Current Trends in Biotechnology and Pharmacy

ISSN 0973-8916

Editors

Prof. K.R.S. Sambasiva Rao, India Prof. Karnam S. Murthy, USAkrssrao@abap.in skarnam@vcu.edu

Editorial Board

Prof. Anil Kumar, India Prof. P. Appa Rao, IndiaProf. Aswani Kumar, India Dr. P. Ananda Kumar, IndiaProf. Carola Severi, Italy Prof. Chellu S. Chetty, USAProf. K.P.R. Chowdary, India Dr. P.V.Diwan, IndiaDr. S.J.S. Flora, India Prof. Huangxian Ju, ChinaProf. H.M. Heise, Germany Dr. K.S. Jagannatha Rao, IndiaProf. Jian-Jiang Zhong, China Prof. Juergen Backhaus, GermanyProf. Kanyaratt Supaibulwatana, Thailand Prof. P.B. Kavi Kishor, IndiaDr. S.P.S. Khanuja, India Prof. M. Krishnan, IndiaProf. P. Kondaiah, India Prof. M.Lakshmi Narasu, IndiaProf. Madhavan P.N Nair, USA Prof. Mahendra Rai, IndiaProf. P. Maruthi Mohan, India Prof. Milan Fránek, Czech RepublicProf. Mohammed Alzoghaibi, Saudi Arabia Prof. Mulchand S. Patel, USAProf. T.V. Narayana, India Dr. R.K. Patel, IndiaDr. Prasada Rao S. Kodvanti, USA Prof. G. Raja Rami Reddy, IndiaProf. T. Ramana, India Dr. Ramanjulu Sunkar, USADr. C. N. Ramchand, India Prof. B.J. Rao, IndiaProf. P. Reddanna, India Prof. Roman R. Ganta, USADr. Samuel JK. Abraham, Japan Prof. Sehamuddin Galadari, UAEDr. Shaji T George, USA Prof. Sham S. Kakar, USADr. B. Srinivasulu, India Dr. N. Sreenivasulu, GermanyProf. A. Subrahmanyam, India Prof. Sung Soo Kim, KoreaProf. B. Suresh, India Prof. Swami Mruthini, USAProf. N. Udupa, India Dr. Urmila Kodavanti, USAProf. Ursula Kuees, Germany Dr. Vikas Dhingra, USA

Assistant Editors

Dr. V.R. Kondepati, Germany Dr. Sridhar Kilaru, UKProf. Chitta Suresh Kumar, India

(Electronic Version)

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Current Trends in Biotechnology and Pharmacy

ISSN 0973-8916

Volume.1 (1) CONTENTS October - 2007

Reviews

Recent developments in multianalyte immunoassayZhifeng Fu, Hong Liu, Zhanjun Yang, and Huangxian Ju 1-17

Effective drug targeting by erythrocytes as carrier systemsV.S. Gopal, A. Ranjith Kumar, A.N. Usha, A. Karthik, and N. Udupa 18-33

Research papers

Screening of supports for Kluveromyces marxianus var.

bulgaricus inulinase immobilization

F.C. Paula, M.L. Cazetta, R. Monti and J. Contiero 34-40

Correct identification of wood-inhabiting fungi by ITS analysisAnnette Naumann, Mónica Navarro-González, Olivia Sánchez-Hernández,

Patrik J. Hoegger and Ursula Kües 41-61

Studies on the influence of penetration enhancers on in vitro permeation ofcarvedilol across rat abdominal skinG. Ramesh, Y. Vamshi Vishnu, V. Kishan and Y. Madhusudan Rao 62-69

Characterization of myostatin gene and identification of SNPs for diversity analysisS. T. Bharani Kumar, Neeraj Dilbaghi, S P S Ahlawat, Bina Mishra, M. S. Tantia

and R. K. Vijh 70-78

Significant medium components for enhanced jasmonic acid production byLasiodiplodia theobromae using Plackett-Burman designP. C. Dhandhukia and V. R. Thakkar 79-86

Characterization of fusarium wilt–resistant and susceptible varieties of ginger(Zingiber officinale) through random amplified polymorphic DNA markersR. Swetha Priya, A. Minaz Khimani and R. B. Subramanian 87-95

Screening for siderophore producing PGPR from black cotton soils ofNorth MaharashtraD. S. Prashant, R. R. Makarand, L. C. Bhushan L and S. B. Chincholkar 96-105

Desiccation tolerance and Starvation resistance exhibit opposite altitudinalcline in Indian populations of Drosophila immigrans

Manvender Singh, Pankaj K.Tyagi and Shruti 116-111

Anticonvulsant Potential of Essential oil of Artemisia abrotanum

S.P.Dhanabal, N.Paramakrishnan, S.Manimaran, B.Suresh 112-116

Abstract

Multianalyte immunoassay has gained increasingattention due to its high sample throughput, shortassay time, low sample consumption and reducedoverall cost per assay. Most of the currentdeveloped approaches for multianalyteimmunoassay are based on spatial-resolved,multilabel or separation mode. This paperreviews the progress of multianalyteimmunoassay and its applications in differentfields with 90 references. The outlook of thispromising technique has been discussed.

Keywords

Immunoassay; Immunosensor; Multianalyteimmunoassay; Array; Review

Introduction

As a promising approach for selective andsensitive analysis, immunoassay has recentlygained increasing attention in different fieldsincluding environmental monitoring, clinicaldiagnosis, food safety, pharmaceutical analysisand bacteria identification. It is often necessaryto monitor or quantitate several components in acomplex system. For example, due to the limitedspecificity and sensitivity of biomarkers forclinical diagnosis, the measurement of a singlebiomarker is usually insufficient for diagnosticpurpose. Some studies have showed that themeasurement of biomarkers panel can avoid false

Recent Developments in Multianalyte Immunoassay

Zhifeng Fu1,2, Hong Liu1, Zhanjun Yang1, Huangxian Ju1*1 Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of

Chemistry, Nanjing University, Nanjing 210093, P.R. China2 College of Pharmaceutical Sciences, Southwest University, 400716, P.R. China

* For correspondance - hxju@nju.edu.cn

positive or false negative results to improve theirdiagnostic value (1). Traditionally, immunoassayof analytes panel is performed as discrete tests,i.e., one analyte per assay run, and several runsare needed to detect all components in a complexsystem. Great consumptions of time, reagent andlabor limit the application. To dissolve theselimitations, multianalyte immunoassay (MAIA)that can measure two or more analytes in a singlerun has become a long-cherished goal ofimmunochemist since simultaneousradioimmunoassay of human insulin and growthhormone in serum sample using I-131 and I-125as labels was reported in 1966 (2). Comparedwith parallel single-analyte immunoassaymethods, MAIA offers some remarkableadvantages, such as high sample throughput,improved assay efficiency, low sampleconsumption and reduced overall cost per assay(3). This review focuses on the progress andapplications of MAIA, including spatial-resolved, multilabel and separation modes.

1. Spatial-Resolved Mode

The spatial resolution of differentimmunoreaction areas using a universal label forfluorescent, chemiluminescent (CL),spectrophotometric, electrochemical, andpiezoelectric detections with array detectorsincluding charge-coupled device (CCD) cameraand multichannel electrochemical workstation isthe most popular MAIA method.

Current Trends in Biotechnology and Pharmacy, Vol.1 (1) 1-21 (2007)ISSN:0973 - 8916

1.1 Optical detection

Antigen and antibody arrays dotted on planarsupports, such as multi-well plate, nylonmembrane and glass slide, combined withfluorescent probes (4-10) and enzymes (9, 11-13) as labels are traditionally adopted to performspatial-resolved MAIA using CCD and laserscanner detector.

Weller’s group (14) proposed a parallel affinitysensor array for the rapid analysis of 10antibiotics in milk. Microscope glass slidemodified with (3-glycidyloxypropyl)trimethoxysilane was used for the preparation ofhapten microarray and inserted into a flow cellto act as an automated flow-through CLmultianalyte immunosensor. After incubationprocess, the horseradish peroxidase labeledimmunocomplexes of the 10 antibioticsgenerated enhanced CL signals, which wererecorded with a CCD camera. The fullyautomated liquid handling and sample processingenabled one analysis cycle to be completed inless than 5 min. With the similar device andprotocol, multiple herbicides (15, 16) andallergen-specific IgE in human serum (17) havebeen assayed in array mode.

Jiang et al. (18) reported a miniaturized,microfluidic version of serial-dilution fluorescentimmunoassay for antibodies in HIV+ humanserum. In this assay, serially diluted solutions ofserum flowed in channels across orthogonal,parallel strips of HIV ENV proteins (gp41 andpg 120) adsorbed on a polycarbonate membrane.The bound antibodies could be measured usinga second, fluorescent labeled antibody. This assayused a microdilutor network to achieve serialdilution and allowed simultaneous, quantitativeanalysis of multiple analytes with highconcentrations on a single chip.

Some immunosensors arrays composed ofrecognition component, fluidics component for

movement of various solutions and detector forcollection of signals produced from positivesamples have been developed at the NavelResearch Laboratory, USA. Sandwichfluoroimmunoassays are performed on thesurface of microscope slides previously patternedwith stripes of capture antibodies. After bothsample and fluorescent tracer antibodies areintroduced in a direction perpendicular patternedwith stripes of capture antibodies, theimmunocomplexes formed can be observed as acheckerboard pattern of fluorescent spots excitedby evanescent wave on the surface. These arrayimmunosensors have been successfully appliedin MAIA of proteins, bacterias and biohazards(19-27).

Barzen et al. (28-30) proposed several opticalmultianalyte immunosensors for environmentpollutants based on flow-injection immunoassaycoupled with total internal reflection fluorescentdetection. They immobilized haptens on thedifferent areas of transducer surface of the flowcell and determined simultaneously multiplepollutants in a spatial-resolved and competitivemode. Rodriguez-Mozaz et al. (31)simultaneously detected atrazine, isoproturonand estrogen estrone in river water using animmunosensor fabricated with a similar protocol(Figure 1). The performance of the developedimmunosensor was evaluated against a well-accepted traditional method based on solid-phaseextraction followed by liquid chromatography-mass spectrometry, and the results obtained fromthe two methods indicated good agreement.

A one-step lateral flow immunoassay on a stripformat for the rapid and simultaneous detectionof free and total prostate specific antigens (f-PSAand t-PSA) and estimation of f-PSA to t-PSAratio (f/t-PSA) in serum has been reported (32).Herein, f-PSA or t-PSA is sandwiched betweenanti-f-PSA or anti-t-PSA monoclonal antibodiesparallel immobilized on the strip and a colloidalgold labeled anti-PSA tracer antibody. The

Zhifeng Fu et al2

presence of f-PSA and t-PSA results in theappearance of two parallel pink colour lines. Twomembrane-based competitive immunoassaysusing gold particles and horseradish peroxidase(HRP) as tracers in lateral flow format have alsobeen developed for MAIAs of carbaryl andendosulfan (33). The visual detection limits forcarbaryl and endosulfan are 100 and 10 µg/L withgold and 10 and 1 µg/L with HRP as labels,respectively.

Yacoub-George et al. (34) designed a portablemultichannel immunosensor for biologicalwarfare agents, which was based on a capillaryELISA technique in combination with aminiaturized fluidics system and used CL as thedetection principle (Figure 2). The fluidic systemallowed three CL immunoassays to be performedsimultaneously within three fused silicacapillaries with three photodiodes as detectors.Koch et al. (35) also presented a portable opticalmultichannel immunosensor for the simultaneousoperation of three flow-through capillary enzymeimmunoassays. The parallel operation wasachieved by stop-flow incubation. When onecapillary was in the process of signal collection,the other two were in incubation procedure. Thiswork represented a versatile tool forimmunoassay of several biological warfareagents in parallel with only one non-arraydetector.

The application of a surface plasmon resonance-based biosensor with four flow channels incombination with a mixture of four specificantibodies resulted in a competitive inhibitionMAIA for the simultaneous detection of fiveaminoglycosides in reconstituted skimmed milk(36). Chung et al. (37) developed a sequentialmethod for the analysis of HRP and bovine serumalbumin using a surface plasmon resonancebiosensor. Non-array fluorescent detector hasalso been used for spatial-resolved detection ofmultiple pesticides (38), hormones (39) andproteins (40) by moving the antigens or

antibodies immobilized affinity microcolumnand capillary immunosensor with a motorizedtranslational stage. Owing to the relativelycomplicated detection device, this strategy needsto be further improved.

1.2 Electrochemical detection

Amperometric immunosensor array fabricatedwith multiple working electrodes sharing onecommon counter electrode and referenceelectrode has been successfully used for MAIAof pepsinogens (41), tumor markers (42-45) andhormones (46). CombiMatrix Corporation (47)developed a microarray of individuallyaddressable electrodes using conventionalCMOS integrated circuitry. This microarraysystem provided a host for MAIA due to the largenumber of electrodes available, which integratedover 1000 electrodes per square centimeter. Theresults for human α 1 acid glycoprotein, ricin,M13 phage, Bacillus globigii spore, andfluorescein indicated that this method was oneof the most sensitive available, with limits ofdetection in the attomole range. Electrochemicalsensor array often suffers from cross-talk due tothe diffusion of electroactive product generatedat one electrode to a neighboring electrode(43,45). Thus, an enough spatial distancebetween adjacent electrodes is necessary tocumber the diffusion procedure. Use of doublesiloxane layer (45) and iridium oxide (42-44)matrix can retard the diffusion of enzyme-generated product to lower cross-talk.

Ju et al. (48,49) proposed two disposableimmunosensor arrays for simultaneouselectrochemical determination of multiple tumormarkers. The low-cost immunosensor arrayswere fabricated simply using cellulose acetatemembrane to co-immobilize thionine as amediator and antigens on different workingelectrodes of a screen-printed chip, on which theimmobilized thionine shuttled electrons betweenHRP labeled to antibodies and the electrodes for

Multianalyte Immunoassay 3

enzymatic reduction of H2O

2 to produce

detectable signals. This chip could avoid theelectrochemical and electronic cross-talksbetween the electrodes, which enabled the arraysto be miniaturized without considering thedistance between immunosensors.

Kong et al. (50,51) proposed two arrays of eightelectrodes for label-free capacitive andconductive immunoassay of liver fibrosismarkers using ultrathin α 1 alumina sol-gel filmsand electrochemically deposited polypyrrole toimmobilize antibodies, respectively.

1.3. Mass-sensitive detection

Luo et al. (52) constructed a 2x5 modelpiezoelectric immunosensor array fabricatedwith disposable quartz crystals for quantificationof microalbumin, 1-microglobulin, α 2microglobulin, and IgG in urine. With thepiezoelectric immunosensor array, 4 urinaryproteins could be quantified within 15 min. Thismethod had an analytical interval of 0.01-60 mg/L. Similarly, a novel simultaneous immunoassaytechnique has been developed for thedetermination of complement factors (C

4,C

5,C

lq

and B factor) by constructing a piezoelectricquartz crystal array system (53). These mass-sensitive piezoelectric immunosensor arrays canprovide a convenient label-free approach toMAIA.

1.4. Optical encoding and addressing

Spatial-resolved arrays are typicallymanufactured by labor-intensive methodsrequiring high precision such as ink-jet printing,micromachining, photolithography, andphotodeposition. Randomly ordered addressablesensor array developed in Walt’s laboratory (54)provided an alternative approach to arrayfabrication. In this approach, micrometer-sizedsensors were produced by immobilizing differentrecognition molecules on the surface ofmicroparticles encoded using two fluorescent

dyes. The addressing procedure was performedby taking the fluorescence intensity at eachemission wavelength and then dividing the twovalues to get the signature of that particular ratio.With this principle, multiple drugs (55), proteins(56-58), biological warfare agents (59) andcytokines (60) were simultaneously detected withCL or fluorescent method and randomly orderedantibodies immobilized copolymer microspheresor metallic particles as microsensors.

Theoretically, thousands of antigens or antibodiescan be spotted onto one single planar support toscreen thousands of analytes. Although this modecan screen large numbers of analytes, accuratequantitative data in these arrays are usuallylimited or difficult to be obtained (44). Requiringof complicated and expensive spotting techniquewith high precision also greatly limits itsapplication. Although optical encoding andaddressing allows randomly ordered sensorarrays to be identified for MAIA, the encodingprocess complicates the manipulation.Furthermore, spatial-resolved MAIA is typicallyperformed with expensive array detector such asCCD camera for optical detection or multi-channel workstation for electrochemicalmeasurement (61).

2. Multilabel Mode

The second dominant mode for MAIA isperformed using different labels to tag antibodiesor antigens (one per analyte), includingradioisotopes, enzymes, fluorescent and metalcompounds. Different analytes can be easilydistinguished using these labels by suchparameters as potential, wavelength, decay timeand so on.

2.1 Wavelength resolution

ELISA for MAIA involves labeling the analyteswith various enzymes, whose catalyzed reactionscan easily be distinguished from each other by

Zhifeng Fu et al4

absorption spectra (62, 63). Selection of theenzyme labels is a key step in the developmentof an ELISA based MAIA. Blake et al. (62)mentioned that the ideal enzyme labels for MAIAshould meet the following requirements: (i) theenzymes should be readily available,inexpensive, and have high turnover numbers;(ii) each enzyme must be stable under theselected simultaneous assay conditions and noteasily to be interfered by other enzyme or itssubstrate; (iii) all enzymes must have similaroptimal assay conditions; (iv) the assay methodfor each enzyme should be simple, sensitive,rapid, and cheap; (v) all enzymes should notoccur in the practical sample to be assayed, andinterfering factors should be absent; (vi) eachenzyme should contain potentially reactivegroups that allow linking to antigen or antibodywhile retaining the enzyme activity; (vii) thespectra of the products of the enzyme-catalyzedreactions should not overlap with each other.

Ihara et al. (64) immobilized a mixture ofantigenic peptides of FAK and c-Myc tonanospheres with red emission, and a mixture ofc-Myc and α catenin to green nanospheres,respectively. As seen in Figure 4 (64), anti-FAKand anti α catenin antibodies could formaggregates with red and green emissions,respectively. The anti-c-Myc antibody couldform aggregate emitting yellow light as a resultof color overlapping. This strategy enabledspecific antibodies to be detected in one-stepprocedure with color-encoded nanospheres.Swartzman et al. (65) proposed a bead-basedtwo-color MAIA for cytokines IL-6 and IL-8using Cy5.5 and Cy 5 as fluorescent labels,respectively. The linear dynamic ranges of themwere 125-4000 pg/mL and 15.6-2000 pg/mL,respectively. This work utilized fluorometricmicrovolume assay technology to image andmeasure bead-bound fluorescence while thebackground fluorescence was ignored.Consequently, no wash steps were required toremove unbound antibody, ligand, and

fluorophore. Goldman et al. (66) used antibody-conjugated quantum dots with emissionmaximums at 510, 555, 590, and 610 nm todemonstrate multiplex assays for four proteintoxins present in the same sample. However, adeconvolution of composite spectra was neededto distinguish the overlapping signals.

2.2. Time resolution

The fluorescence of lanthanide chelates has theadvantages of high quantum yield, long decaytime, exceptionally large Stoke’s shift, andnarrow emission peak. Specific chelatefluorescence can be easily distinguished from thesample matrix fluorescence and the scatteredlight, and the fluorescence from differentlanthanides can also be easily discriminated dueto their difference in decay time and emissionwavelength, which makes the lanthanide chelatespreferable to any other probes for developingmultilabel-based time-resolved MAIA. Of the 15lanthanide ions, Eu3+, Sm3+, and Tb3+ are the mostcommonly employed probes, and have beenwidely used for time-resolved fluorescent MAIAof multiple tumor markers (67), hormones (68),recombinant proteins (69) and antibodies (70).

Ito et al. (71) developed a simple and rapid time-resolved fluoroimmunoassay for simultaneousdetermination of alpha-fetoprotein (AFP), humanchorionic gonadotropin (hCG) and estriol (E3)using Eu3+ and Sm3+ chelates. In this proposedmethod, a 96-well microtiter plate for AFP andhCG assay and a transferable solid phase platefor E3 assay were combined to perform MAIAof the three analytes with only two probes. Themeasurable ranges for AFP, hCG and E3 were3.91-1000 ng/mL, 877-250 000 IU/L and 0.39-100 ng/mL, respectively.

2.3. Potential resolution

The dual-analyte homogeneous immunoassay ofphenobarbital and phenytoin was carried outsimultaneously at physiological pH by square-

Multianalyte Immunoassay 5

wave voltammetry on Nafion-loaded carbonpaste electrode. Phenobarbital and phenytoinwere labeled by cobaltocenium salt andferroceneammonium salt with standard redoxpotentials of -1.05V and 0.26 V, respectively.Detection limits of 0.25 and 0.2 mu wereachieved for the two antiepileptic drugs,respectively (72). As seen in Figure 5 (73), anelectrochemical stripping immunoassay protocolusing different inorganic nanocrystal as tracersand magnetic beads as support has beendeveloped for the simultaneous measurementsof proteins. Each biorecognition event yields adistinct voltammetric peak, whose position andsize reflect the identity and concentration of thecorresponding analyte, respectively. Thisprotocol has been used for a simultaneousimmunoassay of β 2 microglobulin, IgG, bovineserum albumin, and C-reactive protein usingZnS, CdS, PbS, and CuS colloidal crystals aslabels, respectively (73). Hayes et al. (74)proposed a MAIA method for human serumalbumin and IgG. Bismuth and indium ions werecoupled to the two proteins through thebifunctional chelating agent diethylenetriamine-pentaacetic acid. Following the competitivereactions between unlabeled and labeled proteinsfor limited amount of specific antibodiesimmobilized on polystyrene, the bound metal ionlabels were released by acidification and detectedby differential pulse anodic strippingvoltammetry with detection limits of 1.8 and 0.6pg/mL for human serum albumin and IgG,respectively.

2.4. M/e resolution

Zhang et al. (75) developed a dual-labelimmunoassay method for the simultaneousdetermination of AFP and free hCG β in humanserum. Monoclonal antibodies immobilized onmicrotiter plates captured AFP and hCG beta,which were detected by Eu3+-labeled AFP andSm3+-labeled hCG β tracer antibodies withinductively coupled plasma mass spectrometry

(ICPMS) after Eu3+ and Sm3+ were dissociatedfrom the plates with HNO

3 solution. However,

this technique could not be used for microarraydetection since it was necessary to dissolve theelemental tags before introducing them into theplasma source. They (76) also reported thedetection of multiple proteins on each spot ofthe immuno-microarray by laser ablationICPMS. AFP, carcinoembryonic antigen (CEA)and human IgG were detected as model proteinsin sandwich format on a microarray with Sm3+-labeled AFP antibody, Eu3+-labeled CEAantibody, and Au nanoparticle-labeled IgGantibody as tracer antibodies. The detection limitswere 0.20, 0.14, and 0.012 ng/mL for AFP, CEA,and human IgG, respectively. This detectionmethod allowed detection of multiple analytesfrom each spot of microarray with a spatialresolution at micrometer range, which couldalleviate the stress to fabricate high-densityarrays.

2.5. Scintillation energy resolution

In 1966, as the founder of MAIA, Morgan (2)proposed an original simultaneousradioimmunoassay of human insulin and growthhormone in serum sample using I-131 and I-125as labels and exploiting the difference inscintillation energy produced from the tworadioisotopes to discriminate the two analyte.Similarly, the simultaneous radioassay ofvitamins (77) and hormones (78) could be carriedout using Co-57 and I-125 as probes. Recently,few attention is paid to the radioimmunoassaybased MAIA due to the damage of radioisotopesto environment and operator.

2.6. Substrate zone resolution

It has been noted that the different labels used inmultilabel mode often need markedly differentoptimal assay conditions, and traditionallysimple combination of multiple labels often leadsto loss of assay performance (2). Furthermore,

Zhifeng Fu et al6

this mode sometimes suffers from signaloverlapping of different labels (66).

Ju et al. (61) designed a substrate zone-resolvedmultianalyte immunosensing system, with whichHRP labeled carcinoma antigen 125 (CA 125)immunocomplex and alkaline phosphataselabeled CEA immunocomplex were sequentiallydetected in their corresponding CL substratezones. This designed technique solved two keyproblems in multilabel mode: one was to obtaindistinguishable CL signals without considerationof wavelength, and the other was to enable eachCL reaction to be catalyzed by the label in itsoptimal assay condition without loss of assayperformance. Unfortunately, as other MAIAmethods based on multilabel mode, thistechnique encountered a difficulty to find moreavailable enzyme labels, which limited thenumber of analytes. In order to overcome thislimitation, this group further proposed a two-dimensional resolution system of channels andsubstrate zones (79). Using CA 125, CA 153, CA199 and CEA as two couples of model analytes,two couples of capture antibodies wereimmobilized in two channels, respectively. Witha sandwich format the CL substrates for alkalinephosphatase and horseradish peroxidase weredelivered into the channels sequentially toperform multiplex immunoassay after the sampleand trace antibodies were introduced into thechannels for on-line incubation. When three orfour channels were used in the flow-throughdevice, the detectable analytes in a single runcould be 6 or 8, respectively, with a 10 s longeranalytical time for each added channel.

3. Separation Mode

Another method coupled with separationtechniques such as capillary electrophoresis (CE)and high-performance liquid chromatography(HPLC) can be used for MAIA. Competitiveimmunoassay combined with fluorescentdetection is generally adopted to perform CE

based MAIA, the analytes includes abused drugs(79, 81) and peptides (82). Obviously thisstrategy often suffers from the adsorption ofimmunoreagents on inner wall of the capillary,which can be prevented by optimization ofseparation buffer type and pH that allowesapplication of high electric field (82). An on-linecoupling of a label-free reflectometricinterference spectroscopic biosensor to a HPLCsystem has been described for MAIA of fourpesticides (83). In this system a highly cross-reactive antibody against the four pesticides isused to bind the pesticides. The eluate of theHPLC is mixed continuously with the antibodies,and the presence of antigens is detected by areduction of the antibody binding to thetransducer.

Roda et al. (84) proposed a field-flowfractionation (FFF)-CL based solid-phasecompetitive immunoassay, in which micrometer-sized beads coated with the capture antibodywere used as solid phase, and analyte-HRPconjugate was used as tracer. Once thecompetitive immunoreaction took place withinthe injection loop of the system, the antibody-bound tracer was separated from tracer insolution in a few minutes by means of FFF. FFF-based MAIA could be developed by use of beadswith different sizes (1-50 mu), each coated withthe specific antibody for one analyte. The beadscould be fractionated by FFF before CL signalscollection to realize detection of multipleanalytes in a single run.

The thermosensitive poly (N-isopropylacrylamide) (PNIP) and magnetic beadshave been widely utilized as the separationcarriers for immunoassays. A fast homogeneousimmunoreaction as well as a simpleheterogeneous separation process is carried outfor MAIA in the light of some certaincharacteristics of water-soluble PNIP andmagnetic beads, and thus, lower nonspecificaffinity and higher sensitivity are accomplished

Multianalyte Immunoassay 7

(85). The results of CL detection of IgG and IgAindicate the detection limits as low as 2.0 and1.5 ng/mL, respectively.

4. Cross-Reactivity

Cross-reactivity is a crucial analytical parameterregarding specificity and reliability of MAIA,which is frequently encountered in MAIA ofsmall molecule analytes. In many cases, theantibodies recognize a variety of analogs andmetabolites of the target analyte, for example,some s-triazines and their metabolites withsimilar structures shown in Figure 6 (86). Evenmonoclonal antibodies are often unable todiscriminate absolutely molecular analogs withsmall structural differences. Efforts to derivemonoclonal antibodies to small analytesgenerally produce panels of antibodies that differin their cross-reactivity for the primary targetanalyte and its analogs and metabolites.Antibodies arrays combined with somechemometric means inclusive of neural networkare often used to overcome the difficulty in exactquantitation resulted from the cross-reactivity(86-90).

5. Conclusion and Outlook

In recent yeas, MAIA has attracted considerableinterest due to its outstanding advantage in assayspeed, cost and labor consuming. So far thespatial-resolved mode has been the most popularmode due to its high analyte throughput and largeinformation amount. The further work needs todevelop arrays with higher density and simplerpreparation protocol using cheaper array detector.Most of the multilabel mode based methods focuson using of lanthanide chelates as labels andtime-resolved fluorescent detection. More labelswith higher signal resolution degree and lessrequirement to assay condition are urgentlyneeded. Various resolution methods in time,space, substrates, reactants, labels and detectionmethods will be designed and developed forMAIA in the future. Military application and

environment monitor are anxious tominiaturized, integrated and portable MAIAsystem fit for field application. MAIA systemwith high sample throughput and rapid assayspeed has great application potential in diseasescreen.

Acknowledgements

This research was financially supported by theNational Science Funds for Distinguished YoungScholars (20325518) and Creative ResearchGroups (20521503), the Key Program(20535010) from the National Natural ScienceFoundation of China and the Science Foundationof Jiangsu (BS2006006, BS2006074).

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Fig 2 : Scheme representing the arrangement of the fluidics components of the CL multichannelimmunosensor for biological warfare agents.

Fig 1 : Scheme of flow-injection immunosensor used for detection of multiple pollutants in riverwater.

Multianalyte Immunoassay 15

Fig 4 : Schematic illustration of the MAIA using selective aggregation of antigenic peptide-modi-fied nanospheres.

Fig 3 : Schematic diagrams of (A) screen-printed four-electrode system and (B) preparation ofimmunosensor array and MAIA procedure: (a) Nylon sheet, (b) silver ink, (c) graphite auxiliaryelectrode, (d) Ag/AgCl reference electrode, (e) W1, (f) W2 and (g) insulating dielectric.

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Fig 6 : Some s-triazines andtheir metabolites with similarstructures.

Fig 5 : Multiprotein electro-chemical stripping immunoassayprotocol using different inorganicnanocrystal tracers: (A) introduc-tion of antibody-immobilizedmagnetic beads, (B) capture of theantigens to the antibodies-immo-bilized magnetic beads, (C) cap-ture of the nanocrystal-labeledsecondary antibodies and forma-tion of sandwich immuno-complexes, (D) dissolution ofnanocrystals and electrochemicalstripping detection.

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