electrochemical enzyme immunoassay using model labels

Trends in Analytical Chemistry, Vol. 27, No. 6, 2008 Trends

Electrochemical enzymeimmunoassay using model labelsXue-Mei Li, Xiao-Yan Yang, Shu-Sheng Zhang

Enzyme immunoassays (EIAs) based on electrochemical detection offer

several potential advantages and have been applied in clinical, medical,

biotechnological, food and environmental analysis. Among the enzyme

labels employed, horseradish peroxidase (HRP), alkaline phosphatase (ALP)

and glucose oxidase (GOx) are the most common. This brief review reflects

recent advances, challenges, and trends of electrochemical EIAs focusing on

HRP, ALP or GOx as labels over the past five years. We especially emphasize

current development of EIAs combined with other developments, including

nanotechnology and miniaturization.

ª 2008 Elsevier Ltd. All rights reserved.

Keywords: Alkaline phosphatase; ALP; EIA; Electrochemical detection; Enzyme

immunoassay; Enzyme label; Glucose oxidase; GOx; Horseradish peroxidase; HRP

Xue-Mei Li, Xiao-Yan Yang,

Shu-Sheng Zhang*

Key Laboratory of Eco-chemical

Engineering,

Ministry of Education,

College of Chemistry and

Molecular Engineering,

Qingdao University of Science

and Technology, Qingdao

266042, P. R. China

*Corresponding author.

Tel.: +86 532 84022750;

Fax: +86 532 84022750;

E-mail: [email protected]

0165-9936/$ - see front matter ª 20080165-9936/$ - see front matter ª 2008

1. Introduction

Immunoassay based on the detection ofantibody-antigen (Ab-Ag) interaction isone of the most important analyticaltechniques, which has good applicationsin clinical diagnoses for certain tumor-associated disease and in experimentallaboratories, since it enables the specificdetection of small amounts of target mol-ecules in complex biological samples. Bycombining selectivity of an Ab, applica-bility for a wide range of analytes and theinherently very low limits of detection(LODs), immunoassay has become a verypopular analytical method for samples ofimportance in clinical, pharmaceutical,environmental and food analysis [1].

Label-free detection of the interactionbetween an Ag and an Ab has becomepossible with optical (including surface-plasmon resonance (SPR) [2] and localizedSPR [3]), piezoelectric (quartz crystalmicrobalance (QCM) [4]) and surface-topology scanning (atomic force micros-copy (AFM) [5]) transducers. Recently,label-free electrochemical immunoassayfor detection of proteins has become animportant topic in bioanalysis [6]. How-ever, the binding of an Ag to the appropriateAb is accompanied by only small physico-

Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.04.002Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.04.002

chemical changes. Lack of sufficient sensi-tivity for detecting analytes at lowconcentrations is a major impediment todevelopment of label-free immunosensors.The utility of biosensing immunosensorswould be greater if there was a properstrategy to amplify the immunologicalinteractions so as to result in morepronounced changes [7]. Enzymes are themost frequently-used labels linked to theAg or the Ab to visualize the binding event[8].

Enzyme-linked immunosorbent assay(ELISA), the most frequently appliedmethod for immunoassay, has beendeveloped extensively during the past fewdecades. However, ELISA has the disad-vantage of needing multiple incubationsand washing steps, all of which inhibitapplication of conventional immunoassaytechniques to fast, on-line, or fully auto-mated analyte determinations. Research-ers have tried to increase sensitivity ofELISA and shorten reaction times.

A wide variety of electrode materialshave been used as supports to fabricateimmunosensor devices (e.g., carbon paste,glassy carbon and gold (Au)). Recently,several immunosensor devices weredeveloped on screen-printed electrodes(SPEs) [9]. Au nanoparticles have beenextensively used in biomimetic interfacesand cytochemical labels for immobilizationand study of macromolecules (e.g., pro-teins and enzymes [10,11]).

An important trend of decentralizingdisease testing is miniaturization of diag-nostic technology [12]. Miniaturizationhas great potential for diagnostic tech-nology (e.g., improved accuracy, lowerpower and sample consumption, and useat point of care).

Immunosensors have been thesubject of expanding interest in the

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Trends Trends in Analytical Chemistry, Vol. 27, No. 6, 2008

immunochemical studies with enormous potential inclinical diagnosis, environmental and food analysis.

Development of specific immobilization of capture Ag/Ab that maintains its biological integrity and bioreac-tivity has been a critical issue in immunoassay. Con-ventional immobilization methods generally requireplasma to be treated, and surface treatment and bioassayfabrication are technically difficult processes. In somecases, these methods can result in loss of requiredimmunological activities.

Tang et al. have demonstrated a special protein-assaysystem based on a highly hydrophilic, non-toxic, con-ductive biomimetic interface [13]. A recent trend in theprotein-assay format is a shift from assay methods withconventional microtiter plates to those with integratedprotein microchips or microarrays [14]. Protein arrayshave been fabricated using mechanical microspotting[15], electrospray deposition [16] and micromosaictechniques [17]. In many cases, the detection of proteinsat the arrays is based on fluorescence (FL) or chemi-luminescence (CL) measurements, which usually requirelarge, expensive equipment.

In this respect, electrochemical enzyme immunoassay(EIA) represents the best combination of portability,sensitivity, selectivity, low cost, and suitability forvarious applications to detection.

There have been some reviews of electrochemical EIAin this journal [18,19] and another journal [8]. In thisarticle, we describe the advances in electrochemical EIAusing HRP, ALP and GOx as labels. We also describe thework of our group on electrochemical EIA in recentyears.

2. Enzymes and their substrates

Different enzymes (e.g., horseradish peroxidase (HRP),alkaline phosphatase (ALP) and glucose oxidase (GOx),urease, catalase, laccase, acetyl cholinesterase (AChE)and galactosidase) are common choices for enzymelabels in ELISA, of which ALP, HRP and GOx are themost common. ALP is a hydrolase that converts ortho-phosphoric monoesters into alcohols with an optimumactivity around pH 8–10 [20]. Substrates, such asphenyl phosphate, p-aminophenyl phosphate, p-nitro-phenyl phosphate, 1-naphthyl phosphate, 2-phospho-L-ascorbic acid (AAP) and 3-indoxyl phosphate, havebeen employed.

Chailapakul et al. [21] selected AAP for the substrateof ALP to detect mouse immunoglobulin (mIgG), as itcan produce L-ascorbic acid (AA), which is sensitive tothe electrochemical detector, does not foul the electrode,and has excellent stability. The LODs of 0.30 ng/mL and3.50 ng/mL for mIgG were obtained at a poly-o-amino-benzoic acid-modified, boron-doped diamond electrodeand a glassy-carbon electrode, respectively.

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Hydroquinone diphosphate (HQDP) has also been apopular ELISA substrate because its enzymatic hydroly-sis product (HQ) does not foul the electrode, even at thehigh pH associated with optimum ALP activity [22].

However, HRP catalyzes the oxidation of a widevariety of organic and inorganic substrates using H2O2.Various substances can act as substrates (e.g., indole-3-acetic acid, 1-methyluric acid, 3,3 0,5,5 0-tetramethylbenzidine (TMB), 2,2 0-azino-di(ethylbenzothiazoline-6-sulfonic acid) (ABTS), uric acid, paracetamol, ascorbicacid, p-phenylene diamine (PPD), o-phenylene diamine(OPD), p-aminophenol (PAP), o-aminophenol (OAP),1,5-dihydroxynaphthalene (DHN) and o-tilidine (OT)).

In ELISA for ALP, Costa-Garcia�s group reported 3-indoxyl phosphate (3-IP) as suitable with adsorptivevoltammetric detection in an EIA system [23]. The samegroup dealt with the use and the analytical optimizationof 3-IP as a substrate for the determination of HRPactivity [24]. The results showed that 3-IP is the firstsubstrate that could be used for ALP and HRP in affinityassays with the catalytic product of indigo blue.

In more recent studies, two new voltammetric EIAsystems (3,4-diaminobenzoic acid (DBA) [25] and 3,3 0-diaminobenzidine (DAB) [26]) were proposed for thedetection of tumor markers in human sera. The assayswere simple, inexpensive, rapid, reproducible and sensi-tive, implying a promising alternative approach forclinical diagnosis.

GOx catalyzes the oxidation of b-D-glucose to D-gluc-ono-1,5-lactone and hydrogen peroxide, using mole-cular oxygen as the electron acceptor. It is commonlyused in biosensors to detect levels of glucose by keepingtrack of the number of electrons passed through theenzyme by connecting it to an electrode and measuringthe resulting charge. Recently, AChE has been reportedto be applied for EIA using a voltammetric measurement[27]. Since AChE is a high-turnover enzyme for thehydrolysis of acetyl thiocholine, applying AChE bio-chemistry makes EIA very sensitive.

3. Protein immobilization

To achieve high specificity, high sensitivity, rapid re-sponse and flexibility of use, research continues to focuson new assembly strategies. Enzyme electrodes fabri-cated using self-assembled monolayers have been studiedextensively with a view to achieving greater control ofenzyme immobilization. Molecular level control has seenmultiple functionalities integrated into a single layer andquite complicated multilayer molecular constructionsfabricated. Recently, the use of layer-by-layer (LBL)assembly for the design of electrochemical biosensors hasattracted extensive attention [28].

Deposition of organized enzyme films on electrodesurfaces attracted increasing attention because of their


broad biotechnological applications, including biosen-sors and bioprocesses [29]. To achieve nanostructuredenzyme multilayer films with a high protein density,various immobilization strategies have been developed(e.g., step-by-step attachment of enzyme layers throughbiospecific interactions [30], covalent attachment [31],or alternate layer assembly of oppositely chargedmacromolecules [32]).

Mavre et al. [33] presented self-assembled biotinylatedenzyme aggregation on an SPE using magnetic iron-oxide nanoparticles and avidin. Two different self-assembly procedures were tested, taking advantage ofthe spontaneous aggregation of the nanoparticles in thepresence of avidin and the multivalent binding of bio-tinylated diaphorase toward avidin. With this approach,diaphorase oxidoreductase was detected to measure thebioelectrocatalytic oxidation of nicotinamide adeninedinucleotide (NADH) in the presence of a ferrocenemediator.

Recently, EIA was combined with other techniques(e.g., QCM) in developing a new electrochemical bio-sensor. A highly sensitive electrochemical EIA for Toxo-plasma gondii-specific IgG (Tg-IgG) in human serum wasdeveloped, based on enzyme-catalyzed amplification dueto the formation of an insoluble precipitate on the sur-face of the QCM. The characteristics of the immuno-reaction and the enzyme-catalyzed precipitation werestudied using electrochemical impedance spectroscopy(EIS), cyclic voltammetry (CV) and QCM [34].

The non-specific adsorption of the enzyme-labeled Absposed a severe problem and set the LOD at a muchhigher level than that defined by the two equilibriumconstants for the formation of the conjugates. Zhanget al. [35] reported a simple method of suppressing thenon-specific adsorption of an immunoreagent on a sur-face. Two polyanionic polymers, poly(acrylic acid-co-maleic acid) and poly(acrylic acid), were used separatelyto neutralize non-specifically binding positively-chargedmicrodomains of the avidin, which attached to the redoxpolymer film. Amperometric enzyme-amplified sand-

Figure 1. (a) 96-well microplate and (b) 96 sensors containing carbon andduced from [38] with permission).

wich-type immunoassays with biotin-labeled anti-rabbitIgG (aRIgG) and HRP-labeled aRIgG as a demonstrationsystem were performed. The LOD was 7 pg/mL for IgG.

4. Detection techniques

4.1. Multi-channel electrochemical detectorFrom the development of ELISA, incorporation ofmicrotiter plates with an EIA reader provides a veryefficient procedure for immunoassay. Due to its versa-tility, the multi-channel electrochemical detector (MED)have been developed and used for chemical and bio-chemical analysis. Application of the MED for immuno-analysis was not reported until Tang et al. [36]constructed an MED system comprising a microcom-puter-assisted, 16-channel potentiostat and eight sets ofplatinum (Pt) electrodes as an efficient detector forimmunoassay. With this MED system, electroactiveenzymatic products produced in eight microtiter wellscan be analyzed simultaneously with the amperometricprocedure developed. RIgG was coated on the microtiter-well surface, competition between the free and immobi-lized RIgG for the goat anti-RIgG (GaRIgG) in microtiterwells proceeded during the incubation period. With theMED system, the dynamic range and the LOD for RIgGwere 10–1000 ng/mL (0.064–6.4 pM) and 1.0 ng/mL(6.4 pM), respectively. A similar system was applied to asmall-molecule compound, 2,4-dichlorophenoxyaceticacid, in environmental analysis, with a LOD of 0.072 ng/mL [37].

The disposable sensor array employed is a devicemanufactured using the screen-printing technology. Itcomprises a 96-well plate whose bottom was modifiedwith an array of 96 screen-printed sensors, each ofwhich comprised a carbon working electrode and anAg/AgCl pseudoreference electrode (Fig. 1). Thismulti-channel electrochemical plate was designed anddeveloped for simultaneous, independent measurementsof nucleic acids, incorporating HRP as a marker.

Ag/AgCl printed electrodes with the comb-type connections (Repro-

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Piermarini et al. first developed the analytical immuno-sensor array, based on a microtiter plate coupled to anMED system using the intermittent pulse amperometry(IPA) technique, for simultaneous, independent mea-surements of aflatoxin B1 (AFB1), incorporating ALP asa marker [38]. The LOD of AFB1 was 30 pg/mL with thissystem, and AFB1 could be distinguished from otheraflatoxins, with the exception for aflatoxin G1 (AFG1),using anti-aflatoxin B1 Ab.

4.2. Scanning electrochemical microscopyScanning electrochemical microscopy (SECM) has beenused extensively in the past decade for visualizinglocalized biocatalytic activity on surfaces [39], andattempts were made to use the spatial information ob-tained to eliminate interference [40]. Only recently wasSECM demonstrated to be a useful microfabrication andmicrocharacterization tool for biomolecules, due to itssmall tip size, the versatility of the tip material and itsdifferent modes of operation [41].

Recently, miniaturized electrochemical devices withseparate electrodes and Ab-immobilized chips demon-strated the simultaneous detection of several proteins[42]. Aguilar et al. [43] produced a self-contained,microelectrochemical EIA device eliminating the need forexternal reference and auxiliary electrodes. The devicehas the advantages of the SECM systems but is bettersuited for small volumes, miniaturization, and integra-tion with microfluidics. With the recessed microdiskdevice, the volumes of the immunoassay analytes weredown to tens of picoliters (Fig. 2).

Yasukawa et al. [44] reported a dual EIA for thedetection of pepsinogen 1 (PG1) and pepsinogen 2 (PG2)based on SECM. An HRP-labeled sandwich immuno-assay on microspots comprising anti-PG1 IgG Ab andanti-PG2 IgG Ab was used as the model system. HRPconverted ferrocene methanol (FcOH) to its oxidized form

Figure 2. Self-contained microelectrochemical immunoas


(Fc+OH) at localized areas corresponding to microspotscontaining both immunocomplexes. The dual imaging ofPG1 and PG2 with SECM was performed on a substratecontaining microspots of PG1 and PG2 immunocom-plexes.

Jin�s group employed SECM-immunoassay to detect anoncofetal Ag, CA15-3, concentrating the sandwich en-zyme immunocomplex (Ab-Ag-Ab*) on the substrate viaa microcell [45]. The LOD of the method was 2.5 U/mL,with a higher sensitivity than that of the SECM-immu-noassay directly on a plane substrate. Wittstock et al.demonstrated the application of the SECM technique ininvestigating different enzymes within a complex andtheir interactions [46].

5. Combination with other technologies

5.1. Paramagnetic microbead-based enzymeimmunoassayImportant, continuing goals in improving immuno-assays are:� to minimize the volumes of reagents that need to be

used;� to minimize the waste produced;� to shorten the assay times; and,� to lower the LODs.

Paramagnetic microbeads can be used as a mobilesolid phase, on which an immunoassay sandwich isassembled, and can easily be used in small volumes tominiaturize a method. Magnetic beads are known to bea powerful, versatile tool in a variety of analytical andbiotechnology applications [47]. The use of non-porousmagnetic beads greatly improves the performance ofthe immunological reaction, due to an increase in thesurface area and the faster assay kinetics achievedbecause the beads are in suspension and the analytical

say device (Reproduced from [43] with permission).


target does not have to migrate very far [48]. Magneticbeads can be easily manipulated magnetically by usingpermanent magnets or electromagnets, so the analysisof samples performed on magnetic beads can easily beachieved without any pre-enrichment, purification orpre-treatment steps, which are normally necessary forstandard methods [49]. When microbeads are dispersedthroughout a solution, the distance that reagents mustdiffuse is minimized. A multilayer of magnetic beads,which form a three-dimensional structure on the sur-face of the biosensor when an external magnet is ap-plied, could enhance the total sensor surface area andimprove the sensitivity of the biosensor [50]. Withthese characteristics, microbeads are good candidatesfor use in a fluidic system [51], as demonstrated for anelectrochemical immunoassay with mIgG as the modelanalyte.

Thomas et al. [52] reported a sensitive, miniaturizedEIA, formed by coupling a microbead-based immuno-assay with an interdigitated array (IDA) electrode. AnIDA electrode amplified the signal by recycling an elec-trochemically redox-reversible molecule (Fig. 3A). Anenzyme-labeled sandwich immunoassay on paramag-netic microbeads with mIgG as the analyte and b-galactosidase as the enzyme label was used as the model

Figure 3. (A) Analytical scheme of the electrochemical assay with interdigidox cycling. (B) Two types of experimental design for microbead-based asswith permission).

system. By positioning the microbeads near the electrodesurface with a magnet, the enzyme reaction was mea-sured continuously with dual-electrode detection,amplifying the signal four-fold compared to single-electrode detection. On a similar principle, comb IDAelectrodes were fabricated, one benefit of which wasthat the beads were larger than the electrode spacing sothat they did not come into direct contact with thesurface and avoided potential fouling of the electrode.The comb IDA produced current three times more thana coplanar IDA (Fig. 3B) [53].

In their ongoing studies of microbead-based immuno-assay with an IDA electrode, Thomas et al. [54] reporteda paramagnetic bead-based electrochemical immunoas-say using a fluidic device for detecting bacteriophageMS2. The bead was coated with streptavidin, to which abiotinylated rabbit anti-MS2 IgG was attached. Thesandwich immunoassay was developed by capturingthe MS2 virus, and then attaching a rabbit anti-MS2IgG-b-galactosidase conjugate to another site on thevirus. The immunoassay was detected amperometricallywith p-aminophenyl galactopyranoside (PAPG) as sub-strate on both a rotating disk electrode (RDE) and an IDAelectrode. The LOD for MS2 on the IDA electrode waslower than that on the RDE.

tated array (IDA) electrodes coupling the enzymatic reaction and re-ay with (a) comb IDAs and (b) coplanar IDAs (Reproduced from [53]



A fully-automated fluid system for bead-based immu-noassay with electrochemical detection was furtherdeveloped for detection of bacteriophage MS2, oval-bumin and carcinoembryonic Ag (CEA) [55].

This strategy offers great promise for rapid, simple,cost-effective and on-site analysis of biological, food andenvironmental samples.

5.2. Nanostructure-based enzyme immunoassayNanostructures (e.g., nanowires, nanoparticles andcarbon nanotubes) have been used as smart buildingblocks for emerging electronic and sensing devices. Inparticular, sensors based on metallic nanowires areinteresting, as they produce higher sensitivity, highercapture efficiency and faster response times, due to theirlarge adsorption surface (large surface-to-volume ratio),high electrical conductivity and short diffusion time.Recently, biosensor technology has had a dramaticimpact on detection of protein-binding events. Greatprogress is being made in microfabrication and nano-technology, and we predict that these technologies willhave a significant impact on biosensors.

Bhansali�s group has successfully developed anddemonstrated the analytical concept based on sandwichimmunoassay and electrochemical detection for deter-mination of cholesterol, cortisol and cytokeratin-7 usingAu nanowires [56] based on a micro electro-mechanicalsystem. We conclude that functionalized Au nanowireswith a micro-fluidic device using enzyme-fragment-complementation technology can provide an easy, sen-sitive assay for detection in serum (Fig. 4).

Figure 4. Electrochemical test set-up with nanowire ali


5.3. Capillary electrophoresis with enzymeimmunoassaySince capillary electrophoresis (CE) in its modern formwas first described by Jorgenson and Luckas in 1981[57], it has become one of the most powerful, concep-tually simple separation techniques for the analysis ofcomplex mixtures, due to its high resolution and rela-tively short analysis times. Bossi and Baldwin reviewedadvances in CE coupled to biosensor detection [58,59].

Since Nielsen [60] demonstrated the ability of CE toseparate an Ag-Ab complex from Ab and free Ag, CE hasproved to be a powerful separation tool, which hastherefore been examined for use with rapid, efficientimmunoassays in the past decade. CE-based immuno-assays (CE-IAs) offered the possibility of high masssensitivity, small sample-volume requirements, rapidseparations, simultaneous determination of multipleanalytes, and compatibility with automation.

Ultrasensitive CE-IA is primarily based on ultraviolet(UV) and laser-induced FL (LIF) detection, although themajor disadvantage of UV detection is the lack of sensi-tivity. Although LIF is very sensitive, high backgroundnoise due to Rayleigh and Raman scattering and fluo-rescent impurities in the solvent are major disadvan-tages. Amperometry is an alternative for detection in CE-IAs, as it provides excellent sensitivity for the smalldimensions associated with CE, while offering a highdegree of selectivity towards electroactive species. Jin�sgroup [61,62] has performed CE-EIAs with electro-chemical detection (CE-EIA-ED) for different compoundsof clinical interest, using HRP as label and TMB as

gnment (Reproduced from [56] with permission).

Figure 5. Immunochip (Reproduced from [65] with permission).


substrate. The system comprised a separation capillary, areaction capillary and an electrochemical detector,which included a carbon-fiber microdisk bundle elec-trode as the working electrode.

The free enzyme-labeled Ab (Ab*) and the bound Ag-Ab* complex produced in the solution were separated bycapillary zone electrophoresis in a separation capillarywhen a high separation voltage was given. They thencatalyzed enzyme substrate TMB(Red) and H2O2 in areaction capillary following the separation capillary. Thereaction product, TMB(Ox), can be determined usingamperometric detection at the outlet of the reactioncapillary. The authors used this system for the ampero-metric detection of cortisol [61] in a competitive immu-noassay format and for carcinoma tumor-marker Ag-125 (CA125) [62] in a non-competitive mode.

Though CE-IA has been extensively applied to singletumor-marker analysis, a limited amount of research hascovered simultaneous detection of multiple tumormarkers. Our group developed the CE-EIA-ED system forsimultaneous multi-analyte immunoassays that detectedthree important tumor markers (i.e. prostate-specific Ag(PSA), CEA, and human chorionic gonadotropin (HCG))on a Pt electrode, with HRP as label and OAP as sub-strate [63]. With a similar system, two other tumormarkers (i.e. a-fetoprotein (AFP) and thyroxine (T4)) inhuman sera were detected with non-competitive andcompetitive models, respectively [64]. Our work ex-tended previous concepts in electrochemical-based ELI-SA, and provided an important foundation for the futuredevelopment of multi-analyte detection.

5.4. Chip-based capillary electrophoresis with enzymeimmunoassayMiniaturized immunoassay methods using on-chip CE asseparation method with optical detection have beenreported as having the advantages of good reproduc-ibility and shorter reaction times, because the immuno-reaction also occurs in solution. However, fluorescentdye-labeled Ab is observed in CE as a broad peak due tothe charge heterogeneity of the Ab, and this affects theLOD by decreasing the signal-to-noise ratio.

Wang et al. [65] described a microfluidic device forelectrochemical EIA for measuring mIgG using chip-based CE as a separation technique. As shown in Fig. 5,the chip allowed integration of the steps of EIA, elec-trophoretic separation, post-column enzyme catalyticreaction and electrochemical detection to create a lab-on-a-chip. Based on a non-competitive format, the ALP-labeled mIgG in the reagent reservoir and the mIgG inthe analyte reservoir were complexed in the immuno-reaction chamber by applying a suitable voltage, fol-lowed by electrophoretic separation of the free Ab andAb-Ag complex. The enzyme conjugate could catalyzethe hydrolytic reaction of 4-aminophenyl phosphate (p-APP), and the liberated 4-aminophenol (p-AP) hydroly-

sate product could be detected at a mass-produced SPEdetector. Based on the well-defined concentrationdependence and low noise level, an LOD of around2.5 · 10�16 g/mL could be reached.

Wang et al. [66] demonstrated a non-competitiveimmunoassay to determine bone morphogenic protein-2(BMP-2) by CE-CL using HRP-labeled Ab2-mAb andBMP-2 as the model. The LOD of BMP-2 was 6.2 pM (75zmol).

Kawabata et al. [67] reported an immunoassay usingDNA-coupled Ab for bound/free separation in a liquid-phase binding-assay format on chip platform by LIF.Since DNA fragments have high charge-to-mass ratio,sufficient to suppress electrophoretic heterogeneity,DNA-coupled Ab and its complex with Ag showedsharper peaks.

5.5. Lab-on-a-chip enzyme immunoassayProtein chips that are used to check blood or urine forabnormal proteins related to cancer, arthritis, or heartdisease will revolutionize clinical analysis. In addition,protein chips will contribute to basic research to revealcomplicated protein-protein interactions in different celltypes and complex chains of chemical intracellularcommunication. The time required for detection will besignificantly reduced because electrochemical reactionsproceeding on the surface of a metal electrode are usedrather than a reaction that proceeds in the bulk of thesolution. Such ‘‘lab-on-a-chip’’ devices can thus dra-matically change the speed and the scale at whichchemical analyses are performed.

While early lab-on-a-chip immunoassay studiesfocused on optical detection, there are few reports of



analogous on-chip EIAs. Kojima et al. [68] designed anelectrochemical protein array comprising 36 Pt workingelectrodes, with different capture Abs, and operated inthe sandwich EIA mode. To solve the problem of con-formational changes and the loss of activity in directimmobilization of biomolecules, two layers of plasma-polymerized films (PPFs) were used in this study. Fordetection of a-1-fetoprotein (AFP) and b2-microglobulin(b2MG), a distinct current increase following the oxi-dation of hydrogen peroxide produced by the enzymaticreaction of glucose oxidase was observed.

6. Electrochemical immunosensor

Conventional electrochemical immunosensors arebased on immobilization of Ab or Ag on the surface ofelectrodes. Among different immunosensors, the mostcommonly used is the amperometric immunosensor,which integrates high specificity due to the immuno-reaction between Ab and Ag with significant amplifi-cation of the signal catalyzed by the labeled enzyme.Numerous immunosensors for single-analyte tumor-marker measurements and for point-of-care cancerdiagnostics have been reported, and were reviewedrecently [69,70].

Figure 6. (A) Biosensor with eight IrOx working electrodes. (B) Substrate cwith permission).


As for construction of an immunosensor, the crucialstep is immobilization of immunoreagent onto the elec-trode surface. The immobilization method will determinethe sensitivity and the reproducibility of the immuno-sensor. The attachment of reactants to the supportthrough sol-gel entrapment or through the specificbiotin-avidin interaction is an effective, reliable approachfor protein immobilization.

A novel immobilization method was proposed forpreparation of a separation-free immunosensor usingadsorption of CA125 on colloidal Au nanoparticlescoated on an electrode, which was stabilized with acellulose acetate membrane [71]. Our group hasdeveloped some biosensors by entrapping HRP in acolloidal Au-nanoparticle-modified chitosan membrane(Au-chitosan) to modify the indium-tin oxide (ITO)electrode [72].

Wilson [73] described a dual-electrode enzymeimmunosensor for simultaneous amperometric measure-ments of two important tumor markers, CEA and AFP.Capture Abs were immobilized on the porous iridium-oxide (IrOx) electrodes by covalent attachment using(3-aminopropyl)triethoxysilane, and glutaraldehyde,analytes were detected using ALP-labeled Abs andamperometric measurement of HQ oxidation. IrOx hasseveral favorable features as a matrix in immunosensor

ontaining multiple sensors and sample wells (Reproduced from [22]


fabrication, providing a porous three-dimensionalhydrous environment for immobilized proteins, andhindering diffusion of electroactive enzyme-generatedproduct in the matrix. This will probably allow thedevelopment of smaller devices containing sensors thatare closer together than could be achieved using planarelectrodes. As indicated in Fig. 6, Wilson�s group furtherdeveloped the simultaneous electrochemical multi-analyte immunoassay (SEMI) sensor to give simulta-neous quantitative detection of four proteins, with eightIrOx electrodes using non-competitive immunoassay[74] and seven important tumor markers in competitiveimmunoassay [22].

7. Simultaneous multi-analyte immunoassay

Simultaneous multi-analyte immunoassays (SMIAs) canquantitatively measure the concentrations of multipleproteins in a single assay and are the basis of importantnew analytical methods. They use less sample, increasetest throughput, reduce the cost per test, and improvetest efficiency compared to single-analyte assays.

Although the development of EIS has focused pri-marily on single-analyte assays, several methods forperforming multi-analyte analysis using parallel single-analyte immunoassays and SEMI have been describedwith multiple labels, such as enzymes, metal ions andnanoparticles. Wang et al. [75] demonstrated a newbiochip strategy for simultaneous measurements of glu-cose and insulin, comprising the reaction of ALP-labeledAb with insulin and the reaction of the glucose dehy-drogenase (GDH) enzyme with its glucose substrate inthe presence of its �-nicotinamide adenine dinucleotide(NAD+) cofactor. After electrophoretic separation of thefree Ab, Ab-Ag complex, and the NADH product of theglucose enzymatic reaction, there followed a post-column reaction of the ALP enzyme with its p-nitro-phenyl-phosphate substrate. Both the b-nicotinamideadenine dinucleotide (NADH) and the p-nitrophenol(p-NP) products were monitored at the downstreamamperometric detector.

While multiple-label SEMI methods often involve acompromise in assay conditions that increase thecomplexity and reduce the convenience of thesemethods, SEMIs that use a single label to detect allanalytes can offer advantages in simplicity compared tomulti-label methods, so several single-label SEMImethods have been reported. Ju�s group proposed adisposable immunosensor array for simultaneous elec-trochemical determination of carbohydrate Ag 19-9(CA19-9) and CA125 with HRP as the single label[76]. The immunosensor array was fabricated usingcellulose-acetate membrane to co-immobilize thionineas a mediator and two kinds of Ags on two carbonelectrodes of a screen-printed chip, respectively. The

corresponding HRP-labeled Abs were captured on themembranes, on which HRP catalyzed the reduction ofH2O2 to produce detectable signals. Electrochemicaland electronic cross-talks between the electrodes couldbe avoided, with the immobilized thionine as a medi-ator to shuttle electrons. Sensitive, precise, and accu-rate multi-analyte assays for measuring proteinmarkers in biological samples will be valuable tools ina wide range of clinical applications.

8. Conclusions and perspectives

In conclusion, electrochemical EIAs have evolvedgreatly in the past five years with respect to highspecificity, high sensitivity, rapid response and low cost.EIA combined with other techniques (e.g., QCM and CE)has been developed to give new electrochemical assays.Miniaturized electrochemical devices with separateelectrodes and Ab-immobilized chips have demonstratedsimultaneous detection of several proteins, so such‘‘lab-on-a-chip’’ devices can dramatically change thespeed and the scale at which chemical analyses areperformed.

Signal amplification and noise reduction are crucialfor obtaining low LODs in clinical immunoassays [77].Amplification strategies will be explored to achieve highprotein immobilization, which will significantly enhancethe detectable signal. Various immobilization methodswill be developed (e.g., LBL attachment of enzymesthrough biospecific interactions of biotin and avidin, or,alternatively, using bioconjugates featuring enzymelabels and secondary Abs linked to nanostructures). Thenanoparticle label is ideal in biotechnological systemsdue to its inherent advantages, such as ease of prepa-ration and good biocompatibility. Hollow Au micro-spheres provide a biocompatible microenvironment forproteins, and greatly amplify the coverage of enzyme onthe electrode surface. Furthermore, the electrochemicalsignal is amplified by both magnetic bionanosphere la-bels and the bound enzyme on the magnetic bionano-spheres toward the catalytic reaction of substrate. Theassay sensitivity using HRP as enhancer could be furtherincreased 100 times compared with that without HRP[78], so there is no doubt that electrochemical EIA willbecome a powerful tool with a wide range of applicationsin the near future.

Acknowledgements

This work was supported by the Natural Science Foun-dation of Shandong Province (No. Y2007B31), theScientific and Technical Development Project of Qingdao(06-3-1-4-yx), and the National Natural ScienceFoundation of China (No. 20775038).



References

[1] G.N. Zhu, M.J. Jin, W.J. Gui, Y.R. Guo, R.Y. Jin, C.M. Wang,

C.Z. Liang, Y.H. Liu, S.T. Wang, Food Chem. 107 (2008) 1737.

[2] C. Boozer, J. Ladd, S.F. Chen, S.Y. Jiang, Anal. Chem. 78 (2006)

1515.

[3] T. Endo, S. Yamamura, N. Nagatani, Y. Morita, Y. Takamura,

E. Tamiya, Sci. Technol. Adv. Mater. 6 (2005) 491.

[4] K. Yano, U.T. Bornscheuer, R.D. Schmid, H. Yoshitake, H.S. Ji,

K. Ikebukuro, Y. Masuda, I. Karube, Biosens. Bioelectron. 13

(1998) 397.

[5] Y. Dong, C. Shannon, Anal. Chem. 72 (2000) 2371.

[6] M. Vestergaard, K. Kerman, N. Nagatani, Y. Takamura, E. Tamiya,

J. Am. Chem. Soc. 127 (2005) 11892.

[7] I.H. EI-Sayed, X.H. Huang, M.A. EI-Sayed, Nano Lett. 5 (2005)

829.

[8] M. Diaz-Gonzalez, M.B. Gonzalez-Garcia, A. Costa-Garcia,

Electroanalysis (N.Y.) 17 (2005) 1901.

[9] L. Micheli, R. Grecco, M. Badea, D. Moscone, G. Palleschi, Biosens.

Bioelectron. 21 (2005) 588.

[10] M. Li, Y.C. Lin, K.C. Su, Y.T. Wang, T.C. Chang, H.P. Lin, Sens.

Actuators, B 117 (2006) 451.

[11] C. Xia, F. Xin, C. Ke, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 20

(2005) 1805.

[12] Z.P. Chen, Z.F. Peng, Y. Luo, B. Qu, J.H. Jiang, X.B. Zhang,

G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 23 (2007) 485.

[13] D. Tang, R. Yuan, Y.Q. Chai, Biosens. Bioelectron. 22 (2007)

1116.

[14] L.J. Kricka, Clin. Chem. Acta 307 (2001) 219.

[15] P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, D.J. Cahill, Anal.

Chem. 75 (2003) 4368.

[16] N.V. Avseenko, T.Y. Morozova, F.I. Ataullakhanov, V.N. Morozov,

Anal. Chem. 74 (2002) 927.

[17] A. Bernard, B. Michel, E. Delamarche, Anal. Chem. 73 (2001) 8.

[18] M. Farre, L. Kantiani, D. Barcelo, Trends Anal. Chem. 26 (2007)

1009.

[19] N.J. Ronkainen-Matsuno, J.H. Thomas, H.B. Halsall, W.R. Heineman,

Trends Anal. Chem. 21 (2002) 213.

[20] W.W. Cleland, A.C. Hengge, Chem. Rev. 106 (2006) 3252.

[21] A. Preechaworapun, T.A. Ivandini, A. Suzuki, A. Fujishima, O.

Chailapakul, Y. Einaga, Anal. Chem. 80 (2008) 2077.

[22] M.S. Wilson, W. Nie, Anal. Chem. 78 (2006) 6476.

[23] C.F. Bobes, M.T.F. Abedul, A. Costa-Garcia, Electroanalysis (N.Y.)

13 (2001) 559.

[24] P. Fanjul-Bolado, M.B. Gonzalez-Garcia, A. Costa-Garcia, Electro-

analysis (N.Y.) 16 (2004) 988.

[25] S. Zhang, P. Du, F. Li, Talanta 72 (2007) 1487.

[26] S. Zhang, J. Yang, J. Lin, Bioelectrochemistry 72 (2008) 47.

[27] H. Matsuura, Y. Sato, O. Niwa, F. Mizutani, Anal. Chem. 77

(2005) 4235.

[28] W. Zhao, J.J. Xu, H.Y. Chen, Electroanalysis (N.Y.) 18 (2006)

1737.

[29] H. Ai, S.A. Jones, Y.M. Lvov, Cell Biochem. Biophys. 39 (2003)

23.

[30] B. Limoges, J.M. Saveant, D. Yazidi, Aust. J. Chem. 59 (2006)

257.

[31] S. Zhang, W. Yang, Y. Niu, C. Sun, Anal. Chim. Acta 253 (2004)

209.

[32] E.J. Calvo, C.B. Danilowicz, A. Wolosiuk, Phys. Chem. 7 (2005)

1800.

[33] F. Mavre, M. Bontemps, S. Ammar-Merah, D. Marchal, B.

Limoges, Anal. Chem. 79 (2007) 187.

[34] Y.J. Ding, H. Wang, G.L. Shen, R.Q. Yu, Anal. Bioanal. Chem. 382

(2005) 1491.

[35] Y. Zhang, A. Heller, Anal. Chem. 77 (2005) 7758.

[36] T.C. Tang, A. Deng, H.J. Huang, Anal. Chem. 74 (2002) 2617.


[37] A.P. Deng, H. Yang, Sens. Actuators, B 124 (2007) 202.

[38] S. Piermarini, L. Micheli, N.H.S. Ammida, G. Palleschi, D. Moscone,

Biosens. Bioelectron. 22 (2007) 1434.

[39] M. Niculescu, S. Gaspar, A. Schulte, E. Cscregi, W. Schuhmann,


[40] F. Turcu, G. Hartwich, D. Schafer, W. Schuhmann, Macromol.

Rapid Commun. 26 (2005) 325.

[41] M. Maciejewska, D. Schafer, W. Schuhmann, Electrochem.

Commun. 8 (2006) 1119.

[42] D. Ogasawara, Y. Hirano, T. Yasukawa, H. Shiku, K. Kobori,

K. Ushizawa, S. Kawabata, T. Matsue, Biosens. Bioelectron. 21

(2006) 1784.

[43] Z.P. Aguilar, W.R. Vandaveer, I. Fritsch, Anal. Chem. 74 (2002)

3321.

[44] T. Yasukawa, Y. Hirano, N. Motochi, H. Shiku, T. Matsue,


[45] X.L. Zhang, X.W. Peng, W.R. Jin, Anal. Chim. Acta 558 (2006)

110.

[46] T. Wilhelm, G. Wittstock, Angew. Chem. Int. Ed. Engl. 42 (2003)

2248.

[47] Y.Y. Lin, G.D. Liu, C.M. Wai, Y.H. Lin, Electrochem. Commun. 9

(2007) 1547.

[48] E. Zacco, M.I. Pividori, S. Alegret, R. Galve, M.P. Marco, Anal.

Chem. 78 (2006) 1780.

[49] E. Zacco, J. Adrian, R. Galve, M.P. Marcob, S. Alegret, M.I. Pividori,


[50] O.G. Tovmachenko, C. Graf, D.J. van den Heuvel, A. van Blaaderen,

H. Gerritsen, Adv. Mater. 18 (2006) 91.

[51] M.M. Miller, P.E. Sheehan, R.L. Edelstein, C.R. Tamanaha,

L. Zhong, S. Bounnak, L.J. Whitman, R.J. Colton, J. Magn. Magn.

Mater. 225 (2001) 138.

[52] J.H. Thomas, S.K. Kim, P.J. Hesketh, H.B. Halsall, W.R. Heineman,

Anal. Biochem. 328 (2004) 113.

[53] S.K. Kim, P.J. Hesketh, C. Li, J.H. Thomas, H.B. Halsall,

W.R. Heineman, Biosens. Bioelectron. 20 (2004) 887.

[54] J.H. Thomas, S.K. Kim, P.J. Hesketh, H.B. Halsall, W.R. Heineman,

Anal. Chem. 76 (2004) 2700.

[55] X.H. Fu, Biochem. Eng. J. 39 (2008) 267.

[56] S. Aravamudhan, A. Kumar, S. Mohapatra, S. Bhansali, Biosens.

Bioelectron. 22 (2007) 2289.

[57] J.W. Jorgenson, K.D. Lukacs, Anal. Chem. 53 (1981) 1298.

[58] R.P. Baldwin, J. Pharm. Biomed. Anal. 19 (1999) 69.

[59] A. Bossi, S.A. Piletsky, P.G. Righetti, A.P. Turner, J. Chromatogr.

892 (2000) 143.

[60] R.G. Nielsen, E.C. Rickard, P.F. Santa, D.A. Sharknas,

G.S.J. Sittampalam, J. Chromatogr. 539 (1991) 177.

[61] M. Jia, Z. He, W. Jin, J. Chromatogr., A 966 (2002) 187.

[62] Z. He, N. Gao, W. Jin, Anal. Chim. Acta 497 (2003) 75.

[63] S.S. Zhang, X.M. Li, F. Zhang, Electrophoresis 28 (2007)

4427.

[64] X.M. Li, F. Zhang, S.S. Zhang, J. Sep. Sci. 31 (2008) 336.

[65] J. Wang, A. Ibanez, M. Chatrathi, A. Escarpa, Anal. Chem. 73

(2001) 5323.

[66] J.H. Wang, W.H. Huang, Y.M. Liu, J.K. Cheng, J. Yang, Anal.

Chem. 76 (2004) 5393.

[67] T. Kawabata, M. Watanabe, K. Nakamura, S. Satomura, Anal.

Chem. 77 (2005) 5579.

[68] K. Kojima, A. Hiratsuka, H. Suzuki, K. Yano, K. Ikebukuro,

I. Karube, Anal. Chem. 75 (2003) 1116.

[69] J. Lin, H. Ju, Biosens. Bioelectron. 20 (2005) 1461.

[70] J. Wang, Biosens. Bioelectron. 21 (2006) 1887.

[71] L. N Wu, J. Chen, D. Du, H.X. Ju, Electrochim. Acta 51 (2006)

1208.

[72] J. Lin, L. Zhang, S. Zhang, Anal. Biochem. 370 (2007) 180.

[73] M.S. Wilson, Anal. Chem. 77 (2005) 1496.

[74] M.S. Wilson, W. Nie, Anal. Chem. 78 (2006) 2507.


[75] J. Wang, A. Ibanez, M.P. Chatrathi, J. Am. Chem. Soc. 125 (2003)

8444.

[76] J. Wu, Z. Zhang, Z. Fu, H. Ju, Biosens. Bioelectron. 23 (2007) 114.

[77] S. Long, E. Campbell, R. Mackinnon, Science (Washington, D.C.)

309 (2005) 903.

[78] D. Tang, R. Yuan, Y. Chai, Anal. Chem. 80 (2008) 1582.


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