Journal of Electroanalytical Chemistry 688 (2013) 20–31

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

Electroanalysis in micro- and nano-scales

Chang Ming Li a,b,c,d,⇑, Weihua Hu a,b

a Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, PR Chinab Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing 400715, PR Chinac School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, Singapored Center for Advanced Bionanosystems, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, Singapore

a r t i c l e i n f o

Article history:Available online 28 July 2012

Keywords:ElectroanalysisMicro-/nano-scales

1572-6657/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jelechem.2012.07.010

⇑ Corresponding author at: School of Chemical aNanyang Technological University, 70 Nanyang Drive,Tel.: +65 67904485; fax: +65 67911761.

E-mail addresses: ecmli@ntu.edu.sg, ecmli@swu.ed

a b s t r a c t

The tremendous advances in nanoscience and nanoengineering have sparked the accelerated develop-ment of electroanalysis towards highly sensitive and specific detections. In particular, novel nanomate-rials with unique physical and chemical properties have been synthesized for electroanalysis in micro-and nano-scales to qualitatively and/or quantitatively examine the changes of compositions and struc-tures for scientific insights and practical applications. In this review, we mainly survey our recent workson electroanalysis in micro- and nano-scales, which covers micro- and nano-fabrication techniques,microelectrodes and powder microelectrodes, direct electrochemistry of proteins, electrochemical anal-ysis of living cells, and electrochemical immunoassay, with emphasis on the performance improvementby engineered functional nanomaterials. These electroanalytical methods have demonstrated their abilityand reliability to study fundamental science and provide practical applications. It is our intention thatthis review could attract increasing interest to explore new electroanalytical methods in micro- andnano-scales and further develop broad important applications for critical chemical/biological sciences.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

The advances of nanoscience make the world getting smaller.Various critical natural events and their fundamental insights areoften discovered at micro- or nano-scales. In particular, all the bio-logical entities including cells, bacteria, fungus and viruses possessmicro- or nano-scaled dimensions, while the biological processessuch as signal transduction, intracellular trafficking, protein degra-dation and DNA repair often occur at micro- or/and nano-meterscales. The fundamental research explores the structures and prop-erties at nano-scales. Thus, innovative electroanalytical approachesin micro- and nano-scales are of ultimate importance not only invarious fundamental research areas, but also for broad practicalapplications spanning from homeland security, food safety, envi-ronmental monitoring, and health-care among others [1–10].

Electrochemical techniques have been extensively employeddue to high sensitivity, low cost, ease operation and good portabil-ity [2,11,12]. Coupled with specially designed sensing interfaces,electrochemical techniques could offer a wide diversity of possibil-ity in analytical science in micro- and nano-scales for more prom-inent advantages. The small probe electrode used in such an

ll rights reserved.

nd Biomedical Engineering,Singapore 637457, Singapore.

u.cn (C.M. Li).

analysis is able to reduce the capacitive response for significantimprovement of signal-to-noise (S/N) ratio, while offering highspatial resolution to observe the micro- and nano-scaled structuraldetails and high temporal resolution in studies of fast redox reac-tions. Further, it can enable invasive detection, leading to implant-able biosensors. However, the electroanalysis in micro- and nano-scales has great challenges. Advanced micro- and nano-fabricationtechnologies should be innovated to produce delicate miniaturizedprobes, and interface modification or/and nanoengineering shouldbe implemented to achieve high sensitivity and specificity. Funda-mentally, the mass transport and charge transfer at a micro- andnano-scaled interface should be different from the conventionalsized ones. New theoretical approaches to explain the results arevery necessary. In recent years, remarkable progress in electroanal-ysis at micro- and nano-scales has been achieved [1–14], in whichthe nano-scaled materials can be tailored with tunable dimensions,high specific surface areas and specific chemical properties for highelectroactivity to achieve improved sensitivity and specificity,while miniaturized electrodes or devices enable in situ electroanal-ysis of biological processes with high spatio-temporal resolution.

Our group has devoted to electroanalysis at micro- and nano-scales accompanying by the utilization of a wide variety of newlydeveloped functional nanomaterials in recent years, which isreviewed here with a particular emphasis on the performanceimprovement by engineered functional nanomaterials. Theseelectroanalytical methods have demonstrated their ability and

C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31 21

reliability to study fundamental science and provide practicalapplications.

2. Micro- and nano-fabrication for electroanalytical devices

The electroanalysis in micro- and nano-scales greatly relies ontechniques for micro- and nano-fabrication, of which the ‘‘hard’’and ‘‘soft’’ lithography are the two main approaches. ‘‘Hard’’ lithog-raphy is known as well-developed mass microfabrication technol-ogy in microelectronics and has been borrowed to fabricate MEMSs(Micro Electro Mechanical Systems) and miniaturized analyticaldevices for years. The materials to make analytical micro- ornano-devices such as microfluidics are varied depending on theapplications, and the majority is based on glass, silicon and plasticsubstrate. ‘‘Hard’’ lithography is actually the conventional one uti-lized in semiconductor industry. In fabrication of analytical de-vices, silicon and glass are used as the substrate through relevantwell-established microfabrication techniques such as patterning,etching, bonding and integration as well [15]. ‘‘Hard’’ lithographyhas been used by us to fabricate various electroanalytical micro-chips. A silicon array chip on an oxidized silicon substrate has awell structure with gold working electrodes located in the bottomof arrayed wells and square strip gold counter electrodes on thetop side of well walls for impedimetrically labelless detection ofDNA hybridization events, in which the identically designed anddelicately fabricated structure could provide low signal variationsfor a high S/N ratio [16]. The conventional lithography is oftenmodified to fabricate microchips for different electroanalyticalapplications. A unique microchip has been made by us on a glasssubstrate, in which dual ring microelectrodes are made with a pat-terned shadow mask to sequential sputtering of a thin film of Tita-nium (Ti) as an adhesion layer followed by gold layer as theelectrode [17]. A cavity centered between the two gold rings is fur-ther made by etching the glass substrate with hydrofluoric acid(HF) solution for immobilization of probe antibody and conductingof antibody–antigen (Ab–Ag) bio-affinity reactions, while the twoconcentric gold rings function as working and counter/referenceelectrode, respectively to electrochemically detect the bio-interac-tions. A micro-piezoelectric diaphragm based immunoassay chiphas been developed to simultaneously detect hepatitis B virus(HBV) and a-fetoprotein (AFP) [18]. The chip is fabricated bymicro-machining technology to have eight individual circular sen-sors with diameters of 800 lm for immobilization of Hepatitis Bsurface antigen (HBsAg), hepatitis C core antigen (HBcAg) andAFP as the probe molecules, respectively. After target antibodybinding, significant frequency shifts can be detected for sensitiveimmunoassay.

Soft lithography has been widely used to fabricate various ana-lytical micro- and nano-devices, with which we use elastomericstamps, molds, and conformable photomasks to pattern two- andthree-dimensional structures with feature sizes in the micrometeror/and nanometer regime for microarrays and microfluidic devices[17,19–28]. The technique offers a simple and inexpensive processto mass-manufacture micro- and nano-devices for a broad range ofanalytical applications. In biomedical applications, diagnostic de-vices directly contacting patient samples are usually disposable.At this point of view, the advantage of devices made by the lowcost soft lithography over the conventional ones is obvious. Micro-fluidic devices, lab-on-a-chip system with integrated laboratoryfunctions on a single chip have been greatly made by the softlithography for high-throughput capability, excellent detectionsimplicity and low manufacturing cost since its introduction inthe early 1990s [20,22,24–26]. A micro-immunoassay device hasbeen fabricated by such an approach not only to simply producea three-dimensional patterned structure, but also to simulta-

neously introduce functional epoxy groups for efficient proteinimmobilization, in which poly (dimethylsiloxane) (PDMS) replicasmade from a patterned silicon master are used as the molds to castthe device by completely filling a UV-curable resin epoxy/acrylatemixture, followed by removing the mold and curing the epoxy [20].After attachment of the probe proteins, the device demonstrateshigh-throughput, good selectivity and high sensitivity for immuno-assays. A facile approach is further developed by flow-throughfunctionalization of molded PDMS microfluidic channels to fabri-cate a lab-on-a-chip device, rendering a method with very low fab-rication cost but high performance [22].

To fabricate electroanalytical devices with soft lithography, it iscritical to develop an economic and reliable process to transfermetallic patterns on a substrate as microelectrodes and furtherfabricate three-dimensional structure for microfluidic systems.Intensive research and development have been devoted to pattern-ing micro- and nano-electrodes and microfluidic structures in ourlab. Printing techniques with inkjet, screen and microcontact(lCP) are often used to fabricate patterned small electrodes andlab-on-a chip [29]. An electrochemical microfluidic immunoassaydevice has been made by thick screen printing techniques, asshown in Fig. 1 [23]. In the fabrication, a commercially availablecarbon paste is first printed on a polyester board to pattern work-ing microelectrodes array and conducting wires by screen-printingwith a patterned mesh stencil. After curing, Ag/AgCl paste is fur-ther printed and dried as counter/reference electrode. Subse-quently, a UV curable dielectric paste is printed and exposed toUV light to cure as an insulating layer for isolating the conductingwires from the solution in the channels. The dielectric layer isrepeatedly printed and dried to produce microfluidic channelswith the designed depth and a prefabricated polymer cover withinlet and outlet ports is pressed on the top of the microfluidicchannels immediately after printing the last dielectric layer but be-fore curing to complete a lab-on-a chip device for sensitive elec-tronic immunoassay.

The screen-printing approach is especially suitable for massproduction of disposable, portable devices due to its low cost man-ufacturing process. However, it can only fabricate micron-resolu-tion structures up to date. The inkjet printing has accomplishedsubmicron scale resolution [30], but needs post-sintering at hightemperature to remove organics introduced from the ink to in-crease the electrode conductivity. Nanotransfer printing (nTP) of-fers a better resolution (�100 nm) comparing to lCP [31], whichutilizes the surface chemistry or non-covalent forces between thereceiving surface and transferred metal to produce high resolutionelectrodes. We have devoted to the research and development offacile and efficient ultraviolet (UV) transfer embossing processesat room temperature for patterned micro- or/and submicron-elec-trodes [32–37]. The UV transfer embossing is fast and has no needfor clean room to achieve small micron- and even nano-resolution[38]. Although the embossing template is fabricated by theconventional lithography, it can be used repeatedly under lessstringent conditions. In addition, spatially controlled oxygeninhibition of acrylate photopolymerization as a new lithographymethod to pattern high performance microelectrodes [32] andadhesive-free transfer of metal electrode patterns to plastic sub-strate with UV embossing transfer technologies have been devel-oped by us, as shown in Fig. 2 [33], with which, we have madetotally printed carbon nanotube networked field effect transistors(FETs) to achieve mobility up to 27 ± 10 cm2 V�1 s�1 and on/off ra-tio up to 104. Such FETs-based sensitive gas sensors are currentlyunder investigations in our lab [36]. Patterned electrode arraysnow can be totally printed on different substrates with a largesurface area [33], thus providing great potential for mass-manufacturing processes to produce micro- or nano-devices forelectroanalysis.

Fig. 1. Thick-film fabrication of microfluidic biochip: (left) schematic illustration of layer-by-layer screen printing procedure; (a) bare PCB board as substrate, (b) patternedcarbon ink as working electrodes and conductive wires, (c) silver/silver chloride ink printed as counter electrode and reference electrode, (d) UV curable dielectriccomposition for coverage of conductive wires, (e) UV curable dielectric composition for microfluidic channels, (f) PDMS layer with inlet and outlet ports; (middle)configuration of the whole device; (right) section figures of the microfluidic device. (Reprinted with permission from Ref. [23]. Copyright � 2007 the Royal Society ofChemistry).

Fig. 2. Schematic of the single-step transfer printing process: (a) drop-coating of the CNT network and bringing it into contact with plastic substrate, (b) water vaportreatment while mold–substrate contact is established. PVA functions as an adhesive for transfer printing during water vapor treatment, (c) source and drain electrodes alongwith CNT network simultaneously transfer printed onto a PVA thin-film, with the CNT network wrapped in a PVA layer to form the finFET structure. The electrodes areintentionally drawn as semitransparent to display the CNT network underneath. (Reprinted with permission from Ref. [36]. Copyright � 2012 Wiley-VCH Verlag GmbH &Co.).

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Recent microdroplet-based devices have been developed to ad-vance the microfluidics technology by further miniaturization,high-throughput and low cost [25–28]. Droplet based microfluidicsmanipulates samples and regents in discrete nanoliter dropletsrather than the continuous liquid flow in the conventional ones,thus referring as digital microfluidics. In our work, the combinationof soft lithography with conventional lithography is used to

fabricate the digital microfluidic device, in which we use soft lith-ographic technique to fabricate the microchannel structure withPDMS as building blocks, while making ITO electrodes on glassby the conventional lithography [28]. Efficient on-demand com-pound droplet formation by a non-uniform electric field is inno-vated for this microfluidics. In the device, Platinum (Pt) wiremicroelectrodes embedded in the microchannels are designed to

C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31 23

generate and manipulate the droplets. During fabrication, the Ptwire is firstly fixed on an ITO glass wafer and the standard photo-lithography process is further used to pattern photoresist along thePt wire centered along the width direction of the channel. Themicrofluidic droplet based device is finally completed by aligningthe PDMS channels with the patterned ITO glass slide with oxygenplasma pretreatment [28]. With the same strategy, we have fabri-cated an integrated device to enable on-demand droplet formation,trapping, fusion and releasing. Together with on-demand dropletmanipulations within one single microfluidic platform [25–28],the technique can offer great broad potential applications in chem-ical and biochemical analyses, chemical and biochemical synthesesand drug screening and validations.

3. Microelectrodes and powder microelectrodes inelectroanalysis

Microelectrodes have played an important role in advancementof electroanalytical chemistry since the pioneer works by Fleish-mann and Wightman [39,40]. A microelectrode normally has atleast one dimension in microns or sub-microns to have low Ohmloses, small double layer capacitance and enhanced mass transportrate, thus allowing fast electrochemistry and measurements innon-aqueous or unsupported electrolytes [41]. A microelectrodecan have various geometries such as disk, ring, ring-disk, shroudedhemispherical or spherical shape and finite conical surface [42],among which the microdisk electrode is the mostly used one.Microdisk electrode is usually fabricated by sealing a metallic wire(gold, platinum, and silver) with several to tens micrometers diam-eter in a glass capillary, followed by mechanical polish to obtain asmooth disk electrode surface [43]. The design, fabrication, charac-terization and theories of microelectrode have been well reviewedby Zoski [42]. Microelectrodes are now able to detect a wide rangeof analytes and have been exploited in many different areas ofelectroanalysis at micro- and even nano-scales including funda-mental science, environmental monitoring, and biomedical analy-sis [44]. The limiting current of a microelectrode measured atsteady-state can be simply expressed as

iL ¼ 4nFDC0r ð1Þ

where n is the number of electron transfer of the electrochemicalreaction, F is the Faraday constant, D is the diffusion coefficient ofthe reactant species in electrolyte solution, C0 and r are the bulkconcentration of the reactant species and the radius of the micro-electrode, respectively. The steady-state measurement is often em-ployed to detect important biological molecules. Cholesterol is a keycomponent in signal transduction in cell functions. A lipid bilayer-modified microelectrode incorporating cholesterol oxidase has beenused to detect cholesterol for its pathways governing the initialsteps in atherogenesis [45]. The applications of microelectrodesare also expanded into electrocatalysis area. The reduction of thio-nyl chloride in dimethyl formamide has been studied by us by usingPt microdisk electrodes. The total number of electrons, two involvedin the reduction is determined from analysis of the limiting currentand the Tafel plot curve of reduction shows that the transfer of thefirst electron in thionyl chloride reduction is totally irreversible[41,46].

A powder microelectrode has been developed and extensivelyused in electroanalytical chemistry and electrocatalysis of variouspowdery materials since its innovation in 1988 by Cha and Li[47–51]. The powder microelectrode is prepared by etching thetip of a prepared conventional ultramicrodisk electrode to form amicro-cavity, which is further filled with a powder material ofinterest by simple grinding on a flat plate such as glass slide forelectrochemical measurements [48]. The powder in the cavity

can be simply removed by sonication for repeatable use. The pow-der microelectrode can study powder catalyst without the use ofbinders often adopted for paste electrodes and gas diffusion elec-trode to eliminate the binder interference in studies of the intrinsicelectrocatalytic behaviors [48,52,53].

The theory of the powder microelectrode has been investigated[47–50]. It is found from the experimental results that the powderultramicroelectrode could be considered as a combination of twodifferent types of electrochemical devices. The outer surface(end-surface) of the powder microelectrode behaves as a planarelectrode of the same dimension, while the porous matrix withthe electrolyte functions as a thin-layer cell with a large reactionsurface area. The limiting diffusion current measured under thesteady-state is controlled by a mass transport process with zerocurrent contribution from the electrolysis of the thin-layer cell,and thus should be determined by the outside apparent surfacearea of the powder microelectrode as the same in Eq. (1) used forthe conventional disk microelectrode [48]. However, the transientcurrent I of a powder microelectrode needs to be calculated as thesum of the responses from a disk-like microelectrode (ID) and athin-layer cell (IT) as follows [48]:

I ¼ ID þ IT ¼ KDm12 þ KTm DEPðDÞ > DEP > DEPðTÞ ð2Þ

With

ID ¼ ðnFÞ32

pDvRT

� �12

Ac�nFRTðEi � EÞ

� �¼ KDv1

2

DEPðDÞ ¼ 59=nmv ð3Þ

IT ¼n2F2vVc�

RTexp½ðnF=RTÞðE� E�0Þ�

f1þ exp½ðnF=RTðE� E�0Þ�g2 ¼ KTv

DEPðTÞ ¼ 0 ð4Þ

where E00 is the formal potential of the redox couple, Ei is the initialscanning potential, DEP(D) and DEP(T) are the potential separationsbetween the anodic and cathodic peaks for the disk and the thin-layer device respectively, v(E) is the normalized dimensionless cur-rent function for a sweep experiment with a reversible system, KD

and KT are constants independent of the scan rate, A is the surfacearea of the disk electrode, m is the scan rate and V is the volumeof electrolyte within the porous matrix of the powdermicroelectrode.

Eqs. (1)–(4) can be used to analyze the experimental results ob-tained by the powder microelectrodes. Due to its characteristicbehaviors combined from both microdisk electrode and thin layerelectrochemical cell, it can achieve significantly enlarged apparentelectrode surface and homogeneous polarization in the porouselectrode for higher reversibility and better developed diffusionlimiting current, thus providing a facile and powerful tool in stud-ies of the electrochemical behaviors of powder materials [48]. Thepowder microelectrode has been successfully used to study thecyclic charge–discharge behavior of electroactive materials andcould conduct a complete charge–discharge cycle within a fewminutes instead of several hours by using a conventionally sizedelectrode [48,53,54]. It has been also used to study the electrocat-alytic processes in various batteries such as lithium ion and thionylchloride batteries [55]. Apparently, the powder microelectrode canbe very convenient and economical to screen powder materialsand to optimize a porous material-based electrode for high electro-catalytic performance in energy and sensor applications due to itssimple preparation and cycle time reduction. A powder microelec-trode can also significantly improve the sensor performance due toenhanced mass transport and miniaturized sizes. It is known thatOs(bpy)2+/3+ is a good electron transfer mediator used in enzymatic

24 C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31

biosensors and its larger load can result in higher sensitivity. Anacetylene black microelectrode modified with Nafion + Os(bpy)2+/3+

possesses a load level of 2 � 10�7 mol cm�2 in terms of theapparent area of microelectrode, which is an order of magnitudehigher than that usually obtained at the planar modifiedelectrodes, thus leading to great improvement of its based glucosebiosensor [51].

4. Nanostructuring and functionalization of electrocatalysts forfast direct electrochemistry

A direct electrochemistry process is known to have direct elec-tron transfer between electrode and proteins or cells [56–61]. Var-ious proteins and cells can catalyze different important biologicalprocesses, which involve electron transfer, and have been inspiredinto important bioenergy and biosensor applications [13,56–67].However, it is very difficult for proteins or cells to directly transferelectrons onto an electrode through electrochemical reactions. Themajor barrier is that the active center of proteins and cells is deeplyembedded and isolated by the protein insulated apoenzyme shelland cell membrane, respectively, and thus the distance betweenreaction centers of the immobilized proteins or cells and the elec-trode surface exceeds the critical electron tunneling distance fordirect electron transfer [13,57,63,68]. Therefore, small diffusiveelectrochemical redox molecules, called electron mediators areadded to the reaction system or immobilized on electrode to assistthe electron transfer, but this is costive and not efficient [51,60,69–71]. Even all electroactive molecules can directly transfer electronsto electrodes through electrochemical reactions, electrocatalysis isalso needed to enhance the sluggish electron transfer rate for effi-cient energy conversions [12,56]. Direct electrochemistry has fun-damental significance in understanding the fast electron transfer incatalysis process and life science, while the innovative enablingapproaches can lead to important applications in highly efficientenergy conversion systems and sensitive biosensors.

Methods to realize the direct electrochemistry have been exten-sively studied. Nanoengineering and functionalization of electrodematerials are often used to promote the direct electron transferprocess. Glucose oxidase (GOD) is an important enzyme used inglucose biosensors and biofuel cells, and it also suffers electrontransfer from its active center onto the electrode during the bio-electrocatalytic process. Different approaches such as reconstitu-tion of GOD by integrating functionalized flavin adeninedinucleotide with apo-glucose oxidase, deglycosylation of GOD toreduce the insulated shell thickness, and development of specificnanostructure to access GOD active centers through shorteningthe electron tunneling distance have been used to realize GOD di-rect electrochemistry [72–75]. Among those approaches, nano-structuring electrode is an effective method [57,58]. Shan et al.have developed graphene, a two dimensional carbon material withone-atomic-thickness to facilitate direct electron transfer of GODfor high performance glucose biosensor [76]. Bao et al. have dem-onstrated a fast direct electron transfer rate of GOD on a uniquemesoporous TiO2 [75]. Horng et al. have investigated the directelectrochemistry on conducting polyaniline nanowires [77]. Wuet al. have studied the effects of ionic liquids on direct electrontransfer related catalytic activity of GOD through both electro-chemical experiment and theoretical quantum calculation [78].These excellent studies have greatly promoted the developmentof direct electron transfer based glucose biosensors. In comparisonto the vacuum deposition method for fabrications of nanomateri-als, ‘‘wet chemistry’’ approach could be more facile and economi-cal. A slack-uniformly porous TiO2 with anatase crystallinity issynthesized by a simple, hydrothermal method employing carbonnanotube (CNT) as a template, as shown in Fig. 3 [75]. GOD enzyme

is immobilized on as-prepared TiO2 via electrostatic attraction, andpasted on a glassy carbon electrode. The estimated direct electrontransfer rate constant, Ks achieves about 3.96 s�1, which is muchlarger than that observed for GOD immobilized on MWNT(1.53 s�1) and SWNT (0.3 s�1) modified electrodes. By calculationwith the integration of the reduction peak and the total amountof attached GOD, it is found that as high as 16.8% of immobilizedGOD is able to directly communicate electrons with the electrode.Various nanostructured enzyme electrodes have been synthesizedby the ‘‘wet chemistry’’ approaches such as carbon-decorated ZnO(C–ZnO) nanowire array electrode, carbonized TiO2 nanotubes, co-electrodeposited conducting polymer/enzyme film, thin-walledgraphitic nanocages, ionic liquid/mesoporous carbon/protein com-posite, and hollow sphere-nanostructured poly(3,4-ethylenedioxy-thiophene) (PEDOT)/NiO composite as enzyme immobilizationmatrices to gain the direct electrochemistry ability [56–61].

It has been revealed that the direct electrochemistry of proteinsnot only simply relies on the electrode nanostructure, but also isgreatly contributed by the specific surface area, conductivity andhydrophilicity, which can be achieved by delicate tailoring of thephysic-chemical properties. Carbonized TiO2 nanotubes have beenprepared by carbonization with organic polymers to possess syner-gic advantages from highly conductive carbon and nanostructuredTiO2 nanotubes, rendering a fast direct electron transfer rate ofHemoglobin (Hb) to catalyze the oxidation of hydrogen peroxidefor a sensitive sensor [56]. A highly hydrophilic ordered mesopor-ous carbon is fabricated by a microwave-assisted template methodwith a mixture of glucose and poly(vinyl alcohol) to have highhydrophilicity, good conductivity and large specific surface areaand is further used to make a direct electrochemistry-based glu-cose oxidase anode for a biofuel cell, which delivers an outputpower density of 110 lW cm�2 with cell voltage of 0.72 V, a perfor-mance much higher than the reported anodes made from SWNT,bi-polymer layer and carbon black at the same or higher glucoseconcentration. This work provides a universal approach to synthe-size functional carbon nanomaterials with desired architecturesand properties for various important applications in enzyme sen-sors and bioenergy systems [79].

Microbial fuel cell (MFC) is a promising ‘‘green’’ energy source,which relies on the entire microorganism as a self-regeneratedelectrocatalyst converting the chemical energy of simple carbohy-drates or organic wastes to electricity at a high energy efficiency[13,63]. However, due to the non- or poor-conductive nature ofthe bacteria as the biocatalysts, the MFC practical applicationsare limited by its low power density. Very much like the early usedenzyme electrodes, electron mediators are used to transfer elec-trons between microbes and electrode, but they cause cell toxicity,high cost, instability and low energy efficiency [62,64,67]. There-fore, the direct electrochemistry is highly desirable. It has beenexperimentally discovered that the Escherichia coli (E. coli) cells un-der electrochemical tension are evolved to a new strain producingendogenous redox mediators for a ‘‘pseudo’’ direct electrochemis-try process, resulting in significantly improved output power den-sity (see CV in Fig. 4) [65]. Further investigation with atomic forcemicroscope (AFM) and field-emission scanning electron micro-scope (FESEM) has discovered that the cell membrane after theevolvement becomes rougher (Fig. 4) to allow the generatedendogenous redox species passing through for the enhanced elec-tron transfer process. The generated redox species has been speci-fied and the proposed enhancement mechanism is shown as inFig. 4. Inspiring from the electrochemically-evolved E. coli cells,gene engineering technique has been adopted to synthesize anew strain of E. coli with over-expressing glycerol dehydrogenase(GldA) for more efficient biocatalyst, leading to lower polarizationand much higher power density than that of both E. coli with theassistance of mediators and electrochemically-evolved one [80].

Fig. 3. (a) FESEM image of TiO2 obtained without using CNTs template, (b) FESEM image of CNTs used in experiment, (c) SEM of TiO2 obtained using CNTs template withouttetraethyl orthosilicate, (d) low-magnification SEM of TiO2 obtained using CNTs template with tetraethyl orthosilicate, Inset of (d) high-magnification FESEM of TiO2 obtainedusing CNTs template with tetraethyl orthosilicate. (Reprinted with permission from Ref. [75]. Copyright � 2008 Wiley-VCH Verlag GmbH & Co.).

Fig. 4. Electrochemical behavior, SEM and AFM topograph analysis of evolved E. coli cells (left) and a hypothetical mechanism for extracellular electron transport of evolved E.coli cells (right). (Reprinted with permission from Ref. [65]. Copyright � 2008 the Royal Society of Chemistry).

C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31 25

A unique nanostructured layer constructed by self-assembly atmolecule scale for nano-scaled coating or deposition acceleratesthe direct electron transfer rate [52,81]. Nitroaromatic explosivessuch as 2,4,6-trinitrotoluene (TNT) are always concerned due totheir toxicity and contamination [82,83]. To sensitively detectthese compounds, the electrode surface should possess specificadsorptive characteristics towards the compounds while enablingfast charge transfer ability [84,85]. An ordered mesoporous carbon(OMC) is synthesized as shown in Fig. 5 to have uniform poresaround a diameter of 3.5 nm and a much higher specific surfaceof 745.9 m2 g�1 than carbon nanotubes and graphene [86]. Themesopores provide abundant adsorption sites for these small mol-ecules while greatly enhancing mass transport to sensitively detectultratrace levels less than 1 ppb, much superior to these reportedin the literatures. Due to its superior physic-chemical properties,

graphene has demonstrated broad promising applications in vari-ous areas [87–93]. It has been also explored to electrochemicallydetect ultratrace explosives [85]. To improve the specific adsorp-tive ability of explosives, porphyrins with the similar aromaticmolecular structure for strong affinity are used to functionalizegraphene surface, which also exhibits high sensitivity, good repro-ducibility and selectivity.

5. Living cell analysis

Cells perform various biological functions via frequent and com-plex energy and signal exchanges by uptake and excretion/secre-tion processes [94]. Living cells analysis is essential not only tofundamentally understand these processes but also to providegreat potentials for a wide variety of practical applications.

Fig. 5. SEM and TEM images of OMC material and the schematic illustration of the electrochemical reduction of 2,4,6-TNT on OMC electrode. (Reprinted with permission fromRef. [86]. Copyright � 2011 Elsevier Inc.).

26 C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31

Signal communications between cells are achieved mainly viareleasing chemical or biochemical messengers from an emittingcell into the target cell to trigger biological responses [4,94], andthus in situ detection of certain messengers secreted by cells arecritical to explore cellular functions, pathology, drug discovery,toxicology, and disease diagnosis [4,94]. However, a secretion pro-cess is very fast (milliseconds to seconds) while the released mes-sengers amount is ultrasmall. It is very challenging to constructsmart sensing transducer to real-time analyze living cells in micro-and nano-scales.

Nitric oxide (NO) as a biological signaling molecule plays animportant role in the regulation of cell function of the nervous, vas-cular and immune systems. Due to the endogenous level and shortlifetime of NO (5 s of half-life time), it is difficult to be sensitivelyand real-time detected. A three-dimensional graphene materialwith large specific surface area and pronounced mesoporosity bya thermal exfoliation method has been synthesized, and is usedto prepare paste electrode using ionic liquid (IL) as a binder. Thegraphene/IL nanocomposite electrode shows porous structureswith much higher inner porous surface area than its outermostsurface area, and high sensitivity and low detection limit with afast response of less than 4 s are achieved for NO detection, whichis much better than other carbon-based sensors.

Dopamine (DA) is an excitatory chemical neurotransmitterubiquitous in the mammalian central nervous system and is asso-ciated with various diseases and body abnormalities. Electrochem-ical determination of DA in a biological environment is ofteninterfered by the coexisting ascorbic acid (AA), which is an elec-tro-active species with a similar redox potential and comparablesensitivity with DA. A gold electrode is modified with a self-assem-bled monolayer of 11-mercaptoundecanoic acid (MUA) followedby further polyethylene glycol (PEG) modification by electrochem-ical esterification with MUA. In the smart modified layer, thedissociated COO� group of MUA could reject or repel AA, whereasthe –OH groups have much higher affinity to positively charged DAfor improvement of its electrochemical reaction on the electrodesurface. The resulted composite electrode shows high sensitivity

for detection of DA and good selectivity to eliminate the interfer-ence from AA.

Real-time electroanalysis of living cells requires an electro-chemical interface with high specificity, sensitivity and fast re-sponse towards the target molecule, while the interface shouldpossess good biocompatibility to promote living cell growth andretain them in native states without change of the physic-chemicaland biological properties of both cells and the surrounding envi-ronment [95–97]. Hydrogen peroxide (H2O2), a reactive oxygenspecies (ROS) is extremely cytotoxic due to its long-time existenceand large diffusion distance to produce various harmful biologicalmodifications. In order to in situ monitor the H2O2 release in livingcells, a smart multifunctional electrochemical biointerface hasbeen constructed on an indium tin oxide (ITO) glass by layer-by-layer electrodeposition of graphene with Prussian blue (PB) andextracellular matrix protein (laminin) as building blocks, as shownin Fig. 6 [95]. In such an interface, the graphene provides good elec-trical conductivity for electrochemical detection and dimensionallycompatible interface for growth of human cells, and PB as an arti-ficial peroxidase (AP) offers the electrocatalytic activity towardsreduction of H2O2, while the extracellular matrix protein is usedto enhance the cell adhesion on the interface. Such nanolayeredmultifunctional graphene–PB–laminin sensor enables quantitativein situ detection of H2O2 generated from living cells with superiorsensitivity and specificity. With this method, the extracellular re-lease of H2O2 stimulated by a drug is investigated by in situ moni-toring the living cells attached on the electrode surface. Upon thedrug injection, an increased cathodic current from the reductionof H2O2 is observed and slowly decays to the original level afterabout 200 s; while for the control experiments, no current re-sponse is observed, clearly showing sensitive and specific detectionability. This work provides a suitable platform for in situ, selectiveand quantitative molecular detection in cell-based assays.

Gold nanoparticle (AuNP) – deposited polyaniline (PANi) nano-wires are coated with Horseradish peroxidase (HRP) enzyme toin situ analyze the released extracellular H2O2 in living ischemiccells, as schematically shown in Fig. 7 [96]. The nanocomposite

Fig. 6. Scheme showing one path for drug-triggered H2O2 production from a human cell and in situ electrochemical detection of H2O2 on the ITO/(graphene–AP–laminin)10electrochem-biointerface. (Reprinted with permission from Ref. [95]. Copyright � 2010 Wiley-VCH Verlag GmbH & Co.).

Fig. 7. Schematics of construction of the HRP–Au NP–PANi sensing biofilm and the in situ detection. (Reprinted with permission from Ref. [96]. Copyright � 2011 the RoyalSociety of Chemistry).

C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31 27

electrode comprises multicomponents, of which the electrochemi-cally polymerized PANi nanowire material has a large specific sur-face area and defect-free interface to significantly reduce highinterfacial resistance, the negatively charged AuNPs (20 nm diam-eter) efficiently link the positively charged HRP molecules forimmobilization, and facilitate the direct electron transfer of HRPenzyme. This composite sensor is able to well in situ distinguishischemic smooth muscle cells (SMCs) from the normal living cellsby amperometric detection of the average number of the extracel-lular H2O2 molecules released per ischemic SMC higher than that ofnormal ones by 2.7 times.

Single cell analysis at a nano-scale spatial resolution in a real-time manner is critical to dynamically monitor fast cellular eventsfor spatial heterogeneities of chemical compositions and profilecell-to-cell variations at a subcellular level, which can provide bet-ter scientific understanding [4] and unique avenues for medicaldiagnosis and disease treatment [4,92,97–100]. Recently an elec-tro-optical nanoprobe has been developed to in situ monitor localbiochemical processes in single cells with simultaneous fluores-cent and electrochemical detection as shown in Fig. 8 [97]. To

fabricate the nanoprobe, the optical fiber is heated and pulled toform a nanotip using a laser based pulling device. The lateral wallof the pulled nanotip is sputtered to coat 100 nm thick gold filmand is connected with a copper wire for electronic measurements.The gold layer is further insulated by coating a thin copolymer filmbut allowing a gold nanoring exposed as a nano electrode [97]. Thefabricated electro-optical nanoprobe has an optical fiber apertureto efficiently deliver the excitation light for high-resolution fluo-rescence detection of intracellular activities, while the nano-scaledgold electrode surrounding the nanotip electrochemically monitorschanges of electroactive biospecies. The bifunctional nanoprobehas been successfully used to observe the oxidant generation andintracellular antioxidation in single cells correlated to the breastcancer. The detected increase in the local concentration of H2O2

in response to the drug injection clearly shows that the cancer cellsproduce much larger quantities of H2O2 in a shorter time upon oxi-dative stress as compared with the normal cells, suggesting thatthe cell malignancy is associated with the strength of oxidativestress. By precise positioning of the nanoprobe at selected locationson the single cell membrane, localized simultaneous electronically

Fig. 8. Schematics of the experimental setup for simultaneously optical–electro-chemical detection of single cell. (Reprinted with permission from Ref. [97].Copyright � 2011 Elsevier Inc.).

28 C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31

and optically detections upon the drug stimulation discover thatthe activated enzymes are responsible for the oxidative stress tar-geting at specific membrane regions. The beauty of this method isthat its optical measurement traces the fluctuation of the intracel-lular redox homeostasis while the amperometric signals can real-time quantify H2O2 release and decay to better analyze the biolog-ical process [97].

6. Electrochemical immunoassay on micro-/nano-structuredinterfaces

Immunoassay is based on the affinity interaction between anti-gen and antibody for excellent specificity and high sensitivity andhas become the main stream in clinical diagnosis of infectiousdiseases. Currently the widely used immunoassay method isenzyme-linked immunosorbent assay (ELISA), in which antigen isphysically absorbed on a solid surface as the probe molecule tocapture the target antibody followed by sandwiching of an en-zyme-labeled antibody for an enzyme-catalyzed optical detection.As an alternative, electrochemical immunoassay has been exten-sively studied to offer portable and miniaturized devices.

Efficient attachment of high density bio-probes with retentionof biological activities on electrode surface is crucial for high per-formance immunosensing, and the access of target proteins specif-ically to the probes are highly demanded [24,101–107]. Methods toattach proteins on various surfaces including plastic, metal, nano-structured metal oxides and microchannels for different immuno-assay devices have been demonstrated [24,101–108]. Proteins onlyhave marginal thermodynamic stability, and may undergo unfa-vorable changes in conformation to lose bioactivity during theimmobilization process. Layer-by-layer (LbL) assembly couldimmobilize high density of probe molecules on a solid surfacewhile providing a friendly environment to maintain proteins’ na-tive conformation as suggested by the in situ Surface plasmon res-onance (SPR) investigation [109]. Conductive polymers such aspolypyrrole (PPy) have been used in electrochemical immunoas-says due to their conductivity, good stability, tailorable

physiochemical properties and biocompatibility [16,110–114].Physical entrapment and direct adsorption have been adopted toimmobilize proteins on PPy surface without needs of functionalgroups as linkers while retaining of their conformation, but suffersfrom weak attachment and low density [115,116]. SPR is used tostudy the kinetics of heterogeneous bio-affinity reactions of PPyfor valuable information of protein immobilization and bio-inter-actions [101,115,116]. A derivative of pyrrole, pyrrole propylic acid(PPa) with a carboxyl group is employed to electrochemically co-polymerize PPy/PPa composite film on an electrode surface forcovalent immobilization of high density proteins, resulting inhighly sensitive electro-immunoassay [117]. A ‘‘non-fouling’’immobilization surface to efficiently suppress the interferencefrom non-specific adsorptions is achieved by using a brush poly-mer film, which is polymerized via surface-initiated atom transferradicals polymerization (SI-ATRP) for good non-fouling properties[24,102,106]. The polymer brush comprises rich epoxy groups tocovalently immobilize probe proteins while blocking the non-spe-cific absorptions for the good sensitivity and specificity in thedetection of multiple biomarkers with a high-throughput SPRimaging technique [102].

An amperometric sandwich-type immunoassay is delicately de-signed by using the copolymerized PPy–PPa film, in which anti-body (anti-mouse IgG) is covalently immobilized by the PPafunctional groups to capture the target antigens (mouse IgG) forthe Ab–Ag interaction, as schematically shown in Fig. 9 [117]. Thenan enzyme-labeled 2nd antibody, alkaline phosphatase (ALP) con-jugated rat anti-mouse IgG is used to bind the captured IgG. Thesubstrate, p-aminophenyl phosphate (PAPP) as a reporter becomeselectroactive only after ALP catalysis, and thus the sandwichedPPy–PPa–anti IgG–IgG–ALP anti IgG event can be amperometrical-ly detected for the electrochemical immunoassay. Since the cova-lent binding of proteins accomplishes a high density of probemolecules and a high concentration of PAPP can be used, high spec-ificity and broad dynamic range are achieved. A PPa film is alsoelectropolymerized onto screen-printed microelectrode arrays inscreen-printed microchannels to attach protein molecules asprobes for a electrochemical microfluidic immunoassay device[23]. The device demonstrates high sensitivity and good specificity.

Electrochemical impedance spectroscopy (EIS) has been utilizedto monitor various bio-affinity interactions since Bataillard’s workin 1988 [118]. Impedimetric immunoassay measures the imped-ance change caused by bio-affinity bindings such as Ab–Ag bind-ings and is a label-free method. An impedance immunosensorhas been fabricated with a self-assembled monolayer on a elec-trode to immobilize recognition capture proteins and the changeof charge transfer resistance (Rct) of a redox pair such asFeðCNÞ3�=4�

6 after the Ab–Ag binding is measured to indirectly mon-itor the concentrations of target proteins [119]. However, the redoxpair may induce denaturation and loss of biological functions of theprobe proteins to negatively affect the sensitivity and specificity.Further, most of thiol monolayers on gold surface are not stablein the presence of FeðCNÞ3�=4�

6 due to the etching effect of freeCN� ion on the gold [120,121]. Complete reporter-free impedimet-ric immunoassays have been investigated [16,110,112,113,122]. Amulti-components nanocomposite film comprising PPy, PPa andgold nanoparticles (AuNPs) is prepared by electrochemical deposi-tion for an electrochemical immunosensor as schematically shownin Fig. 10 [112]. The deposition mechanism is also investigated byin situ AFM measurements [123]. In this immunosensor, the multi-components in the nanocomposite play different roles in the elec-tronic recognition of the Ab–Ag bindings, of which PPa provideslinkers for probe immobilization, PPy offers a conductive matrixfor sensing and AuNPs improve the conductivity of the sensinglayer for high S/N ratio [112]. The Rct change represents decreasedions doping–dedoping ability of the conductive polymer film after

Fig. 9. Schematic of the amperometric enzyme immunosensor based on the polypyrrolepropylic acid (PPA) film. The electroactive product, p-aminophenol (PAP), wasconverted from the enzymatic conversion of PAPP by alkaline phosphatase (ALP). (Reprinted with permission from Ref. [117]. Copyright � 2006 American Chemical Society).

Fig. 10. Schematic of electrochemical polymerization of PPy�PPa�Au nanocomposite film and probe immobilization via EDC activation of carboxylic functional groups.(Reprinted with permission from Ref. [112]. Copyright � 2008 American Chemical Society).

C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31 29

the Ab–Ag binding [114]. This sensor exhibits an excellent sensitiv-ity of 10 fg mL�1 of human interleukin 5 (IL-5) in PBS and a highdynamic range of three orders of magnitude. A reusable impedi-metric immunosensors based on the probe-immobilized PPy/PPa/Au film has been reported [110]. To regenerate the immunosensingsurface, M glycine buffer is used to rinse the electrode for eluting ofthe probe antibody and captured target protein, which can achievemore than about 95% dissociation of both probe and target proteinsand keep almost the same baseline of Rct after the regeneration[110]. It is worthy of a note for a work. In impedimetric measure-ments, to eliminate or reduce the variations for better precision, anormalized dimensionless unit change is introduced to analyze theimpedance data [16,113] as

DRN ¼ ðR2 � R1Þ=R1

where R1 and R2 are the Rcts measured before and after the Ab–Agbinding, respectively.

7. Conclusions and prospects

In recent years, substantial progress in electroanalysis in micro-and nano-scales has been achieved. Nanostructuring and function-

alization of electrodes with unique physic-chemical properties isshedding new lights on the traditional electrochemistry scienceto accelerate the development of electroanalytical devices towardshigh sensitivity, good specificity, low cost, high-throughput andminiaturization. In particular, micro- and nano-fabrication playan important role in promoting the analytical advances in micro-and nano-scales. These accomplishments not only advance sci-ences significantly, but also provide various important applicationsin clinic diagnosis, drug discovery and environmental controls.

It is expected that the nano-fabrication will be still important forfuture electroanalysis in micro- and nano-scales. The main focusesshould be on the low cost and reproducibility. It is very likely thatweb-to-web nanoprinting will be the promising approach to havelow cost nanodevices while providing mass-manufacturability.

Innovative nanomaterials for electroanalysis especially at anano-scale will be further developed with creative syntheticapproaches. Different from other analytical methods, the nanoma-terials used in electroanalysis are required to possess high conduc-tivity, high specific surface area and unique physicochemicalproperties for detection of specific target molecules.

A smart, multifunctional sensing transducer is possibly needed.Novel nanocomposite electrodes may be one of the most promisingapproaches to provide multifunction for high sensitivity and good

30 C.M. Li, W. Hu / Journal of Electroanalytical Chemistry 688 (2013) 20–31

specificity. Feynman’s lecture on ‘‘There’s Plenty of Room at theBottom’’ mainly introduces interesting ramifications of a generalability to manipulate matter on an atomic scale. In practice, the po-tential to use the bottom-up approach to build a smart sensinglayer should be tremendous.

Electroanalysis in micro- and nano-scales opens great potentialsin various applications. The exploration of important applicationsmay firstly come from biology and electrocatalysis at nano-scales,mainly due to their great importance and complexity and thushighly demanded quantitative analysis in nano-scale. Analysis insingle cells and single biomolecules such as proteins and their mod-ifications could be the research hot points. Direct electrochemistryof non-conductive biomolecules needs more experimental effortsto understand their fundamental insights. Its success could leadto highly sensitive nano-scaled devices.

When electroanalysis comes to micro- or nano-scaled worlds,we will face great theoretical puzzles. The fundamental in tradi-tional electrochemistry is indeed needed to be advanced intonano-worlds with experimental trials and theoretical calculations.For example, the structure of electrode/electrolyte interface atnano-scales will be different from the double layer explanation.The electron transfer at nano-scale or/and sub-nanoscale and masstransport in nano-scale avenues could be different. This work cansignificantly promote the science advances while providing meth-ods or models to analyze the data gained from nano-scales forpractical applications.

Acknowledgments

This work is dedicated to Professor Chuansin Cha in WuhanUniversity and Professor Zhaowu Tian in Xiamen University onthe occasion of their receiving inaugural Chinese ElectrochemistryAchievement Awards, and also is to express our deep thanks per-sonally to them for their profound knowledge, remarkable practiceand invaluable advices to guide us in electrochemistry research foryears.

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