalcells achieved using dual-barrelled theta capillary SECM-SICM probes. Electrochemicalresponse reflects the oxidation of 0.5 mM ferrocenemethanol at a tip potential at 0.5 V vs.Ag/AgCl. Reproduced from ref. 158 with permission from Wiley VCH.

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electrochemically active species formed locally in a substrate generation tipcollection experiment. The idea of using two different redox mediators forde-convoluting topography and activity is not new, but by combining theelectrode potential switch with a hopping movement (see Fig. 2.18a) andminimising the steady-state response time with the aid of small carbonelectrodes (with radii as small as 6.5 nm), this mode enables both to beundertaken within a single experiment. Whilst there may be some limi-tations associated with the need for multiple redox active species within thesame solution, subverting the requirement for complex probes or hardwaregives this approach a clear advantage.

As in many of the above examples, the value of this method was dem-onstrated by imaging living cells, in this case to visualise epidermal growthfactor receptors on the membrane of epidermoid carcinoma (A431) cells.The expression of this protein on the surface of cells is associated with thedevelopment of cancer, but monitoring this process is very difficult toachieve. As demonstrated earlier by this group, labelling the protein with analkaline phosphatase tag enables its electrochemical recognition, since it

Fig. 18 (a) Schematic depiction of VSM-SECM. (b) Simultaneous topographical (left) andelectrochemical (right) images of A431 cells. Topographical detection was achieved using ru-thenium hexamine reduction at a tip potential of � 0.5 V vs. Ag/AgCl whilst observation ofcellular activity was achieved by oxidation of liberated p-aminophenol, at a tip potential of0.35 V. Reproduced from ref. 159 with permission from The National Academy of Sciences.

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catalyses the generation of p-aminophenol from p-aminophenyl phosphate,which can be detected anodically at the tip. Fig 2.18b depicts topographicaland electrochemical images obtained in this way. The authors were also ableto detect neurotransmitter release from PC12 cells and undertake simul-taneous confocal fluorescence microscopy to monitor neuron activity.

Conclusions and outlook

The unique properties of nanoelectrodes present electrochemists with adistinctive challenge. On the one hand, the inherent benefits of nanoelec-trode devices are very clear, and we have seen a host of examples demon-strating the various associated advantages, such as enhanced mass transportand its impact on sensing and fundamental applications, and the use ofhighly localised measurement for imaging and cellular studies. On the otherhand, realising the true potential of these benefits requires us to further ourunderstanding of how interfacial processes operate on the nanoscale andbeyond. The successful application of nanoelectrodes relies on the solidfoundation provided by fundamental studies, which attempt to address thenumerous complications arising from electrode dimensions and diffusionfields becoming comparable in size to double layers and Debye lengths. Weare beginning to see more and more examples of the stochastic phenomenaassociated with nanoelectrodes, wherein individual molecular events can beresolved within an electrochemical measurement. As the scale of our elec-trochemical systems shrink, our ability to comfortably measure such smallcurrents will eventually be stretched, and so further advances in electronicswill begin to play a key role.

Progress within the field of electrochemistry at nanoelectrodes has beenfacilitated by a number of key advances. Notably, new fabrication tech-nologies have enabled much tighter control over nanoelectrode dimensionsand geometry, which has given experimentalists greater confidence in theinterpretation of electrochemical data. Similarly, this has allowed us tomore reliably compare experiment with theory, which itself continues todevelop through advances in computational methods and hardware cap-acity. Momentum in each of these areas is expected to continue to grow, andfundamentals, theory and applications will likely become increasingly in-tegrated, but the greatest advances in electrochemistry at nanoelectrodes areexpected to result from innovative approaches to experimental design.

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