Electric field-driven strategy for multiplexed detection of protein biomarkers using a disposable reagentless electrochemical immunosensor array

A fast, simple, sensitive, and low-cost method for electrochemical multianalyte immunoassay was developed by combining newly designed electric field-driven incubation with a screen-printed reagentless immunosensor array. The disposable array was prepared by immobilizing respectively horseradish peroxidase (HRP)-labeled antibodies modified gold nanoparticles in biopolymer/sol-gel modified electrodes to obtain direct electrochemical responses of HRP. Upon the formation of immunocomplexes, the responses decreased due to increasing spatial blocking and impedance. At a driving potential of 0.5 V, the incubation process could be accomplished within 2 min. Under optimal conditions, this method could simultaneously detect carbohydrate antigens 153, 125, and 199 and carcinoembryonic antigens ranging from 0.084 to 16, 0.11 to 13, and 0.16 to 15 U mL(-1) and 0.16 to 9.2 ng mL(-1) with a detection time of less than 5 min, and the detection limits corresponding to the signals of 3SD were 0.06, 0.03, and 0.10 U mL(-1) and 0.04 ng mL(-1), respectively. The disposable immunosensor array and simple detection system for fast measurement of panels of tumor markers show significant clinical value for application in cancer screening and provide great potential for convenient point-of-care testing and commercial application.     

Reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen-125

A novel strategy for immunoassay and the preparation of reagentless immunosensors was proposed. This strategy was based on the immobilization of antigen and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody. A reagentless immunosensor for carcinoma antigen-125 (CA 125) determination was developed. The immunosensor was prepared by immobilizing CA 125 with titania sol-gel on a glassy carbon electrode by the vapor deposition method. The incubation of the immunosensor in phosphate buffer solution (PBS) including HRP-labeled CA 125 antibody led to the formation of a HRP-modified surface. The immobilized HRP displayed its direct electrochemistry with a rate constant of 3.04 +/- 1.21 s(-1). With a competition mechanism, a differential pulse voltammetric determination method for CA 125 was established by the peak current decrease of the immobilized HRP. The current decrease resulted from the competitive binding of the CA 125 in sample solution and the immobilized CA 125 to the limited amount of HRP-labeled CA 125 antibody. Under optimal conditions, the current decrease was proportional to CA 125 concentration ranging from 2 to 14 units mL(-1) with a detection limit of 1.29 units mL(-1) at a current decrease by 10%. The CA 125 immunosensor showed good accuracy and acceptable precision and fabrication reproducibility with intraassay CVs of 8.7 and 5.5% at 8 and 14 units mL(-1) CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units mL(-1). The storage stability was acceptable in a pH 7.0 PBS at 4 degrees C for 15 days. The proposed method provided a new promising platform for clinical immunoassay.     

Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited by gold nanoparticles and enzymatic reaction

A novel ultrasensitive multiplexed immunoassay method was developed by combining alkaline phosphatase (ALP)-labeled antibody functionalized gold nanoparticles (ALP-Ab/Au NPs) and enzyme-Au NP catalyzed deposition of silver nanoparticles at a disposable immunosensor array. The immunosensor array was prepared by covalently immobilizing capture antibodies on chitosan modified screen-printed carbon electrodes. After sandwich-type immunoreactions, the ALP-Ab/Au NPs were captured on an immunosensor surface to catalyze the hydrolysis of 3-indoxyl phosphate, which produced an indoxyl intermediate to reduce Ag(+). The silver deposition process was catalyzed by both ALP and Au NPs, which amplified the detection signal. The deposited silver was then measured by anodic stripping analysis in KCl solution. Using human and mouse IgG as model analytes, this multiplexed immunoassay method showed wide linear ranges over 4 orders of magnitude with the detection limits down to 4.8 and 6.1 pg/mL, respectively. Acceptable assay results for practical samples could be obtained. The newly designed strategy avoided cross talk and the need of deoxygenation for the electrochemical immunoassay and, thus, provided a promising potential in clinical applications.     

Multifunctional gold-silica nanostructures for ultrasensitive electrochemical immunoassay of streptomycin residues

A facile and simple electrochemical immunoassay for ultrasensitive determination of streptomycin residues (STR) in food was designed by using nanogold-assembled mesoporous silica (GMSNs) as bionanolabels on a three-dimensional redox-active organosilica-functionalized sensing interface. To construct such a sensing interface, we initially synthesized organosilica colloids by using wet chemical method, and then utilized the prepared colloidal organosilica nanocomposites for the immobilization of monoclonal anti-STR antibodies on a glassy carbon electrode based on a sol-gel method. The bionanolabels were prepared based on coimmobilization of horseradish peroxidase (HRP) and STR-bovine serum albumin conjugates (STR-BSA) on the GMSNs. With a competitive-type immunoassay format, the assay toward STR analyte was carried out in pH 5.5 acetate acid buffer (ABS) by using redox-active organosilica nanocomposites as electron mediators, biofunctionalized GMSNs as traces, and hydrogen peroxide (H(2)O(2)) as enzyme substrate. Under optimal conditions, the reduction current of the electrochemical immunosensor decreased with the increase in STR level in the sample, and displayed a wide dynamic range of 0.05-50 ng mL(-1) with a low detection limit (LOD) of 5 pg mL(-1) at 3s(B). Intra- and interassay coefficients of variation were less than 8.7 and 9.3% for STR detection, respectively. In addition, the methodology was validated with STR spiked samples including honey, milk, kidney, and muscle, receiving a good correspondence with the results obtained from high-performance liquid chromatography (HPLC).     

Sequential determination of two proteins by temperature-triggered homogeneous chemiluminescent immunoassay

A novel protocol for performing a sequential dual-protein immunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to current multilabel-based detection techniques, a single HRP label is employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different targets by taking advantage of thermal response, as demonstrated by sequential detection of human IgG and IgA. PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31 degrees C; thus, it can be separated from supernatant by centrifugation. Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed by primary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in the sample, and HRP-labeled second antibodies. Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Within the calibrated amount, the protocol had excellent precision within 11% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual analyte in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serum samples.     

Piezoelectric immunosensor based on gold nanoparticles capped with mixed self-assembled monolayer for detection of carcinoembryonic antigen

It is very important for a piezoelectric immunosensor to increase specific binding and decrease nonspecific adsorption. This study presents the development of such a piezoelectric immunosensor for the detection of carcinoembryonic antigen. An AT-cut quartz crystal's Au electrode surface was first modified with homogenous self-assembled monolayer of cysteamine (CE). Gold nanoparticles capped with mixed self-assembled monolayer of CE and MH (6-mercapto-1-haxanol) were then attached to the CE monolayer via glutaraldehyde (GA). Antibodies were immobilized onto a mixed self-assembled monolayer of CE and MH with GA as a reactive intermediate too. The binding of target antigens onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the antigen concentration. The stepwise assembly of the immunosensor was characterized by means of cyclic voltammetry technique. This immunoassay was shown to be specific and sensitive, thus providing a viable alternative to carcinoembryonic antigen detection method.     

Sensitive chemiluminescent imaging for chemoselective analysis of glycan expression on living cells using a multifunctional nanoprobe

A novel sensitive chemiluminescent (CL) imaging method was developed for in situ monitoring of cell surface glycan expression through chemoselective labeling of carbohydrate motifs and then binding to a multifunctional nanoprobe. The nanoprobe was fabricated by assembling biotin-DNA and a large amount of horseradish peroxidase (HRP) on gold nanoparticles (AuNPs). The chemoselective labeling was performed by selective oxidization of the hydroxyl sites of sialyl and galactosyl groups on cell surfaces into aldehydes by periodate and galactose oxidase, respectively, and then aniline-catalyzed hydrazone ligation with biotin hydrazide for specific recognition to avidin. With the biotin-avidin system, the multifunctional nanoprobe could conveniently be bound to the glycan sites on the cell surface. The DNA chain presenting between the AuNPs and biotin assembled on the nanoprobe could obviate the steric effect, and HRP acted to trigger the CL emission of the luminal-H(2)O(2) system. Therefore the expression of both sialyl and galactosyl groups could be selectively monitored by CL imaging with high sensitivity due to the high amount of HRP. Using human liver cancer HCCC-9810 cells as a model, this CL imaging strategy could detect HCCC cells ranging from 6 × 10(2) to 1 × 10(7) cells mL(-1) with a detection limit down to 12 cells. More importantly, this method could be used for distinguishing cancer cells from normal cells and monitoring of dynamic carbohydrate expression on living cells, providing promising application in clinical diagnosis and treatment of cancer.     

Immunophenotyping of acute leukemia using an integrated piezoelectric immunosensor array

Immunophenotyping evaluation is of particular importance for the clinical diagnosis, therapy, and prognosis of acute leukemia. In this paper, an integrated piezoelectric immunosensor array has been developed for the first time to detect the differentiated leukocyte antigens for immunophenotyping of acute leukemia. The probes (crystals) of the array were fabricated with plasma-polymerized n-butylamine film and nanometer-sized gold particles on which the Fab'-SH fragments obtained by the reduction of leukemic lineage-associated monoclonal antibodies (markers) were subsequently immobilized. Investigation results showed that the developed immunosensor array could rapidly identify normal cells from leukemic blasts and define the leukemic blasts within certain phenotypic groups (lineages) by only one analysis of the sample purified or unpurified. It permits the detection of unpurified leukocytes in the dynamic concentration range of 2 orders of magnitude (10(4)-10(6) cells mL(-1)). Up to 17 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with 8 M urea. Moreover, the piezoelectric immunoassay system was applied to evaluate a number of practical specimens with immunophenotyping results in acceptable agreement with those clinically classified. The newly proposed multiparameter analysis technique provides a rapid, simple, and direct alternative tool for clinical immunophenotyping of acute leukemia.     

Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.     

Sensitive and simultaneous detection of cardiac markers in human serum using surface acoustic wave immunosensor

We present a rapid and sensitive surface acoustic wave (SAW) immunosensor that utilizes gold staining as a signal enhancement method. A sandwich immunoassay was performed on sensing area of the SAW sensor, which could specifically capture and detect cardiac markers (cardiac troponin I (cTnI), creatine kinase (CK)-MB, and myoglobin). The analytes in human serum were captured on gold nanoparticles (AuNPs) that were conjugated in advance with detection antibodies. Introduction of these complexes to the capture antibody-immobilized sensor surface resulted in a classic AuNP-based sandwich immunoassay format that has been used for signal amplification. In order to achieve further signal enhancement, a gold staining method was performed, which demonstrated that it is possible to obtain gold staining-mediated signal augmentation on a mass-sensitive device. The sensor response due to gold staining varied as a function of cardiac marker concentration. We also investigated effects of increasing operating frequency on sensor responses. Results showed that detection limit of the SAW sensor could be further improved by increasing the operating frequency.     

Versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels

A versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels has been developed for sensitive protein detection. This sandwich-type sensor is fabricated on an indium tin oxide chip covered with a well-ordered gold nanoparticle monolayer. Gel imaging systems were successfully introduced to develop a novel high-efficient optical immunoassay, which could perform simultaneous detection for the samples with a series of different concentrations of a target analyte. The linear range of this assay was between 0.1 and 500 ng/mL, and the assay sensitivity could be further increased to 0.005 ng/mL with the linear range from 0.005 to 100 ng/mL by stripping voltammetric analysis. The immunosensor showed good precision, high sensitivity, acceptable stability, and reproducibility and could be used for the detection of real sample with consistent results in comparison with those obtained by the ELISA method.     

Highly sensitive photoelectrochemical immunoassay with enhanced amplification using horseradish peroxidase induced biocatalytic precipitation on a CdS quantum dots multilayer electrode

Herein we demonstrate the protocol of a biocatalytic precipitation (BCP)-based sandwich photoelectrochemical (PEC) horseradish peroxidase (HRP)-linked immunoassay on the basis of their synergy effect for the ultrasensitive detection of mouse IgG (antigen, Ag) as a model protein. The hybrid film consisting of oppositely charged polyelectrolytes and CdS quantum dots (QDs) is developed by the classic layer by layer (LbL) method and then employed as the photoactive antibody (Ab) immobilization matrix for the subsequent sandwich-type Ab-Ag affinity interactions. Improved sensitivity is achieved through using the bioconjugates of HRP-secondary antibodies (Ab(2)). In addition to the much enhanced steric hindrance compared with the original one, the presence of HRP would further stimulate the BCP onto the electrode surface for signal amplification, concomitant to a competitive nonproductive absorption that lowers the photocurrent intensity. As a result of the multisignal amplification in this HRP catalyzed BCP-based PEC immunoassay, it possesses excellent analytical performance. The antigen could be detected from 0.5 pg/mL to 5.0 ng/mL with a detection limit of 0.5 pg/mL.     

Channel and substrate zone two-dimensional resolution for chemiluminescent multiplex immunoassay

A two-dimensional resolution system of channels and substrate zones was proposed for multiplex immunoassay performed with a designed multichannel chemiluminescent (CL) detection device coupled with a single photomultiplier. Using carcinoma antigen 125 (CA 125), carcinoma antigen 153 (CA 153), carcinoma antigen 199 (CA 199), and carcinoembryonic antigen (CEA) as two couples of model analytes, two couples of capture antibodies were immobilized in two channels, respectively. With a sandwich format, the CL substrates for alkaline phosphatase and horseradish peroxidase were delivered into the channels sequentially to perform a multiplex immunoassay after the sample and tracer antibodies were introduced into the channels for on-line incubation. CA 125, CA 153, CA 199, and CEA could be assayed in the ranges of 0.50-80, 2.0-100, and 5.0-150 U/mL and 1.0-70 ng/mL with limits of detection of 0.15, 0.80, and 2.0 U/mL and 0.65 ng/mL at 3sigma, respectively. The whole assay process including regeneration of the device could be completed in 37 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for a simple, automated, and near-simultaneous multianalyte immunoassay.     

Ultrathin alumina sol-gel-derived films: allowing direct detection of the liver fibrosis markers by capacitance measurement

A capacitive immunoassay based on antibody-embedded ultrathin gamma-alumina sol-gel films (approximately 20 to 40 nm) was successfully prepared in this work. The nanofilms greatly increased the capacitance change initiated by the recognition between the immobilized antibody and the target antigen, which allowed capacitive measurements capable of directly determining the antigen more sensitive than that of thick films. Meanwhile, the inorganic films with high permittivity significantly increased the time constant (i.e., RC value) of the films, which rendered the potentiostatic step method with acceptable S/N ratio. These two advantages enabled the immunosensor to be readily employed in a multichannel capacitance analysis system. An eight-channel hIgG capacitive sol-gel-derived immunoassay based on this system was constructed to illustrate the application. Compared with the detection limits of SiO2 sol-gel-derived hIgG capacitive immunosensors or the conventional ELISA immunoassay, the immunoassay based on thin alumina gel film showed a lower detection limit of 1 ng mL(-1). The novel immunoassay was employed to co-determine two liver fibrosis markers (hyaluronan and laminin) in mixed samples from approximately 0.5 to 50 ng mL(-1). The little derivation caused by the interfered antigen indicated that the sensitive, specific, low-cost sol-gel-derived multichannel immunosensors might be a promising approach in the application of screening disease markers.     

Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers

A novel flow-through immunosensing system for performing a multianalyte chemiluminescent determination in a single run was designed. A new analytical strategy of substrate zone-resolved technique was proposed. Using carcinoma antigen 125 (CA 125) and carcinoembryonic antigen (CEA) as model analytes, the capture antibodies for CA 125 and CEA were immobilized on an UltraBind aldehyde-activated membrane to act as an immunoreactor, to which the mixture of CA 125, CEA, and their corresponding tracers, horseradish peroxidase (HRP)-labeled anti-CA 125 and alkaline phosphatase (ALP)-labeled anti-CEA, was introduced for on-line incubation. The substrates for HRP and ALP were then delivered into the detection cell sequentially to perform substrate zone-resolved immunoassay by a sandwich format. Under optimal conditions, CA 125 and CEA could be assayed in the ranges of 5.0-100 units/mL and 1.0-120 ng/mL, respectively. The whole assay process including incubation, wash, detection, and regeneration could be completed in 35 min. The serum samples from the clinic were assayed with the proposed method, and the results were in acceptable agreement with the reference values. This method and the strategy of substrate zone-resolved technique could be further developed for high-throughput multianalyte immunoassay.     

Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags

A hemin bio-bar-coded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin bio-bar-code. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an electrochemiluminescent (ECL) immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs), and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon-nanotube-enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in bio-bar-code electrocatalyzed reduction of dissolved oxygen. Using α-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL(-1) to 1 ng mL(-1) and a detection limit of 1.0 fg mL(-1). The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the bio-bar-code. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.     

An immunosensor based on the glucose oxidase label and optical oxygen detection

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen sensor and phase measurements. The oxygen sensor membranes prepared on microporous filters were used as a solid phase on which the immunoassay was carried out. These sensing materials in combination with a new measurement setup provided high sensitivity for the detection of oxidase enzymes, being at nanogram per milliliter level, i.e., 10(-11)-10(-12) M, with respect to glucose oxidase and its conjugates. Experimental data on the sensitivity were validated using theoretical equations and calculations. Using the new measurement setup and IgG-anti-IgG as a model, a number of different sensing materials were studied aimed to optimize the immunosensor and evaluate its performance. This approach was then applied to a practical system for the detection of human lactate dehydrogenase isoenzymes. It provided similar sensitivity of approximately 1 ng/mL, which is comparable to that of standard ELISA. The attributes of the new immunosensor approach are discussed with respect to performance and versitility.     

Magnetic bead-sensing-platform-based chemiluminescence resonance energy transfer and its immunoassay application

A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody-antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H(2)O(2)) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (R = I(425)/I(525)) and human IgG concentration in the range of 0.2-4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as R = 1.9871C + 2.4616, and a detection limit of 2.9 × 10(-11) M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.     

Multiplex measurement of seven tumor markers using an electrochemical protein chip

An electrochemical immunosensor for performing multianalyte measurements of tumor markers is described. The sensor consisted of an array of immunosensing electrodes fabricated on a glass substrate. Each electrode contained a different immobilized antigen and was capable of measuring a specific tumor marker using electrochemical enzyme-based competitive immunoassay. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. The biosensor was used to measure the concentrations of seven important tumor markers: AFP, ferritin, CEA, hCG-beta, CA 15-3, CA 125, and CA 19-9. The sensor had excellent precision and accuracy and was comparable in performance to single-analyte ELISAs (1.9-8.1% interassay CV; <2 ng/mL (or units/mL) detection limit for most analytes). Multianalyte assays provide significant advantages over single-analyte tests in terms of cost per test, labor, test throughput, and convenience. We anticipate that chip-based sensors, as described herein, will be suitable for the mass production of economical, miniaturized lab-on-a-chip devices that will have applications in a wide range of clinical, environmental, and biodefense applications.     

Carbon nanotube microwell array for sensitive electrochemiluminescent detection of cancer biomarker proteins

This paper describes fabrication of a novel electrochemiluminescence (ECL) immunosensor array featuring capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests residing in the bottoms of 10-μL wells with hydrophobic polymer walls. Silica nanoparticles containing [Ru(bpy)(3)](2+) and secondary antibodies (RuBPY-silica-Ab(2)) are employed in this system for highly sensitive two-analyte detection. Antibodies to prostate specific antigen (PSA) and interleukin-6 (IL-6) were attached to the same RuBPY-silica-Ab(2) particle. The array was fabricated by forming the wells on a conductive pyrolytic graphite chip (1 in. × 1 in.) with a single connection to a potentiostat to achieve ECL. The sandwich immunoassay protocol employs antibodies attached to SWCNTs in the wells to capture analyte proteins. Then RuBPY-silica-Ab(2) is added to bind to the captured proteins. ECL is initiated in the microwells by electrochemical oxidation of tripropyl amine (TprA), which generates excited state [Ru(bpy)(3)](2+) in the 100-nm particles, and is measured with a charge-coupled device (CCD) camera. Separation of the analytical spots by the hydrophobic wall barriers enabled simultaneous immunoassays for two proteins in a single sample without cross-contamination. The detection limit (DL) for PSA was 1 pg mL(-1) and for IL-6 was 0.25 pg mL(-1) (IL-6) in serum. Array determinations of PSA and IL-6 in patient serum were well-correlated with single-protein ELISAs. These microwell SWCNT immunoarrays provide a simple, sensitive approach to the detection of two or more proteins.