DOI: 10.1002/elan.201400037

Protein Based Electrochemical Biosensors for H2O2

Detection Towards Clinical DiagnosticsAjay Kumar Yagati[a] and Jeong-Woo Choi*[a, b]

1 Introduction

Hydrogen peroxide (H2O2) is a reactive oxygen metabolicby-product that serves as a key regulator for a number ofoxidative stress related state [1]. H2O2 mediated pathwayshave been linked to asthma, inflammatory arthritis, athe-rosclerosis, diabetic vasculopathy, osteoporosis, anda number of neurodegenerative diseases [2–4]. H2O2 isa by-product of several enzymatic reactions that can beused as diagnostic tools for detection of the onset of vari-ous biological conditions [5]. H2O2 is not only involved inphagocytosis but also acts as insulin that helps the trans-port of sugar through the body [6,7]. Another importantapplication of H2O2 is it creates intracellular thermogene-sis, a warming of cells which is absolutely essential tolife�s processes [8]. Hence, it is considered to be an im-portant analyte because of its importance in clinical diag-nosis. H2O2 is produced by all cells of the body for manydifferent physiological reasons, the granulocytes produceH2O2 as a first line of defense against bacteria, yeast,virus, parasites, macrophages, and most fungi [9, 10]. It isinvolved in metabolic pathways which utilize oxidases,peroxidases, cyclooxygenase, lipoxygenase, myeloperoxi-dase, catalase and many other enzymes. H2O2 is oftenused in therapy, as well as other treatment modalities, ona routine basis, usually given by intravenous injection[11,12]. H2O2 would help arthritis because of its ability tosupply oxygen to oxygen-hating organisms causing arthri-tis (Streptococcus viridans). However, it is usually treatedas an intermediate or by-product of metabolism and con-sidered of minor significance in metabolic pathwaysexcept as it relates to biochemical disruption, tissue orcellular damage [13,14]. Wlassoff et al. [15] reporteda new treatment of cancer based on the innate overpro-duction of H2O2 in cancer cells. It is described that, Hy-drogen peroxide serves as a prodrug in the presence oftransition metal ions, such as iron delivered by ferrocene.

Under the effect of ferrocene, hydrogen peroxide is splitinto hydroxyl anions and highly reactive hydroxyl radi-cals. These radicals cause�s oxidative DNA damage whichinduces apoptosis leading to elimination of cancer cells.Maramag et al. [16] reported the influence of H2O2 onthe treatment of prostate cancer. Many studies describethe protective role of vitamin C (ascorbic acid) againstcancer development and in treatment of establishedcancer. Vitamin C inhibits cell division and growththrough the production of hydrogen peroxide, which dam-ages the cells probably through an as yet unidentifiedfree radical(s) generation/mechanism. Further, the role ofH2O2 and hydroxyl radical formation plays a major rolein the killing of Ehrlich tumor cells [17]. Also, the pro-duction of potent oxidizing species, including the hydrox-yl radical (OH), has been demonstrated during treatmentof intact human MCF-7 breast cancer cells with doxorubi-cin [18]. H2O2 has wide application in surgery and den-tistry, particularly in the debridement of wounds [19]. Itsuse in sensitive areas such as carotid artery and vein indi-cates its value in safety cleaning wounds as shown inFigure 1. Thus direct or indirect detection of hydrogenperoxide is one of the central themes in the design andfabrication of various biosensors.

Abstract : Hydrogen peroxide (H2O2) has a diverse arrayof physiological and pathological effects within the livingcells depending on the extent, timing, and location of itsproduction. Detection of H2O2 is important in food indus-try, clinical diagnostics, and environmental monitoring atlowest levels. Electrochemical biosensors are efficient asthey can analyze biological sample by direct conversioninto an electrical signal. Electrochemical sensors based on

direct electron transfer (DET) of proteins were devel-oped to achieve fast electron transfer by avoiding free-diffusing redox species with improved sensitivity. Wesummarize the prerequisites for the DET of proteins forimmobilization on the electrode surfaces with recent de-velopments in development of H2O2 sensors and futureprospects in this field.

Keywords: Proteins · Clinical diagnostics · Biosensors · Hydrogen peroxide

[a] A. K. Yagati, J.-W. ChoiResearch Center of Integrated Biotechnology,Sogang University35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul, Republic ofKoreatel: +82-2-705-8480; fax: +82-2-3273-0331*e-mail: jwchoi@sogang.ac.kr

[b] J.-W. ChoiDepartment of Chemical & Biomolecular Engineering,Sogang University35 Baekbeom-ro (Sinsu dong), Mapo-gu, Seoul, Republic ofKorea

Specia

lIssu

eD

IAG

NO

STIC

S

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1259

Review

In electrochemistry, H2O2 can be either oxidized or re-duced directly at ordinary solid electrodes. However,these processes in analytical applications are limited byslow electrode kinetics and high overpotential which willreduce the sensing performance and may suffer fromlarge interferences from other existing electroactive spe-cies in real samples such as ascorbate, urate, and nitrates[20,21]. Hence, the research on H2O2 detection is mainlyfocused on electrode modifications in order to decreasethe overpotential and increase the electron transfer kinet-ics. Towards this, several types of nano-biomaterials suchas redox proteins [22], transition metals [23], metaloxides [24], metal porphyrin [25], redox polymers [26],and carbon nanotubes [27] have been employed to per-form electrocatalytic H2O2 detection. Furthermore, thesize and structure of biomaterials (proteins) can be tail-ored for designing a novel sensing platform and enhanc-ing sensing performance [28]. Highly sensitive and selec-tive determination of single chemical at extremely lowconcentrations is important for environmental monitor-ing, drug screening and for clinical diagnostics. Recently,

a detection method which is based on the development ofnanopore for the precise detection of single chemicals hasbeen reported [29–31]. It consists of a hub of the bacter-iophage phi29 DNA packaging motor is a connector con-sisting of 12 protein subunits encircled into a 3.6 nm chan-nel for the passage of dsDNA to enter during packagingand to exit during infection.

The electron-transfer rate between a redox protein andelectrode surface is usually slow, which is the major obsta-cles of the electrochemical system. Significant effortshave been made to understand the biological systemscontrol which electron transfer processes are possible(i.e., reduction potentials) and how fast they will occur(i.e., rate constants) [32]. To achieve efficient electricalcommunication between redox protein and electrodethere are many challenging objects has to overcome [33].The factors that affect the properties of these importantbiological electron transfer sites are generally consideredas either intrinsic (an inherent behavior or property ofitself) or extrinsic (modulated external factors) to theactive site [34]. Apart from electron mediators the inher-ited property of direct electron transfer property of pro-tein has received much attention to understand the bio-chemical process and also towards development of novelbioelectronic devices [35,36].

Here in this review, we analyze different protein immo-bilization methods for better electron transfer betweenprotein to surface and also study the properties of pro-teins based direct electron transfer towards the electrocatalytic reduction of H2O2. Also this study summarizesthe recent advances in protein based direct electron trans-fer systems and gain an insight into the materials used inthe electrocatalytic sensing of H2O2 towards clinical diag-nostics.

2 Methods of Protein Immobilization

Understanding protein adsorption at solid surfaces is im-portant for the development of many fields in nanotech-nology, such as biomaterial production for medical devi-ces, drug delivery systems, or in vitro diagnostics. Proteinadsorption to solid surfaces in aqueous environments iscrucial and very complex process which is driven by theprotein-surface forces including van der Waals, hydropho-bic and electrostatic forces. Proteins are usually adsorbedon the surface of solid surface in a disordered orientationand suffer from conformational changes that lead to theinaccessibility of the metal center to the underlying elec-trode surfaces for enzymatic reactions [37]. The confor-mational change of protein in the adsorbed state willdiffer with the type of protein, depending on the substrateincluding hydrophobicity/hydrophilicity, environmentalconditions such as temperature, pH and ionic strength ofthe solution. Hence, controlled immobilization methodsare required to retain the native properties of the protein[38]. The basic requirements for a successful immobiliza-tion requires; 1) stability and specific affinity of the pro-teins for the surface 2) uniformity on the surfaces 3) pos-

Ajay Kumar Yagati received hisPh.D. in 2011 at Sogang University,Korea and M.Tech. (Bioelectronics)in 2006 from Tezpur University,Assam, India also he obtained M.Sc.(Electronics) in 2001 from AndhraUniversity, India. He worked atSogang University as a Post-doctoralresearcher at nanobioelectronics lab,with Professor Jeong-Woo Choi. Healso worked as a Senior Project As-sistant at the School of Medical Sci-ence and Technology (SMST), IndianInstitute of Technology, Kharagpur, India with Professor SujoyK. Guha. He is currently working as Assistant Professor atGachon University of Medical Campus, Incheon, Korea. His re-search interests include electrochemistry; biosensors based onchemically modified electrodes and working on the developmentof nanoscale bioelectronic devices.

Jeong-Woo Choi received his Ph.D.in 1990 at Rutgers University, USA.He worked at IBM Almaden Re-search Center and Mitsubishi Elec-tronics Advanced Technology R&DCenter as a visiting researcher in1993 and 1996, respectively. He alsoobtained a D.Eng. at Tokyo Instituteof Technology, Japan in 2003 as wellas an MBA degree at University ofDurham, UK in 2007. He has beena professor of Department of Chemi-cal & Biomolecular Engineering,Sogang University, South Korea, for over 25 years. He is a leadingresearcher in the field of nanobioelectronics including nanobio-electronic device, cell chip and organ-on-a chip. He has pub-lished over 330 peer-reviewed papers in Adv. Mater., Ange-

Specia

lIssu

eD

IAG

NO

STIC

S

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1260

Review

sibility to control the immobilization density of the immo-bilized proteins 4) preserving the native biological func-tions of proteins 5) orientation of the protein to achievemaximum binding density.

Most immobilization methods developed so far in-volves in the modification or coating of the surface withproper linkers to change/enhance the surface property toprovide functional groups for binding the protein. Alter-natively, immobilization of proteins on a bare surfacewith no modification exploits specific interactions be-tween the protein and the surface, such as immobilizationon the Au surface via thiol (sulfhydryl) groups and neces-sitates using an affinity peptide that is specific to the par-ticular surface.

2.1 Physisorption

This method of immobilization of protein is based on thephysical adsorption of protein on the solid surfaces andhence it causes little or no conformational change of pro-tein or loss of its properties. Physisorption is based on thevan der Waals interactions between the adsorbate and thesubstrate and also between the adsorbed molecules [46].Physical adsorption generally leads to dramatic changesin the protein microenvironment, and normally involvesmultipoint protein adsorption between a single proteinmolecule and a number of binding sites on the immobili-zation surface. Moreover, even if the surface has a uni-form distribution of binding sites, physical adsorptioncould lead to heterogeneously populated immobilizedproteins. This has been known for unfavorable lateral in-teractions among bound protein molecules. In general,this kind of adsorption is completely nonspecific, i.e. ,almost all gases can physisorb under the certain condi-tions to almost all surfaces. Further, physisorbed mole-cules can leave the surface after certain amount of time.The session of physisorption is an exothermic process

occurs at lower magnitudes of temperature at an enthalpyof the order ~20 kJmol�1 due to weak van der Waalsforces of attraction. This method has advantages such as,the procedure require no expertise or special equipmentalso it does not require surface treatment but it suffersfrom protein leaching from the immobilization support[47].

2.2 Chemisorption

Chemisorption requires a specialized substrate thatallows for a chemical linkage between the biomoleculeand the surface and this method often requires the exper-tise of a chemist. Here in this case, the enthalpy is aroundan order of magnitude ~200 kJ mol�1 which is muchhigher than physisorption. Bifunctional silane/thiol cou-pling reagents are usually used to form a chemical bondwith the side functional group of the amino acid/ carbox-ylic acid residue of the protein at one end and with theglass/Au surface at the opposite end [48]. Basically thereare three different procedures in coupling protein to theunderlying surface. In the first method, the substrate isactivated with some chemical linker and then the proteinis allowed to bind the substrate through the chemicalbonding. In the second method, biomolecules such as pro-teins or antibodies are activated to sulfur containing mol-ecules and allowed to self-assemble on the substrate. Fi-nally, the third method involves in the protein binding tosubstrate by ligand-receptor mechanism such as lock andkey interaction. The chemical attachment involves moredrastic conditions for the immobilization reaction thanthe attachment through adsorption [49]. The covalent andcoordinate bonds formed between the protein and thesupport can lead to a change in the structural configura-tion of the immobilization protein. Such a change in theenzymatic structure may lead to reduced activity, unavail-

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 1. Schematic representation depicts how the enzyme catalase protects the cell and breaks down the hydrogen peroxide (H2O2)into water (H2O) and singlet oxygen (O2). The single oxygen acts as a powerful oxidizing agent that kills/inhibits harmful organisms.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1261

Review

ability of the active site of an enzyme for the substrates,altered reaction pathways or a shift in optimum pH.

2.3 Site-Directed Immobilization

In both the physisorption and chemisorption techniques,proteins attach to surface in different orientations leadsto considerable activity loss. Hence, good orientationtechniques have been proposed to increase the efficiencyof biotransformation and to enhance the sensitivity ofbiological reactions. To achieve well-oriented immobiliza-tion site-specific immobilization was proposed. The ap-proach to site-specific immobilization requires functional-ization of the target molecule, tailoring the surface, orboth. The strategy is to develop recombinant proteins in-corporating histidine residues in either amino or carboxylend of the protein or cysteine residues on specific parts ofthe protein [50,51]. When protein has cysteine residues inthe correct regions, protein could be immobilized and ad-dressed on the Au surface with good orientation and cov-erage. Table 1 presents the various protein immobilizationmethods on the solid surfaces to achieve intrinsic proper-ties of the protein for better performance towards novelelectrochemical sensors.

3 Direct Electron Transfer of Proteins

Many of the fundamental processes in nature depend onupon the redox processes of constituent biomolecules.Cell respiration involves the stepwise oxidation of organicsubstrates through a chain of redox reactions. To under-stand these biological processes, electrochemical methodsoffers as a prominent technique for examining the elec-tron transfer properties. The molecules of particular inter-est are redox-active proteins and enzymes. Now-a-days,studies of direct electrochemistry of redox proteins at theelectrode-solution interface are of great interest. Thereare two main approaches that can be imagined in explor-ing suitable and effective coupling between a protein andan electrode: a) direct and unmediated electron transfer;b) indirect and mediated electron transfer [52]. The mostimportant and challenging, is certainly, the first option asit is based on the aspect of protein electrochemistry that

efforts have been concentrated till date. Electrode surfa-ces have been sought at which a freely diffusing redoxprotein can take part in rapid and direct electron transfer.

The studies on direct electron transfer are a simple andinformative means for understanding the kinetics andthermodynamics of biological redox processes. Further, itprovides a model for the study of the mechanism of elec-tron transfer between enzymes in biological systems, andestablishes a platform for developing new kinds of bio-sensors.

There are several factors that influence the direct elec-tron transfer between the electrodes and proteins. Gener-ally these factors include; a) electroactive prostheticgroups buried deep within the protein structure; b) ad-sorptive denaturation of proteins onto electrodes, c) un-favorable orientations of proteins at electrodes. Severalstrategies and works have been reported for the directelectron transfer of proteins from its first reports on thedirect electrochemistry of redox protein in 1977 [53,54].Since then achieving reversible, direct electron transferbetween redox proteins and electrodes without using anymediators and promoters had made great accomplish-ments. Chemical modification of electrode surfaces is notthe only route towards rapid and reversible electrontransfer between an electrode and redox protein. A gen-eral requirement for such an interaction is that the sur-face of the electrode is electrostatically compatible withthe surface of the protein, particularly that part implicat-ed in the electron transfer process. In fact, direct electrontransfer has now been achieved between a variety of elec-trode surfaces and a range of redox proteins.

3.1 Direct Electron Transfer of Cytochrome c

Mitochondrial cytochrome c (cyt c hereafter) is a proteinubiquitous to all eukaryotic organisms and is the mostwidely studied protein with regard to its electrochemicalproperties because of its high solubility in water com-pared with other redox-active proteins. Cyt c transfers anelectron from complex III to complex IV, membrane-bound components of the mitochondrial electron-transferchain. Cyt c takes part in electron transfer within the res-piratory chain in mitochondria. Electrons are transferred

Specia

lIssu

eD

IAG

NO

STIC

S

Table 1. Some selected coupling methods for biomolecules commonly used for developing sensor devices.

Immobilization method Immobilized surface Protein modifications Binding mechanism Reference

Physical adsorption Polystyrene, Glass surface Wild proteins Physical Adsorption [39]Chemisorption Au/ITO surface anchored with

linker materialsWild proteins Chemisorption with EDC-

NHS linking[40]

Recombinant proteintechnology

Au surface Recombinant protein withcysteine (Cys) introduced

Chemisorption of SH-groupson Au

[41]

Silane coupling method Glass or ITO modified with silanecoupling reagents having aldehyde

Wild proteins/enzymes Schiff�s base linkage betweenaldehyde and amino groups

[42]

Streptavidin-mediated im-mobilization or vice versa

Nanowires/nanostructures Biotinylated protein Protein-ligand binding affinitybetween streptavidin-biotin

[43, 44]

Recombinant DNA tech-nology

In vivo or in Cellulo Single Chain FragmentVariable (scFv)

Binding of scFv with rabbitIgG antigen

[45]

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1262

Review

from NADH and FADH2 to dioxygen by proteins locatedin the inner mitochondrial membrane. These systems areorganized into multi-protein complexes, of which com-plexes I (NADH-ubiquinone reductase) and II (succinic-ubiquinone reductase) accept electrons from matrix sub-strates (NADH and FADH2, respectively) and reduceubiquinone to ubiquinol, complex III (ubiquinol-cyto-chrome c oxidoreductase) catalyzes the transfer of elec-trons from ubiquinol to cyt c, and complex IV ( cyto-chrome c oxidase) transfers electrons from cyt c to dioxy-gen. This whole process is coupled in the synthesis ofATP [55]. It is known that the lysine residues surroundingthe heme crevice of the protein plays a vital role in bind-ing interactions and electron transfer with its redox part-ners. Cyt c is an excellent model for studying the electrontransfer of typical enzymes from a structure point of view.Further, cyt c is known to have some intrinsic peroxidaseactivity due to its close similarity to peroxidase. In addi-tion to these properties, cyt c has several advantages foruse as a biocatalyst. (a) The heme prosthetic group is co-valently bound to the protein. This property may be im-portant for catalysis in the presence of organic solvents;cyt c does not lose its heme catalytic group in these sys-tems, while peroxidases do; (b) cyt c is active over a widerange of pH [from pH 2–pH 11]. No other enzyme isactive over such a pH range; (c) cyt c is able to performbiocatalytic reactions even at higher temperatures andafter chemical modification its thermo stability will behighly increased; and (d) cyt c is inexpensive. Cost andstability are the two main factors for biocatalysis ona large scale. The direct electrochemical measurementsindicated that the reduction potentials (E8’) at pH 7 and25 8C is in the range of 0.260–0.280 V [56].

3.2 Direct Electron Transfer of Myoglobin

The biological function of myoglobin (Mb) is to bufferthe oxygen concentration in respiring tissues. The affinityof myoglobin for oxygen lies between that for hemoglo-bin, which releases oxygen during its passage through re-spiring tissues, and for the cytochromes that make use ofmolecular oxygen in oxidative respiration. It is a kind ofheme protein containing a single polypeptide chain withan iron heme as its prosthetic group [57]. The physiologi-cal function of Mb is to store dioxygen and increase thediffusion rate of dioxygen in the cell. Although Mb doesnot function physiologically as an electron carrier, it un-dergoes the oxidation and reduction process in the respi-ratory system. Thus, its electron-transfer reactions playessential roles in biological processes. It is an ideal modelmolecule for the study of electron transfer reactions ofheme proteins, biosensing, and electrocatalysis. Towardsthe usage of Mb in electrochemical detection systems, Mbis a water-soluble heme-containing protein present inmost mammals and vertebrates, and has been used exten-sively for investigating electron transfer and electrocataly-sis with the protein immobilized on an electrode surface.Its main function is to store oxygen and enhance diffusion

in the muscle. This globular protein has a single 153 or154 amino acid chain with a molecular weight of ~17 kDa[58]. It is a versatile protein with high tolerance for chem-ical and mechanical environments and can be easily ex-pressed and purified in large quantities in E. coli. Anoth-er important feature is, it can accommodate various muta-tions without any adverse effects on its conformationaland functional properties. Mb contains a single iron-por-phyrin center that can accommodate ferrous, ferric orferryl oxidation states within this heme active site moiety.

Even though a wide range of reports is available forimmobilization of proteins for carrying out direct electrontransfer and electrocatalysis with electrodes, a direct com-parison involving active site orientation of monolayer andmultilayer covalent attachment of proteins is absent. Thisis mainly due to the difficulty to achieve a site-directedcovalent tagging of the native conformation of Mb on thesurface with the full control over active site orientation.Furthermore, formation of both a monolayer as well asa multilayer on the electrode surface is a challengingtask. However, several researchers are working on Mbimmobilized electrodes to achieve the direct electronproperties towards the development of novel electro-chemical sensors.

3.3 Direct Electron Transfer of Hemoglobin

Hemoglobin (Hb) is a heme-containing protein that con-sists of “globin fold” and it reversibly binds to molecularoxygen. Hb are found in human erythrocytes (red bloodcells) with a concentration approximately 30 % (w/v) or20 mM in heme. Erythrocytes take up approximately 40–45% of the blood volume (hematocrit) and hence 100 mLof human blood contains approximately 15 g of Hb. It hasa molar mass of approximately 67 000 g/mol and compris-es of two a- and two b-subunits, each containing a hemeor Fe(II)-protoporphyrin IX. Since each heme can bindto oxygen molecules, four molecules of oxygen bind toHb tetramer. Several studies were performed on the elec-trochemical behavior of heme proteins to understand theproperties and their biological activity. Hb is ideally usedfor the study of electron transfer reactions of heme pro-teins because of its commercial availability and its cost[59,60]. Further, Hb possesses functional groups that canbe readily oxidized or reduced by chemical redox agents;it does not easily undergo facile redox reactions at elec-trodes. Unlike other small heme proteins such as cyt c, it,however, is difficult for Hb to exhibit heterogeneous elec-tron-transfer process in most cases, which means Hb ex-hibits such a slow electron transfer that no useful currentsappear at conventional electrodes, even when rather largeovervoltages are applied due to its extended three dimen-sional structure and resulting inaccessibility of the elec-troactive centers as well as its strong adsorption onto theelectrode surface for subsequent passivation [61].

Specia

lIssu

eD

IAG

NO

STIC

S

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1263

Review

3.4 Direct Electron Transfer of Ferredoxin

Ferredoxins (Fdx) from are one electron carrier iron-sulfur proteins that function as electron transport chainsand oxidoreduction reactions. The Fe-S center takes partin single electron transfer reaction in which one Fe atomundergoes oxidoreduction between Fe2+ and Fe3+ . Fdxare small, soluble, generally very acidic proteins that areinvolved mainly as electron carriers of low oxidation-re-duction potential in fundamental metabolic process. Iron-sulfur proteins (non-heme iron proteins, Fe-S) which maycontain one, two, three or four Fe atoms linked to inor-ganic sulfur atoms and/or through cysteine-SH groups tothe protein molecule. Rubredoxins (Rd) comprises ofsingle iron-sulfur cluster without acid-labile sulfur thatare characterized by having iron in typical thiolate coor-dination, i.e. an iron center surrounded by four cysteineresidues or sulfur-containing ligands. Oxidized rubredoxinusually exhibits the characteristic EPR spectrum of high-

spin Fe(III) ion in a rhombic ligand field, g=4.3; the re-duced form gives no discernible EPR signal. Only nega-tive redox potentials at pH 7 have been noted for thoserubredoxins presently characterized. Desulforedoxin isa variant rubredoxin with a higher symmetry and distinc-tive EPR spectrum with g-values of 7.7, 4.1 and 1.8. TheSpinach Ferredoxin contains Fe atoms in [2Fe-2S] redoxsite which are bridged by two sulfides and the tetrahedralcoordination of each iron completed by cysteine residues.Fdx contains clusters of two iron atoms and representedas [2Fe-2S]2 + with 97 amino acid residues (AA) whichhave a molecular weight ranging from 11000 to 11900[62]. One electron reduction of the [2Fe-2S]2+ site occursat one of the iron atoms to give the reduced [2Fe-2S]+

site. In the oxidized state, both iron atoms are in a similarchemical state, which appears from the chemical shift andquadrupole splitting to be high-spin Fe3+ . In the reducedstate the iron atoms are different and the molecule ap-pears to contain one high-spin Fe2+ and one high-spin

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 2. Schematic diagrams for Iron-sulfur [Fe-S] proteins a) Iron site of Clostridium pasteurianum rubredoxin in which one Fe isbound by four cysteines (b) [2Fe-2S] redox site in Spinach Ferredoxin; c) [3Fe-4S] redox site from Desulfovibrio gigas ferredoxin andd) Proposed model for electron transfer of the iron-sulfur group in spinach ferredoxin.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1264

Review

Fe3 + atom. In the oxidized state the two high-spin ferriciron atoms where only one of these iron atoms accepts anelectron on reduction. The spins of the iron atoms arecoupled in such a way that there is no net spin in theground state (S=0).When the ferredoxin is reduced oneelectron is transferred to the specific iron atom, whichthen becomes high-spin ferrous; thus in the reduced statethere is one high-spin ferrous and one high-spin ferriciron atom [63]. Figure 2 illustrates the model envisagesfor the iron-sulfur clusters in different ferredoxin proteinmolecules. The Desulfovibrio gigas ferredoxin, a sulfatereducing organism, has a major role in sulfate reducingbacteria (SRB) metabolism and contains a [3Fe-4S] redoxsite. The [3Fe-4S]1 + cluster undergoes a typical redox pro-cesses, accepting a total of 3 electrons: [3Fe-4S]1+![3Fe-4S]0!3Fe-4S]2�. The electrochemical behavior of thisprotein is complex and several transitions are observed inthe different redox regions (one at �130 mV and at�690 mV). Apart from Fe in the cluster the disulfide (S�S) bridge can accept two electrons [64].

3.5 Direct Electron Transfer of Horseradish Peroxidase

Horseradish peroxidase is an important heme-containingenzyme obtained from plant source. It has attracted theattention of many researchers from a variety of disci-plines because of its practical and commercial applica-tions. Advances in understanding the structure and cata-lytic mechanism of horseradish peroxidase have beenmade using protein engineering and other techniques.Horseradish peroxidase is not one enzyme, but a group oflarge family of isoenzymes which have different molecu-lar forms of the same enzyme that catalyze the same bio-chemical reaction but have distinct physical, chemical andkinetic properties arising from small differences in theiramino acid sequence. HRP-C is the most abundant isoen-zyme isolated from horseradish root. Horseradish Perox-idase is a heme protein with 308 amino acid residues. TheN-terminal residue is blocked by a pyrrolidenecarboxylresidue that appears to be buried inside the polypeptidechain. The C-terminal peptides were sequenced with andwithout a serine residue, indicating a rather labile Asn-Ser peptide bond [65]. HRP-C contains iron (III) proto-porphyrin IX (ferri-protoporphyrin IX), i.e. a hemegroup, as a prosthetic group in its active site. In additionto the four coordination positions with the nitrogenatoms of the porphyrin pyrrole rings, the heme iron hastwo axial coordination sites (the fifth and sixth positions)where binding can also occur. Horseradish peroxidase isone of the most widely used enzymes in analytical appli-cations. Due to its characteristics, HRP meets all the re-quirements for a successful use in analytical systems,apart from that the ability of HRP to catalyze the oxida-tion of numerous chromogenic substrates enables the useof spectrophotometric detection systems, including fluo-rescence and luminescence, opening way to a wide rangeof procedures.

4 Applications of H2O2 Biosensors Based on DirectElectron Transfer of Protein

Biosensors have shown great potential towards healthcare and environmental monitoring systems. The crucialelements in the performance of the biosensor mainly de-pends on the components, among which is the activematrix materials (the layer between the recognition layerof biomolecules and transducer) plays a vital role in es-tablishing good stability, sensitivity and the endurance ofa biosensor. Several studies have led to the rapid develop-ment of wide range of biosensors with improved detec-tion limits [66]. Further, with the advancements in nano-technology and in the field of engineering much improvedwith miniature biosensing systems have made to possible.The operation principle of a biosensor is shown inFigure 3.

The biosensors which are based on direct electrontransfer of proteins, the redox state of the analyte or asso-ciated species is altered by either intermediately storingthe redox equivalents in a redox protein-integrated pros-thetic group or transfer of electrons between a suitableco-substrate within the active site. The advantage of bio-sensors which are based on direct electron transfer ismainly the absence of mediators which makes these bio-sensors having better selectivity and less prone to inter-ference [67]. Further they have an advantage that there isa prospect to modulate the desired properties by proteinmodification by recombinant engineering towards novelbiosensor devices.

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 3. Operation principle of biosensing detection system.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1265

Review

Electron transfer is a fundamental reaction in biologi-cal systems and it is very important to understand the bio-logical function and to design synthetic energy transduc-tion systems. Basically, electron transfer can be consid-ered as a transition between two electronic states, donor(D) and acceptor (A). According to Marcus theory, theelectron transfer rate is determined by the electronic cou-pling between D and A (VDA), the reaction free energy(DG) and the reorganization energy (l) by the followingEquation 1 [68]

kET ¼2p

�hV2

DA

1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

4plkBTp exp � lþ DG�ð Þ2

4lkBT

� �

ð1Þ

Three key parameters, electronic coupling V, drivingforce DG, and reorganization energy l, determine the ETrate. DG is defined through the difference of the D andA redox potentials. Further, these quantities may signifi-cantly deviate from their values measured in aqueous sol-utions or in organic solvents. The electronic couplingVDA decay exponentially with the distance R between Dand A

V � V0 exp � b

2R� R0ð Þ

� �

ð2Þ

The parameter b is determined by the superexchangeinteraction of D and A with their surrounding [69]. ETcan also occur through incoherent hopping including tran-siently populated electronic states localized on a bridgealong with direct and bridge-mediated super exchange be-tween donor and acceptor. Further, if the prostheticgroup is deeply buried inside the molecule then the directelectron transfer with high rate is unlikely because of theexponential decrease of the tunneling probability with theincrease of the distance between the redox partners.

There are in principle two experimental approaches toestablishing whether DET is occurring between redox en-zymes and electrodes [70]:

a) Indirect evidence based on observing a catalytic re-sponse current in the presence of the enzyme sub-strate.

b) Direct evidence from observation of independentelectrochemical activity of the redox cofactor compris-ing the active site in the absence of substrate.

The electrochemical reactions occur at bare workingelectrode often suffer from interferences or surface foul-ing byproducts arising in the follow-up reactions, linkedwith the main electrochemical process. Modified electro-des can be a solution towards this which avoids the draw-backs of bare electrodes such as adsorption of molecules,unpredictable surface reactivity and sluggish kinetics.

Electrochemical biosensors consisting of enzymes illus-trating the three generations in the development of bio-sensor (a) first generation electrode utilizing the H2O2

produced by the reaction; (b) second generation elec-trode utilizing a mediator (ferrocene) to transfer the elec-trons, produced by the reaction, to the electrode; (c)third generation electrode directly utilizing the electronsproduced by the reaction [71] are shown in Figure 4. Thefollowing reaction occurs at the enzyme in all three bio-sensors:

Substrate ð2HÞ þ FAD-oxidase!Productþ FADH2-oxidase

ð3Þ

This is followed by the processes:

ðaÞ Biocatalyst : FADH2-oxidaseþO2 !FAD-oxidaseþH2O2

ð4Þ

Electrode : H2O2 ! O2 þ 2Hþ þ 2e� ð5Þ

ðbÞ Biocatalyst : FADH2-oxidaseþ 2 Ferriciniumþ !FAD-oxidaseþ 2 Ferroceneþ 2Hþ

ð6Þ

Electrode : 2 Ferrocene! 2 Ferriciniumþ þ 2e� ð7Þ

ðcÞ Biocatalyst=Electrode :

FADH2-oxidase! FAD-oxidaseþ 2Hþ þ 2e�ð8Þ

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 4. Schematic diagram shows the developments of three different biosensor generations.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1266

Review

4.1 Based on Direct Electron Transfer of Cytochrome C

Cytochrome c which contains iron centered porphyrinand can easily undergo oxidation and reduction overa wide range of potentials which are varied by the proteinenvironment around heme groups [72]. Due to the redoxcapability of heme proteins, cyt c widely used to study themechanism of the catalytic process between redoxenzyme and substrate and used in enzyme-based biosen-sors, especially H2O2 biosensors. However, electron trans-fer between cyt c and solid electrodes is usually slow [73].Thus, it is necessary to search for a way to develop a cytc modified electrode that will enhance electron transferto the solid surface, while still maintaining well-behavedelectrochemistry and good stability. Recently cyt c immo-bilized on Au nanoparticles on ITO substrate (cyt c/AuNP/ITO) was applied for the detection of H2O2 asshown in Figure 5 [74].

Generally, the direct electron transfer of cyt c atnormal Au bulk electrode is difficult; however with theincorporation of gold nanoparticle the electron transferproperties of the protein are enhanced. Experimental re-sults showed that AuNP�s can act as tiny conduction cen-ters, which can facilitate the rapid transfer of electronswhich have a very high surface to volume ratio. The de-veloped cyt c/AuNP/ITO electrode showed a couple ofwell-defined and quasi-reversible redox peaks because ofthe Fe3+ /2+ redox center, which was responsible for elec-tron exchange. With increment with scan rates both the

anodic and cathodic peaks were increased linearly shownin Figure 6.

The electrocatalytic response for the detection of H2O2

based on the cyt c/AuNP/ITO electrode can be expressedby the following equation

cyt c�FeðIIIÞ þ e� ! cyt c�FeðIIÞ ð9Þ

2cyt c�FeðIIÞ þ 2Hþ þH2O2 ! 2cyt c�FeðIIIÞ þ 2H2O

ð10Þ

I�t curves were obtained for cyt c/AuNP/ITO for addi-tions of 20 mL of 200 mM H2O2 at a potential of �0.1 Vvs. Ag/AgCl. The response was also confirmed on thebare ITO electrode and cyt c/ITO electrode which showsvery less response and slight increment in the currentcomparative to cyt c/AuNP/ITO towards H2O2. As shownin Figure 7 the developed electrode cyt c/AuNP/ITOshowed linear response up to a concentration of 6 mMhaving a detection limit of 0.5 mM.

A comparison Table 2 is provided for cyt c immobilizedon various modified electrode surfaces based on thedirect electron transfer properties of the protein towardsthe detection of H2O2.

4.2 Based on Direct Electron Transfer of Myoglobin

Myoglobin (Mb) generally has a slow electron transfer(~10�5 cm s�1) rate hence efforts have been made to en-

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 5. A schematic route for the fabrication of ITO/AuNP/cyt c electrode consisting of cyt c and AuNP�s by layer-by-layer assemblyon MPTMS functionalized indium tin oxide (ITO) surface. Figure reproduced with permission from: Reference [74], � 2012 Elsevier.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1267

Review

hance the electron transfer rate [84]. Proteins immobi-lized on metal oxide structures will not only providestable immobilization with complete retention of theirbiological cognition properties and also provides largesurface area and its surface free energy can absorb pro-teins strongly which plays a vital role in developing betterbiosensor devices. Recently many articles have been pro-posed for the detection of H2O2 where proteins/enzymeswere immobilized on metal oxide surfaces. A comparisonTable 3 is provided for the Mb adsorbed on modifiedelectrode surfaces and its important parameters for H2O2

sensor. Metal oxide surfaces can provide a favorable mi-croenvironment for proteins and enhance the direct elec-tron transfer rate between the proteins and electrodes.The electrochemical catalytic reduction for H2O2 by Mb/CeO2/ITO electrodes was examined by cyclic voltamme-try (CV) [85]. The Mb/CeO2/ITO electrode shows twopairs of quasi-reversible reduction and oxidation peakswhich correspond to CeO2 and Mb shown in Figure 8.

The redox peaks (Epa =0.6 V and Epc =0.3 V) appearedcorresponds to the formation of Ce3+/Ce4+ couple and

the peaks at �0.38 and �0.2 V corresponded to the Fe3 +

/Fe2+ redox center of Mb. The electron transfer rate con-stant (Ks) was calculated to be 1.57 s�1 with a surface cov-erage of 5.142 � 10�11 molcm�2.

The mechanism of the electrocatalytic reduction ofH2O2 is expressed as:

Mb ðFe3þÞ þ e� !Mb ðFe2þÞ ð11Þ

H2O2 þ 2 Mb ðFe2þÞ ! 2 Mb ðFe3þÞ þ 2 H2O ð12Þ

The catalytic current is linearly increased with the addi-tions of 10 mL aliquots of H2O2 and a calibration plot isestablished for the accurate determination of H2O2. Theamperometric response of the Mb/CeO2/ITO electrode toH2O2 was recorded through successively adding H2O2 toa continuous stirring PBS solution. The amperometric re-sponse has linear relationship with the concentration ofH2O2 shown in Figure 9.

The developed sensor based on Mb/CeO2/ITO elec-trode has a sensitivity of 5.4 mAmM�1 cm�2 with a detec-tion limit of 0.6 mM. The calculated apparent Michaleisy�Menten constant (Km

app), which indicate the catalytic ac-tivity of the protein to its substrate, can be obtained fromthe Lineweaver�Burk equation:

Iss�1 ¼ Imax

�1 þKmapp ðImaxCÞ�1 ð13Þ

where Iss is the steady current obtained after adding sub-strate, which can be obtained from amperometric experi-ments. C is the bulk concentration of the substrate, andImax is the maximum current measured under the saturat-ed substrate condition. The value of the apparent Michae-lis�Menten constant (Km

app) was 3.15 mM, suggesting thatthe biosensor exhibited high affinity for H2O2. The selec-tivity of the developed sensor is examined with differentinference compounds such as Ascorbic acid, uric acid,sodium nitrite and sodium bicarbonate at 0.2 mM whichis evident that there was minimal influence of interferingspecies on the H2O2 response.

Zhang et al. [89] reported a modified electrode of Mbimmobilized on Au nanoparticles on ITO surface. Theelectrodes shows pair of redox peaks at �0.23 and�0.09 V related to Myoglobin. This Mb/Au/ITO electrodehas good electrocatalytic response towards H2O2 witha linear range of 2.5–500 mM with the detection limit of0.48 mM. The value of the apparent Michaelis�Mentenconstant (Km

app) was 1.3 �10�4 M shows a high biologicalaffinity to H2O2.

4.3 Based on Direct Electron Transfer of Hemoglobin

Many H2O2 sensors have been proposed during severalyears based on Hemoglobin (Hb) because of its perox-idase activity and commercial availability. Direct electro-chemistry of proteins on an electrode surface has beenstudied to sensitively detect H2O2 without the additional

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 6. (a) Cyclic voltammogram for (i) bare ITO, (ii) AuNP/ITO, (iii) cyt c/AuNP/ITO electrode in HEPES buffer solution atpH 7 at a scan rate of 0.05 Vs�1; (b) cyclic voltammogram of cytc/AuNP/ITO electrode in 10 mM, pH 7.0, HEPES buffer with in-creasing scan rate from 0.01 to 0.1 V s�1. Figure reproduced withpermission from: Reference [74], � 2012 Elsevier.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1268

Review

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 7. (a) Current-time curve obtained for ITO/AuNP/cyt c electrode upon successive addition of 20 mL aliquots of 200 mM H2O2 to5 mL stirred 10 mM HEPES buffer at pH 7 with an applied potential of �0.1 V under nitrogen atmosphere; chronoamperometriccurve obtained for (b) cyt c/ITO and (c) AuNP/ITO obtained by the addition of 20 mL aliquots of 200 mM H2O2 in 5 mL stirred solu-tion of 10 mM HEPES buffer at the potential of �0.1 V under nitrogen atmosphere. Figure reproduced with permission from: Refer-ence [74], � 2012 Elsevier.

Table 2. Summarizes the values of selected quantities measured from cyt c immobilized electrode towards H2O2 sensing that were re-ported in the literature.

Electrode matrix Detection limit (mM) Linear range (mM) ks (s�1) Reference

cyt c/AuNP/ITO 0.5 – 0.69 [74]cyt c/RTIL-PDDA-AuNPs/MUA-MCH/Au 5.0 0.04–3.45 2.65 [75]cyt c/PFS-DNA/Au 0.72 0.003–1.83 0.78 [76]cyt c/MWNTs/GCE 350 0.002–0.42 4.0 [77]cyt c/Nanoporous Au 6.3 0.01–12 – [78]cyt c/Au/Chit 9.8 0.85–13 – [79]cyt c/MPCE 0.146 0.02–24 17.6 [80]cyt c/PAN-PDA/GCE 7.3 0.002–0.38 – [81]cyt c/GNPs/RTIL/MWNTs/GCE 3.0 0.05–11.5 0.78 [82]cyt c/MPA/Au 1.0 0–0.25 1600 [83]

Table 3. Summarizes the values of selected quantities measured from Mb immobilized electrode towards H2O2 sensing that reportedin literature.

Electrode matrix Detection limit (mM) Linear Range (mM) Reusability Reference

Mb/CeO2/ITO 0.6 3.0–3000 2 weeks [85]Mb/HMS/GCE 0.062 4.0–124 3 months [86]Nafion/Mb/IL/GCE 0.14 1.0–180 – [87]Nafion/Mb/CGNs/GCE 0.5 1.5–90 – [88]Mb/Au/ITO 0.48 2.5–500 – [89]Mb/HSG/SN-CNTs/GCE 0.36 2.0–1200 – [90]GNRs@SiO2-Mb/RTIL-sol-gel/GCE 0.12 0.2–180 3 weeks [91]Mb/EMIM-BF4/HA/GC 0.6 2.0–270 [92]Mb/clay-IL/GCE 0.73 3.9–259 – [93]Mb/SDS-GNPs-GR/BPG 0.012 0.5–7.5 4 weeks [94]

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1269

Review

electron transfer mediator. Several studies were per-formed by immobilizing Hb on different electrode surfa-ces, such as glassy carbon electrode [95], metal oxides[96], nanoparticles [97], carbon nanotubes [98] and gra-phene [99,100]. However, there is a great need to furtherenhance the direct electron transfer rate of the mediator-less H2O2 sensor for not only further increasing the sensi-tivity, but also improving the response time, since thesensor often suffers from slow response time due to itslimited direct electron transfer rate between redox pro-teins and electrode.

The mechanism for electrocatalytic reaction of Hb to-wards H2O2 can be expressed as follows [101]:

HbðFe3þÞ þH2O2 ! Compound I ðFe4þ¼OÞ þH2O

ð14Þ

Compound I ðFe4þ¼OÞ þ e� þHþ ! Compound II ð15Þ

Compound IIþ e� þHþ ! Hb ðFe3þÞ þH2O ð16Þ

Chen et al. [102] proposed a strategy for modified elec-trode (Hb/Au/Hb/MWNT/GC) by the preparation nano-hybrid film composed of multiwall carbon nanotubes(MWNT) and gold colloidal nanoparticles (GNPs) byusing proteins as linker materials towards the detection ofH2O2. The strategy is negatively charged MWNT was firstmodified on the surface of glassy carbon (GC) electrode,then, positively charged Hb was adsorbed onto MWNTfilms by electrostatic interaction. The {Hb/GNPs}n multi-layer films were finally assembled onto Hb/MWNT filmthrough layer-by-layer assembly technique. This Hb/Au/Hb/MWNT/GC electrode give a pair of well-definedredox peaks at �0.26 and �0.37 V at scan rate of 50 mV/s, characteristic of heme Fe(III)/Fe(II) redox couples of Hb,suggesting that direct electron transfer has been achieved

between Hb and underlying electrode are shown inFigure 10.

As compared to those H2O2 biosensors only based oncarbon nanotubes, the proposed biosensor modified withMWNT and GNPs displays a broader linear range anda lower detection limit for H2O2 determination. Thelinear range is from 2.1 �10�7 to 3.0 �10�3 M with a detec-tion limit of 8.0 �10�8 M at 3s. The Michaelies�Mentenconstant KM

app value is estimated to be 0.26 mM. More-over, this biosensor displays rapid response to H2O2 andpossesses good stability and reproducibility as shown inFigure 11. Table 4 summarizes the Hb immobilized onvarious modified electrode surfaces towards the electro-catalytic reduction of H2O2.

Specia

lIssu

eD

IAG

NO

STIC

S

Fig. 8. Cyclic voltammogram obtained from Mb/CeO2/ITOelectrode showing redox properties of both CeO2 and Mb layerson ITO surface. Figure reproduced with permission from: Refer-ence [85], � 2013 Elsevier.

Fig. 9. (a) Amperometric (I/t) curve obtained for Mb/CeO2/ITO electrode upon successive additions of 10 mL aliquots of100 mM H2O2 to 5 mL of 10 mM PBS with constant stirring at anapplied potential of �0.3 V under nitrogen purging. Inset (i) Cali-bration curve; (ii) Response time of Mb/CeO2/ITO electrodetoward the detection of H2O2. (b) I/t curve obtained for Mb/CeO2/ITO electrode at �0.3 V upon successive additions of 5 mLaliquots of (0.2 mM) uric acid (UA), L-ascorbic acid (AA),sodium nitrite (NaNO2) and sodium bicarbonate (NaHCO3)along with 5 mL of H2O2 with constant stirring of 5 mL PBSbuffer pH 7.0. Figure reproduced with permission from: Refer-ence [85], � 2013 Elsevier.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1270

Review

4.4 Based on Direct Electron Transfer of Ferredoxin

Many biosensors have been reported based on the directelectrochemistry of protein towards the electrochemicaldetection of H2O2. Until now, many proteins such as cytc, Mb, Hb has been extensively studied for the detection.These heme proteins have some disadvantages for exam-ple, Hemoglobin, which is a redox active protein consist-ing of four electroactive-iron (III) hemes as prostheticgroups enables reversible conversion of Hb-Fe(III) toHb-Fe(II) but the rate of electron transfer from the pro-tein to the surface of the electrodes modified directly byhemoglobin is slow; moreover a large, three dimensionalstructure of hemoglobin leads to inaccessibility of theredox centers that are located inside the protein. There-fore, direct electron transfer between the hemoglobin andelectrode is difficult. Towards the development of newkind of protein based sensor, spinach ferredoxin has been

successfully utilized for the electrocatalytic detection ofH2O2 [112].

The Fdx/MUA/Au modified electrode shows good elec-trocatalytic activity towards the reduction of H2O2. Theelectrocatalytic response of the Fdx/MUA/Au towardsH2O2 was investigated and the principle of catalyzingH2O2 was presented by the following equation.

Fdx½2Fe�2S�2þ þ e� ! Fdx½2Fe�2S�þ ð17Þ

Fdx½2Fe�2S�þ þH2O2 !Fdx½2Fe�2S�þ�H2O2�Fdx½2Fe�2S�þ !Fdx½2Fe�2S�2þ þ 2OH�

ð18Þ

From the above equations, in the bio-catalytic cycle ofFdx, at first it forms an enzymatic reducing agent asFdx[2Fe-2S]+. This is catalytically active and can form an

Specia

lIssu

eD

IAG

NO

STIC

SSpecia

lIssu

eD

IAG

NO

STIC

S

Fig. 10. Cyclic voltammogram obtained from Hb/GNPs/Hb/MWNT/GC electrode at a scan rate of 50 mV s�1 in 0.1 M PBS atpH 6.0 (a) without H2O2; (b) 0.53 mM H2O2 and c) 1.27 mMH2O2. Figure reproduced with permission from: Reference [102],� 2007 Elsevier.

Table 4. Summarizes the values of selected quantities measured from Hb immobilized electrode towards H2O2 sensing that reportedin literature.

Electrode matrix Detection limit (mM) Linear range (mM) KmApp (mM) Reference

Hb/GNPs/Hb/MWNT/GC 0.08 0.21–3000 0.26 [101]Hb/P123/PGE 0.5 1.0–500 0.51 [102]Hb/Au-MFIOH/GCE 0.8 1–18 000 – [103]Hb/chitosan@Fe3O4/Au 1.1 2.3–9600 3.7 [104]Hb/graphene/Fe3O4/GCE 6.0 0.25–1.7 – [105]Nafion/Hb/Ni/ITO 0.5 0.8–122 0.325 [106]Hb/PHD/MWCNTS/GCE 0.35 1.0–1.500 0.51 [107]Hb/ZnO/MWCNT/GCE 0.02 – – [108]Hb/PAM-P123/GCE 0.4 1.0–30 – [109]Hb/SDS/TiO2/GCE 0.087 0.5–40 – [110]Hb/IL/MWILE 3.18 20.0–4500 0.51 [111]

4500–13000

Fig. 11. Amperometric respone of Hb/GNPs/Hb/MWNT/GCelectrode towards H2O2 at an applied potential of �300 mVupon successive additions of H2O2. Inset: Linear calibrationcurve. Figure reproduced with permission from: Reference [102],� 2007 Elsevier.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1271

Review

adduct with H2O2, Fdx[Fe-2S]+�H2O2�Fdx[Fe-2S]+ sub-sequently it forms OH� radical and then back to the rest-ing state of the native enzyme, Fdx[Fe-2S]2+.

The Fdx/MUA/Au immobilized electrode showeda couple of well-defined and quasi-reversible redox peaksfor the reduction of the FeIII ion in the [2Fe-2S]2+ cluster,with the formation of [2Fe-2S]+. The anodic peak poten-tial (Epa) and cathodic peak potential (Epc) are located at�0.12 and �0.17 V (vs. Ag/AgCl), respectively. Theformal potential (E8’) is ca. �0.15 V and peak-to-peakseparation (DEp) of 60 mV was observed. The developedsensor showed good amperometric response for the selec-tive determination of H2O2 without any interference ef-fects as shown in Figure 12.

The stability of the sensor have been examined by CVsweeps over a potential range of �0.4 to 0.2 V at0.05 Vs�1 for 50 cycles which retains its original valueand the signal decreased 3.2 % after storing at 4 8C for10 days.

4.5 Direct Electron Transfer of Horseradish PeroxidaseTowards H2O2

HRP is an enzyme that catalyzes the oxidation of a widevariety of organic and inorganic substances, with H2O2 aselectron accepter. In peroxidase based biosensors, H2O2 isreduced at low over potential due to the direct electrontransfer between the electrode and HRP redox center.Thus, it is widely used enzyme for H2O2 detection biosen-sors. But, the redox center of HRP is surrounded in the

protein matrix, which reduces the electrical conductivityof the enzyme and thus leads to poor electron transferrates. Meanwhile the redox center is hidden inside theprotein matrix of the enzyme; it can avoid interference ofmany molecules with large size. The catalytic center ofthe enzyme is deeply embedded in an insulating proteinshell which results in sluggish electron-transfer kinetics,further it loses its bioactivity when it adsorbed directlyonto the electrode surface. Therefore, appropriate pro-moters should be employed to facilitate the electrontransfer and retain the biological activity of the immobi-lized enzyme. With the fast growth of nanotechnology,various nanomaterials such as metal nanoparticles [113],metal oxide materials [114], nanofibers [115], nanotubes[116], nanowires [117] have been widely used to modifyelectrodes, which are conductive to realize the direct elec-tron transfer of the enzyme.

The general catalytic cycle proceeds through a sequenceof reactions which can be described by the followingequations

HRP ðFe3þÞ þH2O2 ! Compound I ½ðFe4þ¼OÞ� þH2O

ð19Þ

Compound I ½ðFe4þ¼OÞ� þ e� þHþ ! Compound II

ð20Þ

Compound IIþ e� þHþ ! HRP ðFe3þÞ þH2O ð21Þ

Specia

lIssu

eD

IAG

NO

STIC

SSpecia

lIssu

eD

IAG

NO

STIC

S

Fig. 12. Amperometric response of Fdx/MUA/Au electrode at �0.2 V upon successive additions of 10 mL aliquots of 200 mM H2O2

in stirring with 5 mL 10mM Tris-HCl, pH 7.0. Inset (upper): i–t curve obtained for Fdx/MUA/Au electrode upon successive additionof 10 mL aliquots of 500mM H2O2 to 5 mL stirred 10 mM Tris-HCl buffer at pH 7 also with three successive additions of 10 mL aliquotsof 200mM of NaNO2 at an applied potential of �0.2 V under nitrogen atmosphere. Inset (lower): i–t curve obtained for MUA/Au elec-trode upon successive addition of 10 mL aliquots of 200 mM H2O2 to 5 mL stirred 10mMTris-HCl buffer at pH 7.0with applied poten-tial of �0.2 V under nitrogen atmosphere; inset shows the i–t response of the curve during 100–300 s. Figure reproduced with permis-sion from: Reference [112], � 2011 Elsevier.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1272

Review

In the first step of this cycle the HRP resting statereact with H2O2 and is get oxidized to a high oxidationstate intermediate known as Compound I, in which oneelectron removed from the ferric iron to the Fe4 + oxyferr-yl center and an electron is removed for the porphyrin togive Porphyrin p-cation radical. In the second step in-volves a single electron oxidation of the substrate by com-pound I and the p-cation radical is reduced to givea second high oxidation state as compound II. Finally thecompound II react with another substrate molecule fromwhich an electron is extracted by the Fe4+ oxyferryl to re-generate the resting state of the enzyme.

Zhang et al. [118] proposed a strategy for modifiedelectrode (HRP/GO/Nafion/GCE) by immobilizing HRPon graphene oxide-Nafion nanocomposite film towardsthe application of H2O2 sensor. GO-based polymer com-posites provide a favorable microenvironment for HRPto realize its direct electron transfer, which allows it to beused for H2O2 sensing with great sensitivity. The cyclicvoltammogram of HRP/GO/Nafion/GCE electrode dis-play a couple of well-defined, stabile and reversible redoxpeaks, which were attributed to the redox reaction ofHRP with a formal potential (E8’) of �369 mV shown inFigure 13.

The electrocatalytic reduction of H2O2 at HRP/GO/Nafion/GCE was studied by cyclic voltammetry in the po-tential range of 0.30 to �0.70 V. When H2O2 was addedreduction peak increases and decrease or no change inoxidation peak current is observed. These results demon-strate the typical electrocatalytic reduction processes ofH2O2. The HRP/GO/Nafion/GCE electrode showed tworeduction peaks at �0.23 V and �0.39 V corresponds tothe reductions of O2 and HRP.

It is clearly shown from the Inset of Figure 14 thatHRP/GO/Nafion/GCE, the response current increasedlinearly with the concentration of H2O2 from 1.0 mmolL�1

to 1.0 mmolL�1 with the linear regression equation I=0.1182+12.82 C (I, mA; C, mmol L�1) (R=0.9986). Thedetection limit for H2O2 at a signal-to-noise ratio of 3 wasfound to be 4.0 �10�7 molL�1. At HRP/Nafion/GCE, thelinear range for the response current to the concentrationof H2O2 was limited from 1.0 mmolL�1 to 0.1 mmolL�1.Michaelis�Menten model yields the apparent Michaelis�Menten constant KM

app =0.684 mmol L�1 for HRP/GO/Nafion/GCE to the reduction of H2O2 (Icat

�1 =�0.099+68.40C�1, Icat, mA; C, mmolL�1; R=0.9999). The stabilityof the electrode was tested, and it was found that HRP/GO/Nafion/GCE could be repeatedly scanned withoutsignificant decrease of peak currents. The cathodic peakcurrent of HRP/GO/Nafion/GCE was decreased by lessthan 5 % after 4 weeks of storage. Table 5 summarizes theHRP immobilized on various modified electrode surfacestowards the electrocatalytic reduction of H2O2.

Specia

lIssu

eD

IAG

NO

STIC

SSpecia

lIssu

eD

IAG

NO

STIC

S

Fig. 13. Cyclic voltagmmograms of (a) HRP/GO/nafion/GCE,(b) HRP/Nafion/GCE and (c) Go/Nafion/GCE in 0.1 mol L�1

pH 7.0 PBS at a scan rate of 100 mVs�1. Figure reproduced withpermission from: Reference [118], � 2007 Elsevier.

Fig. 14. (A) Cyclic voltagmmograms of HRP/GO/nafion/GCEin 0.1 molL�1 pH 7.0 PBS containing H2O2 upon increasing con-centrations (B) Amperometric response of (a) GO/Nafion/GCE,(b) HRP/Nafion/GCE and (c) HRP/GO/Nafion/GCE in0.1 molL�1 pH 7.0 PBS at �0.57 V on successive additions ofH2O2. Inset: Calibration curve of the electrocatalytic currentversus H2O2 concentration. Figure reproduced with permissionfrom: Reference [118], � 2007 Elsevier.

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1273

Review

Similarly, Catalase (Cat) is also a heme enzyme, whichis present in all aerobic organisms having a molecularweight of ~240 000 composed of four identical subunitswith each containing a single heme prosthetic group. Theheme group consists of a protoporphyrin ring and a cen-tral Fe atom, where iron is usually in the ferric oxidationstate as its stable resting state. As a catalyst, the enzymefunctions either in the catabolism of H2O2 or in the per-oxidatic oxidation of small molecule substrates by H2O2.Under normal physiological conditions, enzyme controlsthe H2O2 concentration so that it does not reach toxiclevels that could bring about oxidative damage in cells.The mechanism of disproportionation of H2O2 catalyzedby cat can expressed as

H2O2 þ CatðFeIIIÞ ! Compound IþH2O ð22Þ

H2O2 þ Compound I! CatðFeIIIÞ þO2 þH2O ð23Þ

where Cat(FeIII) is the resting state of the enzyme, Com-pound I is a two-equivalent oxidized form of Cat(FeIII)containing an oxyferryl heme (FeIV=O) and a porphyrinp-cation radical. H2O2 first oxidizes Cat(FeIII) to formCompound I and H2O, and then reduces Compound I toCat(FeIII) and produces O2. This enzyme can act either asa reductant or as an oxidant in the reactions, and returnsto its resting state after one catalytic cycle, while H2O2

undergoes dismutation to produce H2O and O2.

5 Conclusions and Future Prospects

In recent years numerous reviews have addressed electro-chemical H2O2 biosensors based on the catalytic proper-ties of redox proteins and enzymes. This review is mainlyconcentrated on recent advances based on the direct elec-trochemistry of protein for electrocatalytic H2O2 determi-nation. Several methods with variety of electrode surfacematerials are used to immobilize the proteins in order toenhance the direct electron transfer rate and provide pro-teins with suitable microenvironment. With the develop-ment of novel electrode surfaces mediator-free biosensorswith greater enhancements in sensitivity and selectivitywere achieved. Many research groups are continuously

working to develop protein based biosensors while, veryfew proteins will display the direct electron transfer whilemost cases mediator is used for better electron transferfunctions. Further, there is not any standard protocol toimmobilize protein on electrode surface to achieve directelectron transfer reactions. Though several studies onconventional electrodes have achieved considerable re-sults for the detection of H2O2, but with help of nanotech-nology much better sensing methodologies towards H2O2

can be developed. Also, artificial enzymes that mimic nat-ural enzymes could be another alternative for efficientway to develop biosensors. Natural proteins/enzymes arevery sensitive to the environment and susceptible to de-nature relatively on unmodified electrodes also with in-crement in temperature or pH of the solution. The poorstability of natural enzymes becomes a barrier towardsthe fabrication process of biosensors and long-time usagein real world applications. Apart from these, as the costof proteins are high which further limits the mass produc-tion of biosensors. Hence, artificial enzymes can be an al-ternative as they are more robust and can be easily tailor-made to the desired properties for constructing andbetter biosensing applications. Novel nanobiomaterialsare expected to come with better understanding of thebiological process and will continue the developments ofH2O2 sensors towards clinical diagnostics, food safety andenvironmental monitoring.

Acknowledgements

This research was supported by the Leading Foreign Re-search Institute Recruitment Program through the Na-tional Research Foundation of Korea (NRF) funded bythe Ministry of Science, ICT & Future Planning (MSIP)(2013K1A4A3055268) and Sogang University ResearchGrant of 2013 (SRF-201314003.01).

References

[1] M. Giorgio, M. Trinei, E. Migliaccio, P. G. Pelicci, Nat.Rev. Mol. Cell Bio. 2007, 8, 722–728

Specia

lIssu

eD

IAG

NO

STIC

SSpecia

lIssu

eD

IAG

NO

STIC

S

Table 5. Summarizes the values of selected quantities measured from HRP immobilized electrode towards H2O2 sensing that reportedin literature.

Electrode matrix Detection limit (mM) Linear range (mM) Sensitivity (mAmM�1 cm�2) Reference

HRP/NanoCeO2/ITO 0.5 1.0–170 8.44 [119]HRP/Au NP/MPA/Au 0.16 0.48–1200 311.72 [120]HRP/Composite-3 0.009 0.01–0.22 7.8 [121]HRP/laponite/chitosan 5 29–1400 19.7 [122]HRP-flower ZnO-AuNP-Nafion/GCE 9.0 15–1100 – [123]HRP/HIL/TNT-GNP 2.2 15–750 – [124]Nafion/HRP-GNSs-TiO2/GCE 5.9 41–630 – [125]HRP/PTMSPA@GNR 0.06 10–1000 0.021 [126]Clay-HRP-Clay/AuCS-GCE 9.0 39–310 – [127]HRP/Au NAE 0.42 0.74–15000 45.86 [128]

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1274

Review

[2] R. Rojking, J.-A. Dominguez-Rosales, N. Nieto, P. Green-wel, CMLS, Cell. Mol. Life. Sci. 2002, 59, 1872–1891.

[3] J. M. Lean, C. J. Jagger, B. Kirstein, K. Fuller, T. J. Cham-bers, Endocrinology 2005, 146, 728 –735.

[4] J. Schalkwijk, W. B. van den Berg, L. B. A. van de Putte,L. A. B. Joosten, Arthritis Rheumatism 1986, 29, 532 –538.

[5] C. Andre, S. W. Kim, X. H. Yu, J. Shanklin, PNAS 2013,110, 3191 –6.

[6] G. Rothe, G. Valet, J. Leukoc. Biol. 1990, 47, 440 –8[7] M. S. Patole, T. Ramasarma, Biochem. Educ. 2010, 16, 58 –

62.[8] W. C. Douglass, Hydrogen Peroxide: Medical Miracle,

Rhino Publishing, S. A. Panama, 2003, pg. 20[9] J. W. Homan-Muller, R. S. Weening, D. Roos, J. Lab. Clin.

Med. 1975, 85, 198 –207.[10] R. K. Root, J. Metcalf, N. Oshino, B. Chance, J. Clin.

Invest. 1975, 55, 945–955.[11] H. M. Dockrell, J. H. L. Playfair, Infect. Immun. 1983, 39,

456–459.[12] A. J. Kemper, J. E. O�Boyle, S. Sharma, C. A. Cohen,

R. A. Kloner, S. F. Khuri, A. F. Parisi, Circulation 1983, 68,603–611.

[13] I. Slesak, M. Libik, B. Karpinska, S. Karpinski, Z. Miszal-ski, Acta Biochim. Pol. 2007, 54, 39–50.

[14] S. G. Rhee, S. W. Kang, W. Jeong, T.-S. Chang, K. S. Yang,H. A. Woo, Curr. Opin. Cell Biol. 2005, 17, 183 –189.

[15] W. A. Wlassoff, C. D. Albright, M. S. Sivashinski, A. Ivano-va, J. G. Appelbaum, R. I. Salganik, J. Pharm. Pharmacol.2007, 59, 1549–1553.

[16] C. Maramag, M. Menon, K. C. Balaji, P. G. Reddy, S. Lax-manan, Prostate 1997, 32, 188 –195.

[17] J. H. Doroshow, Proc. Natl. Acad. Sci. 1986, 83, 4514 –4518.

[18] B. K. Sinha, J. R. F. Muindi, G. Batist, C. E. Myers, Proc.Am. Assoc. Cancer Res. 1985, 26, 225.

[19] V. Patel, M. Kelleher, M. McGurk, Br. Dent. J. 2010, 208,61–64.

[20] M. S. Lin, B. I. Jan, Electroanalysis 1997, 9, 340.[21] L.-C. Jiang, W.-D. Zhang, Electroanalysis 2009, 21, 988.[22] H. Wang, R. Guan, C. Fan, D. Zhu, G. Li, Sens. Actuators

B 2002, 84, 214.[23] I. L. de Mattos, L. Gorton, T. Laurell, A. Malinauskas,

A. A. Karyakin, Talanta 2000, 52, 791.[24] L. Li, Z. Du, S. Liu, Q. Hao, Y. Wang, Q. Li, T. Wang, Ta-

lanta 2010, 82, 1637.[25] J. M. Vago, V. C. Dall�Orto, E. Forzani, J. Hurst, I. N. Re-

zzano, Sens. Actuators B 2003, 96, 407.[26] M. G. Gargulio, N. Huynh, A. Proctor, A. C. Michael,

Anal. Chem. 1993, 65, 523–528.[27] J. Wang, Electroanalysis 2005, 17, 7–14.[28] K. Cui, Y. Song, Y. Yao, Z. Huang, L. Wang, Electrochem.

Commun. 2008, 10, 663.[29] F. Haque, J. Lunn, H. Fang, D. Smitthrud, P. Guo, ACS

Nano 2012, 6, 3251 –3261.[30] S. Wang, F. Haque, P. G. Rychahou, B. M. Evers, P. Guo,

ACS Nano 2013, 7, 9814–9822.[31] F. Haque, J. Li, H.-C. Wu, X.-J. Liang, P. Guo, Nano Today

2013, 8, 56 –74.[32] R. H. Holm, P. Kennepohl, E. I. Solomon, Chem. Rev.

1996, 96, 2239.[33] S. A. Trammell, A. Spano, R. Price, N. Lebedev, Biosens.

Bioelectron. 2006, 21, 1023.[34] R. M. Mueller, M. A. North, C. Yang, S. Hati, S. Bhatta-

charyya, J. Phys. Chem. B 2011, 115, 3632.[35] A. K. Yagati, T. Lee, J. Min, J.-W. Choi, Biosens. Bioelec-

tron 2013, 40, 283.

[36] A. K. Yagati, T. Lee, J. Min, J.-W. Choi, Bioelectrochemis-try 2012, 83, 8.

[37] S. V. Rao, K. W. Anderson, L. G. Bachas, Mikrochim. Acta1998, 128, 127.

[38] J. K. Myers, C. N. Pace, J. M. Scholtz, Protein Sci. 1995, 4,2138.

[39] A. Folch, M. Toner, Annu. Rev. Biomed. Eng. 2000, 2, 227.[40] X. Wang, M. Zhou, Y. Zhu, J. Miao, C. Mao, J. Shen, J.

Mater. Chem. B 2013, 1, 2132.[41] S.-U. Kim, A. K. Yagati, J. Min, J.-W. Choi, Thin Solid

Films 2009, 518, 682.[42] X. Liu, Z. Ma, J. Xing, H. Liu, J. Magn. Magn. Mater.

2004, 270, 1.[43] W. Shu, E. D. Laue, A. A. Seshia, Biosens. Bioelectron.

2007, 22, 2003.[44] X. Duan, Y. Li, N. K. Rajan, D. A. Routenberg, Y. Modis,

M. A. Reed, Nature Nanotech. 2012, 7, 401.[45] R. Asano, Y. Watanabe, H. Kawaguchi, H. Fukazawa, T.

Nakanishi, M. Umetsu, H. Hayashi, Y. Katayose, M. Unno,T. Kudo, I. Kumagai, J. Biol. Chem. 2007, 282, 27659.

[46] M. Rzepka, P. Lamp, J. Phys. Chem. B 1998, 102, 10894.[47] F. Zaera, C. S. Gopinath, J. Chem. Phys. 2002, 116, 1128.[48] K. Nakanishi, T. Sakiyama, Y. Kumada, K. Imamura, H.

Imanaka, Curr. Proteomics 2008, 5, 161.[49] B. Bonanni, D. Alliata, A. R. Bizzarri, S. Cannistraro,

Chem. Phys. Chem. 2003, 4, 1183.[50] A. K. Yagati, M. Jung, S.-U. Kim, J. Min, J.-W. Choi, Thin

Solid Films, 2009, 518, 634.[51] A. K. Yagati, S.-U. Kim, J. Min, J.-W. Choi, Biosens. Bio-

electron. 2009, 24, 1503.[52] J. E. Frew, H. A. O. Hill, Eur. J. Biochem. 1998, 172, 261.[53] M. J. Eddowes, H. A. O. Hill, J. Chem. Soc. Chem.

Commun. 1977, 21, 771.[54] P. Yeh, T. Kuwana, Chem. Lett. 1977, 1145.[55] A. Messerschmidt, R. Huber, T. Poulos, K. Wieghardt,

Handbook of Metalloproteins, Vol. 1, Wiley, Chichester,2001.

[56] R. Vazquez-Duhalt, J. Mol. Catal. B: Enzyme 1999, 7, 241.[57] J. T. Sage, D. Morikis, P. M. Cahmpion, Biochemistry 1991,

30, 1227.[58] K. Chu, J. Vojtchovsky, B. H. McMahon, R. M. Sweet, J.

Berendzen, I. Schlichting, Nature 2000, 403, 921.[59] A. R. Fanelli, E. Antonini, Biochim. Biophys. Acta 1958,

30, 608.[60] M. Nagai, Y. Sugita, Y. Yoneyama, J. Biol. Chem. 1969,

244, 1651.[61] J. J. Mieyal, R. S. Ackerman, J. L. Blumer, L. S. Freeman,

J. Biol. Chem. 1976, 251, 3436.[62] H. Matsubara, R. M. Sasaki, R. K. Chain, J. Biol. Chem.

1968, 243, 1725.[63] K. K. Rao, R. Cammack, D. O. Hall, C. E. Johnson, Bio-

chem. J. 1971, 122, 257.[64] C. Moreno, A. L. Macedo, I. Moura, J. LeGall, J. J. G.

Moura, J. Inorg. Biochem. 1994, 53, 219.[65] N. C. Veitch, Phytochemistry 2004, 65, 249–259.[66] L. Gorton, A. Lindgren, T. Larsson, F. D. Munteanu, T.

Ruzgas, I. Gazaryan, Anal. Chim. Acta 1999, 400, 91.[67] Y. Degani, A. Heller , J. Phys. Chem. 1987, 91, 1285.[68] A. A. Voityuk, J. Phys. Chem. B 2011, 115, 12202.[69] D. N. Beratan, J. N. Betts, J. N. Onuchic, Science 1991, 252,

1285.[70] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A. I. Yaropo-

lov, J. W. Whittaker, L. Gorton, Biosens. Bioelectron. 2005,20, 2517.

[71] R. S. Freire, C. A. Pessoa, L. D. Mello, L. T. Kubota, J.Braz. Chem. Soc. 2003, 14, 230.

Specia

lIssu

eD

IAG

NO

STIC

SSpecia

lIssu

eD

IAG

NO

STIC

S

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1275

Review

[72] W. Chen, S. Cai, Q. Q. Ren, W. Wen, Y.-D. Zhao, Analyst2012, 137, 49.

[73] H. Z. Zhao, Q. Du, Z.-S. Li, Q.-Z. Yang, Sensors (Basel).2012, 12, 10450.

[74] A. K. Yagati, T. Lee, J. Min, J.-W. Choi, Colloids Surf. B2012, 92, 161.

[75] Y. Song, H. Liu, L. Wan, Y. Wang, H. Hou, L. Wang, Elec-troanalysis, 2013, 25, 1400.

[76] Y. Song, L. Wan, K. Cui, L. Liu, M. Zhang, J. Liao, L.Wang, Z. Li, J. Electroanal. Chem. 2011, 656, 206.

[77] G.-C. Zhao, Z.-Z. Yin, L. Zhang, X.-W. Wei, Electrochem.Commun. 2005, 7, 256.

[78] A. Zhu, Y. Tian, H. Liu, Y. Luo, Biomaterials 2009, 30,3183.

[79] J.-J. Feng, G. Zhao, J.-J. Xu, H.-Y. Chen, Anal. Biochem.2005, 342, 280.

[80] L. Zhang, Biosens. Bioelectron. 2008, 23, 1610.[81] L. Zhang, Z. Shi, Q. Lang, J. Pan, Electrochem. Acta 2010,

55, 641.[82] C. Xiang, Y. Zou, L.-X. Sun, F. Xu, Electrochem.

Commun. 2008, 10, 38.[83] G. Suarez, C. Santschi, O. J. F. Martin, V. I. Slaveykova,

Biosens. Bioelectron. 2013, 42, 385.[84] I. Taniguchi, K. Watanabe, M. Tominaga, F. M. Hawkridge,

J. Electroanal. Chem. 1992, 333, 331.[85] A. K. Yagati, T. Lee, J. Min, J.-W. Choi, Biosens. Bioelec-

tron. 2013, 47, 385.[86] Z. Dai, X. Xu, H. Ju, Anal. Biochem. 2004, 332, 23.[87] A. Safavi, F. Farjami, Anal. Biochem. 2010, 402, 20.[88] W. Yang, Y. Li, Y. Bai, C. Sun, Sens. Actuators B 2006, 115,

42.[89] J. Zhang, M. Oyama, J. Electroanal. Chem. 2005, 577, 273.[90] C.-Y. Liu, J.-M. Hu, Biosens. Bioelectron. 2009, 24, 2149.[91] W.-L. Zhu, Y. Zhou, J.-R. Zhang, Talanta 2009, 80, 224.[92] Y. Zhang, J. Zheng, Electrochem. Commun. 2008. 10, 1400.[93] Z. Dai, Y. Xiao, X. Yu, Z. Mai, X. Zhao, X. Zou, Biosens.

Bioelectron. 2009, 24, 1629.[94] M.-Q. Xu, J.-F. Wu, G.-C. Zhao, Int. J. Electrochem. Sci.

2013, 8, 6502.[95] Y. Wang, M. Tang, X. Lin, F. Gao, M. Li, Microchim. Acta

2012, 176, 405.[96] A. Salimi, R. Hallaj, S. Soltanian, Biophys. Chem. 2007,

130, 122.[97] A. Sun, Q. Sheng, J. Zheng, Appl. Biochem. Biotechnol.

2012, 166, 764.[98] C. Liu, J. Hu, Electroanalysis 2008, 20, 1067.[99] L. Xie, Y. Xu, X. Cao, Colloids Surf. B Biointerf. 2013, 107,

245.[100] N. Hui, R.-F. Gao, X.-Q. Li, W. Sun, K. Jiao, J. Braz.

Chem. Soc. 2009, 20, 252.[101] N. Jia, Q. Lian, Z. Wang, H. Shen, Sens. Actuators B,

Chem. 2009, 137, 230.[102] S. Chen, R. Yuan, Y. Chai, L. Zhang, N. Wang, X. Li, Bio-

sens. Bioelectr. 2007, 22, 1268.[103] L. Ren, J. Dong, X. Cheng, J. Xu, P. Hu, Microchim. Acta

2013, 180, 1333.

[104] Y.-H. Wang, C.-M. Yu, Z.-Q. Pan, Y.-F. Wang, J.-W. Guo,H.-Y. Gu, Microchim. Acta 2013, 180, 659.

[105] Y. Wang, H. Zhang, D. Yao, J. Pu, Y. Zhang, X. Gao, Y.Sun, J. Solid State Electrochem. 2013, 17, 881.

[106] H. Tian, F. Liang, J. Jiao, J. Hu, J. Electrochem. Soc. 2013,160, B125.

[107] Z. Wang, J. Yi, S. Yang, Sens. Actuators B, Chem. 2013,176, 211.

[108] S. Palanisamy, S. Cheemalapati, S.-M. Chen, Anal. Bio-chem. 2012, 429, 108.

[109] J. Li, J. Tang, L. Zhou, X. Han, H. Liu, Bioelectrochemistry2012, 86, 60.

[110] R. Wang, J. Zhang, Y. Hu, Bioelectrochemistry 2011, 81,34.

[111] N. Moheimanian, J. B. Raoof, A. Safavi, R. Ojani, Electro-analysis 2012, 24, 1386.

[112] A. K. Yagati, T. Lee, J. Min, J.-W. Choi, Bioelectrochemis-try 2011, 80, 169.

[113] C. Ren, Y. song, Z. Li, G. Zhu, Anal. Bioanal. Chem. ,2005, 381, 1179–1185

[114] Y. Astuti, E. Topoglidis, A. G. Cass, J. R. Durrant, Anal.-Chim. Acta 2009, 648, 2–6.

[115] A. Devadoss, H. Han, T. Song, Y. P. Kim, U. Paik, Analyst2013, 138, 5025–5030.

[116] M. Magyar, K. Hajdu, T. Szabo, B. Endrodi, K. Hernadi,E. Horvath, A. Magrez, L. Forro, C. Visy, L. Nagy Phys.Status Solidi B 2013, 250, 2559–2563.

[117] M. Yang, F. Qu, Y. Lu, Y. He, g. shen, R. Yu, Biomaterials2006, 27, 5944–5950.

[118] L. Zhang, H. Cheng, H.-M. Zhang, L. Qu, Electrochim.Acta, 2012, 65, 122–126.

[119] Anees A. Ansari, Pratima R. Solanki, B. D. Malhotra, J.Biotechnol. 2009, 142, 179–184.

[120] Q. Wan, H. Song, H. Shu, Z. Wang, J. Zou, N. Yang, Col-loids Surf. B, Biointerf. 2013, 104, 181–185.

[121] Y. Umasankar, B. Unnikrishnan, S.-M. Chen, T.-W. Ting,Anal. Meth. 2012, 4, 3653 –3660.

[122] D. Shan, Q. B. Li, S. N. Ding, J. Q. Xu, S. Cosnier andH. G. Xue, Biosens. Bioelectron. 2010, 26, 536–541.

[123] C. Xiang, Y. Zou, L.-X. Sun, F. Xu, Sens. Actuators B 2009,136, 158 –162.

[124] X. Liu, H. Feng, J. Zhang, R. Zhao, X. Liu, D. K.Y. Wong,Biosens. Bioelectron. 2012, 32, 188–194.

[125] Y. Wang, X. Ma, Y. Wen, Y. Xing, Z. Zhang, H. Yang, Bio-sens. Bioelectron. 2010, 25, 2442 –2446.

[126] S. Komathi, A. I. Goplan, S.-K. Kim, G. S. Anand, K.-P.Lee, Electrochim. Acta 2013, 92, 71–78.

[127] X. Zhao, Z. Mai, X. Kang, X. Zou, Biosens. Bioelectron.2008, 23, 1032–1038.

[128] J. Xu, F. Shang, J. H.T. Luong, K. M. Razeeb, J. D. Glen-non, Biosens. Bioelectron. 2010, 25, 1313–1318.

Received: January 24, 2014Accepted: April 4, 2014

Published online: May 19, 2014

Specia

lIssu

eD

IAG

NO

STIC

SSpecia

lIssu

eD

IAG

NO

STIC

S

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 1259 – 1276 1276

Review