A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen

A new simple immunoassay method for carcinoembryonic antigen (CEA) detection using a disposable immunosensor coupled with a flow injection system was developed. The immunosensor was prepared by coating CEA/colloid Au/chitosan membrane at a screen-printed carbon electrode (SPCE). Using a competitive immunoassay format, the immunosensor inserted in the flow system with an injection of sample and horseradish peroxidase (HRP)-labeled CEA antibody was used to trap the labeled antibody at room temperature for 35 min. The current response obtained from the labeled HRP to thionine-H(2)O(2) system decreased proportionally to the CEA concentration in the range of 0.50-25 ng/ml with a correlation coefficient of 0.9981 and a detection limit of 0.22 ng/ml (S/N=3). The immunoassay system could automatically control the incubation, washing and current measurement steps with good stability and acceptable accuracy. Thus, the proposed method proved its potential use in clinical immunoassay of CEA.     

A sensitive amperometric immunosensor for carcinoembryonic antigen detection with porous nanogold film and nano-Au/chitosan composite as immobilization matrix

A sensitive amperometric immunosensor for carcinoembryonic antigen (CEA) was prepared. Firstly, a porous nano-structure gold (NG) film was formed on glassy carbon electrode (GCE) by electrochemical reduction of HAuCl₄ solution, then nano-Au/Chit composite was immobilized onto the electrode because of its excellent membrane-forming ability, and finally the anti-CEA was adsorbed onto the surface of the bilayer gold nanoparticles to construct an anti-CEA/nano-Au/Chit/NG/GCE immunosensor. The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry (CV). The gold colloid, chitosan and nano-Au/Chit were characterized by transmission electron microscopy and UV–vis spectroscopy. In addition, the performances of the immunosensor were studied in detail. The resulting immunosensor offers a high-sensitivity (1310 nA/ng/ml) for the detection of CEA and has good correlation for detection of CEA in the range of 0.2 to 120.0 ng/ml with a detection limit of 0.06 ng/ml estimated at a signal-to-noise ratio of 3. The proposed method can detect the CEA through one-step immunoassay and would be valuable for clinical immunoassay.     

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody-glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.     

Electrochemical DNA base pairs quantification and endonuclease cleavage detection

A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.     

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au(shell)@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab(2)) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au(shell)@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au(shell)@GOD were performed by one-pot assembly of Fc@Ab(2) and GOD on the surface of Au(shell)@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL(-1) with the detection limit down to 6.7 pg mL(-1). The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease.     

A novel electrochemical immunoassay based on diazotization-coupled functionalized bioconjugates as trace labels for ultrasensitive detection of carcinoembryonic antigen

In this article, a novel sandwich-type electrochemical immunosensor based on the signal amplification strategy of diazotization-coupling concept for ultrasensitive detection of carcinoembryonic antigen (CEA) was reported. It operates through physisorption of monoclonal anti-CEA on 4-aminothiophenol (4Atp) functionalized gold electrode interface as the detection platform. Diazo-4Atp-coupled-thionine (Thi)-conjugated gold nanoparticles (GNPs) were prepared for immobilization of horseradish peroxidase (HRP) and secondary anti-CEA to form core-shell bioconjugates that were used as electrochemical signal amplification reagent. The sensitivity of the immunosensor was greatly amplified by a dual amplification: one is that a large number of thionine and HRP was introduced on the electrode surface through sandwich immunoreaction, the other is that HRP as enhancer could catalyze the oxidation reaction of thionine by H(2)O(2), which results in great enhancement of the reduction peak current. Thus, the bioconjugates-based assay provided an amplification approach for detecting CEA at trace levels and led to a detection limit as low as 0.7 pg/mL (at a three times signal-to-noise ratio) that is well-below the threshold value of 2.5 ng/mL for clinical diagnosis. The assay was evaluated for clinical serum samples with various CEA concentrations and received in excellent accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA).     

Effects of particle characteristics on magnetic immunoassay in a thin channel

The effects of size and porosity of particles on magnetic immunoassay in a thin channel were studied. Experimental parameters were investigated and compared using a model immunoassay complex of carcinoembryonic antigen (CEA)/anti-CEA. The rate constant for the affinity reaction between functional particles increased as the size of magnetic nanoparticles (800-80 nm) decreased. The affinity reaction between functional particles had no significant effect on the sizes of microparticles (1.0-4.4 μm) at commonly used thin channel flow-rates of 0.001-0.025 ml/min. Competitive and sandwich reactions of CEA/anti-CEA were studied for CEA detection. Microparticles of different porosities produced similar linear ranges of detection and limits of detection. The limits of detection for CEA were 0.29 pg/ml and 0.21 pg/ml for competitive and sandwich reactions, respectively. The linear ranges of detection were from 0.49 pg/ml to 4.9 ng/ml for both competitive and sandwich reactions. The detection limits were lower, and the linear ranges were wider than those of literature. There was a 9% difference in CEA detection measurements between competitive and sandwich magnetic immunoassay. The measurements of two magnetic immunoassays differed by less than 13% from the ELISA reference measurements. The running time was less than 30 min. Magnetic immunoassay in a thin channel has great potential for biochemical analysis and immunoassay-related applications.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

Direct Electrochemical Immunoassay Based on Immobilization of Protein-Magnetic Nanoparticle Composites on to Magnetic Electrode Surfaces by Sterically Enhanced Magnetic Field Force

A direct electrochemical immunoassay system based on the immobilization of α-1-fetoprotein antibody (anti-AFP), as a model system, on the surface of core-shell Fe₂O₃/Au magnetic nanoparticles (MNP) has been demonstrated. To fabricate such an assay system, anti-AFP was initially covalently immobilized on to the surface of core-shell Fe₂O₃/Au MNP. Anti-AFP-modified MNP (bio-nanoparticles) were then attached to the surface of carbon paste electrode with the aid of a permanent magnet. The performance and factors influencing the performance of the resulting immunosensor were studied. α-1-Fetoprotein antigen was directly determined by the change in current or potential before and after the antigen–antibody reaction versus saturated calomel electrode. The electrochemical immunoassay system reached 95% of steady-state potential within 2 min and had a sensitivity of 25.8 mV. The linear range for AFP determination was from 1 to 80 ng AFP ml⁻¹ with a detection limit of 0.5 ng AFP ml⁻¹. Moreover, the direct electrochemical immunoassay system, based on a functional MNP, can be developed further for DNA sensor and enzyme biosensor. Revisions requested 2 November 2005; Revisions received 17 January 2006     

Simultaneous detection of dual proteins using quantum dots coated silica nanoparticles as labels

Simultaneous detection of multianalytes associated with a particular cancer is beneficial for disease diagnosis. Here, a facile immunosensing strategy was designed to allow simultaneous electrochemical detection of dual proteins, in a single run. CdSe and PbS water-soluble quantum dots (QDs) were prepared and coated on monodisperse silica nanoparticles as labels for proteins detection. Rabbit immunoglobulin G antigen (IgG) and carcinoembryonic antigen (CEA) were chosen as model proteins for analysis. After a typical sandwich immunoassay, CdSe and PbS QDs labels were introduced onto the Au substrates' surface, which were then dissolved and could be simultaneously monitored by square-wave-voltammetric (SWV) stripping measurements. Under selected conditions, IgG and CEA could be assayed in the ranges of 0.05-40 ng mL(-1) and 0.05-25 ng mL(-1), respectively. The proposed method possessed high sensitivity, good precision, and satisfactory reproducibility and regeneration.     

Bimetallic AuPt nanochains: Synthesis and their application in electrochemical immunosensor for the detection of carcinoembryonic antigen

The recent development in the nanotechnology has paved the way for a large number of alloyed nanomaterials and devices of desirable properties which have useful functions for electrochemical sensor and biosensor applications. In this paper, bimetallic AuPt nanochains were synthesized through a mild chemical method, with which anti-horseradish peroxidase-conjugated anti-carcinoembryonic antigen (HRP-anti-CEA-NCAuPt) was developed for electrochemical detection of carcinoembryonic antigen (CEA) in a sandwich-type immunoassay format. The alloyed nanocrystals exhibit not only sound signal amplification effect of Au nanoparticles, but also further new combination of interfacial, electrical and structural properties arising from the disparate AuPt components. As a result, the electrochemical signal was significantly amplified by using the HRP-anti-CEA-NCAuPt as tracer and hydrogen peroxide as enzyme substrate. The linear range of the developed immunosensor is 0.01-200ng/mL and the detection limit is 0.11pg/mL of CEA, which makes the biometallic nanochains promising candidates for the next-generation sandwich-type electrochemical immunoassays.     

A rapid competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) test strip for microcystin-LR (MCLR) determination

A competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) is described for the determination of microcystin-LR (MCLR) using a double-sided microporous gold electrode in cartridge-type cells. A gold film sputtered on one side of porous nylon membrane constitutes a working electrode, while another gold film formed on the opposite side serves as a pseudo reference electrode. After immobilizing MCLR antibody on working electrode by physical adsorption, the double-sided electrode was placed simply in a diffusion U-type or within a dry strip-type cell with a conjugate pad pre-loaded with a glucose oxidase labeled MCLR (GOx-MCLR) on working electrode side. Assays were performed in two steps: an MCLR-containing sample mixed with a known amount of GOx-MCLR conjugate either in buffer solution or in pre-loaded dry pad was incubated for an appropriate period (about 10 min) to induce competitive reaction with an immobilized anti-MCLR antibody on working electrode, and a fixed concentration of glucose solution (substrate) was then added to the backside of the working electrode. Due to the competitive nature of the assay, enzymatically generated product, hydrogen peroxide (H2O2), was detected at the working gold electrode (at +800 mV versus Au) by oxidation, and the magnitude of amperometric current was inversely proportional to the concentration of MCLR in the sample. The response time after substrate addition was about 30s. Mean recovery of MCLR added to tap water was 93.5%, with a coefficient of variation (CV) of 6.6%. The proposed competitive NEEIA system is in general comparable to existing heterogeneous enzyme immunoassays with a similar detection limit (100 pg/mL MCLR), and suitable for developing a disposable type biosensor for on-site monitoring of environment.     

Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.     

Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection

This paper presents an electrochemical immune sensor for label free detection of epidermal growth factor receptor (EGFR) by immobilizing anti-EGFR antibody (Anti-EGFRab) on dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) on gold (Au) electrode. Electrochemical studies show that increased surface concentration of redox moieties onto Anti-EGFRab/DTSP immuno-electrode leads to high electron transport and improved sensing performance. The antigen-antibody complex demonstrates a high association constant (5×10(12)L/mol) that results in high affinity of Anti-EGFRab to EGFR, confirming that the DTSP-SAM provides a conducive environment for anti-EGFR immobilization. The electrochemical response of EA/Anti-EGFRab/DTSP/Au electrode as a function of EGFR concentrations exhibits a linear range from 1pg/mL to 100ng/mL, a detection limit of 1pg/mL at a sensitivity of 2.02μAM(-1)at a regression coefficient of 0.99.     

Optimized ferrocene-functionalized ZnO nanorods for signal amplification in electrochemical immunoassay of Escherichia coli

A novel amplified electrochemical immunoassay based on ferrocene (Fc)-functionalized ZnO nanorods (NRs) was developed in the present work. The detection antibody ((d)Ab) and Fc were immobilized onto the surface of ZnO NRs, denoted as {(d)Ab-ZnO-Fc} bioconjugates. The amount of (d)Ab and Fc in the bioconjugates was investigated using the copper reduction/bicinchoninic acid reaction (BCA protein assay) and inductive coupled plasma-atomic emission spectroscopy (ICP-AES), respectively. Greatly amplified signal was achieved in the sandwich-type immunoassay when (d)Ab and Fc linked to ZnO NRs at a proper ratio. Using Escherichia coli (E. coli) as a model antigen, the designed immunoassay showed an excellent analytical performance, and exhibited a wide dynamic response range of E. coli concentration from 10(2) to 10(6)cfu/mL with a detection limit of 50 cfu/mL (S/N=3). By introducing a pre-enrichment step, the detection of 5 cfu/10 mL E. coli in hospital sewage water was realized. This proposed signal amplification strategy was promising and could be easily extended to monitor other biorecognition events.     

Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels

An electrochemical method for the simultaneous detection of two different tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), in one-pot, using CdS/DNA and PbS/DNA nanochains as labels was developed. Herein, magnetic beads (MBs) as bimolecule immobilizing carriers, were used for co-immobilization of primary anti-CEA and anti-AFP antibodies. The distinguishable signal labels were synthesized by in situ growth of CdS and PbS nanoparticles on DNA chains, respectively, which were further employed to label the corresponding secondary antibodies. A sandwich-type immunoassay format was formed by the biorecognition of the antigens and corresponding antibodies. The assay was based on the peak currents of Cd(2+) and Pb(2+) dissolved from CdS and PbS nanoparticles by HNO(3) using square wave stripping voltammetry. Experimental results show that the multiplexed electrochemical immunoassay has enabled the simultaneous monitoring of CEA and AFP in a single run with wide working ranges of 0.1-100ngmL(-1) for CEA and 0.5-200ngmL(-1) for AFP. The detection limits reach to 3.3pgmL(-1) for CEA and 7.8pgmL(-1) for AFP.     

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe(3)O(4), which was synthesized with easy handling feature of Fe(3)O(4) by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe(3)O(4) magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 μg mL(-1), with the detection limit of 0.025 μg mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c.     

Multianalyte immunoassay based on insulating-controllable PoPD film at arrayed electrodes integrated on a silicon chip

A novel, simple and label-free multianalyte immunoassay system is presented here by integrating arrayed electrodes on a silicon chip via MEMS. The chip is consisted of six Au disk electrodes, an Au counter electrode and an Ag/AgCl reference electrode. Semi-insulating poly(o-phenylenediamine) (PoPD) was utilized to co-polymerize and immobilize antibodies at the arrayed Au electrodes, and wider linear detection range was obtained than those prepared with completely insulating PoPD. Electrochemical cyclic voltammogram (CV), AC impedance spectroscopy, AFM and fluorescence microscopy were employed to characterize the system. The arrayed electrodes offered exact control of deposition position via electrochemical operation, allowing selectively immobilization of different antibodies at desired positions on a single chip. Specific recognition of antibody (Ab) to corresponding antigen (An) was quantitatively monitored by cyclic voltammograms in the presence of electrochemical redox probe, ferrocene methanol. The proposed immunoassay chips showed sensitive response to three liver fibrosis markers, hyaluronic acid (HA), collagen type IV (IV-C) and lamin (LN) at ng/mL level simultaneously and specifically in a tiny amount of volume, usually 50 μL. The results obtained via chips were well consistent with those obtained by commercial radio immunoassays (RIA).     

Signal enhancement of surface plasmon-coupled emission (SPCE) with the evanescent field of surface plasmons on a bimetallic paraboloid biochip

We present a novel approach to the enhancement of surface plasmon-coupled emission (SPCE) using surface plasmon excitation in a bimetal (Ag/Au) layer and we validate the enhancement by presenting the results of a model human IgG immunoassay. Theoretical calculations using Fresnel's equations have been carried out to determine the optimum bimetallic composition and the resulting electric field enhancement. Signal enhancement of SPCE was confirmed using a range of bimetallic layers which were deposited on the surface of a high collection efficiency polymer array biochip and subsequently immobilized with Alexa Fluor 647 labeled anti-human IgG. The bimetallic film of Ag/Au (36/10nm) was determined as an optimum substrate for maximum SPCE signal which was a compromise between the long-term stability of the metal layer and the optimized evanescent field enhancement. An enhanced dose-dependent response was also demonstrated which was ～3 times greater than that detected with a pure gold layer. A human IgG immunoassay showed a dose-dependent response yielding a limit of detection of 1pg/ml by the 3σ rule. The improved performance of the bimetal layer compared to that of an assay carried out on a pure gold layer is attributed to the enhanced evanescent field intensity of surface plasmons in the bimetal combination which excites more fluorescence hence producing an enhanced SPCE signal. This result demonstrates the potential of the SPCE-based array biochips as a sensitive and high-throughput analysis platform for biomolecular interactions.