[Show abstract][Hide abstract] ABSTRACT: A multiplexed electrochemical immunoassay method was developed for simultaneous ultrasensitive measurement of tumor markers based on electrochemical stripping analysis of silver nanoparticles (Ag NPs). The Ag NPs were deposited on a disposable immunosensor array with a reduction reaction catalyzed by nanogold labels. The immunosensor array was prepared by covalently immobilizing capture antibodies on chitosan modified screen-printed carbon electrodes. Through a sandwich-type immunoreaction, antibody-functionalized Au NPs were captured onto immunosensor surface to induce the silver deposition from a silver enhancer solution. The deposited Ag NPs could be directly measured by anodic stripping analysis in KCl solution. The catalytic deposition enhanced the analytical sensitivity for detection of protein markers. The interference of dissolved oxygen could be avoided as the detection was performed with positive stripping potential range. Using carcinoembryonic antigen and α-fetoprotein as model analytes, the proposed multiplexed immunoassay method showed wide linear ranges of three orders of magnitude with the detection limits down to 3.5 and 3.9 pg mL(-1), respectively. The localized silver deposition, as well as the stripping detection process, eliminated completely the electrochemical cross talk between adjacent immunosensors. The immunosensor array exhibited acceptable reproducibility, stability and accuracy, showing a promising potential in multianalyte determination for clinical application.
[Show abstract][Hide abstract] ABSTRACT: A palladium nanoparticle decorated carbon nanotube was designed as a label for preparation of a highly sensitive disposable immunosensor. The immunosensor was constructed by assembling the capture antibody on gold nanoparticles decorated graphene nanosheets modified screen printed carbon working electrode. With a sandwich immunoassay mode, the palladium nanoparticle decorated carbon nanotubes were captured to the immunocomplex and showed strong electrocatalytic activity toward oxygen reduction. The use of carbon nanotube carrier offered a high amount of palladium nanoparticles on each immunoconjugate, hence amplified the detectable signal from the electro-reaction of dissolved oxygen. The graphene nanosheets and gold nanoparticles improved the electronic conductivity and the hydrophilicity of electrode surface for immobilization of the capture antibody, respectively. Under optimal conditions, a linear detection range from 50 pg/mL to 10 ng/mL and a limit of detection of 44 pg/mL (0.3 pM) were achieved for human IgG. Using dissolved oxygen as a signal reporter, the detection process avoided deoxygenation. The immunosensor showed acceptable stability, precision and accuracy, indicating potential applications in clinical diagnostics.
[Show abstract][Hide abstract] ABSTRACT: A streptavidin-functionalized silver-nanoparticle-enriched carbon nanotube (CNT/Ag NP) is designed as trace tag for ultrasensitive multiplexed measurements of tumor markers using a disposable immunosensor array. The CNT/Ag NP nanohybrid is prepared by one-pot in situ deposition of Ag NPs on carboxylated CNTs. The nanohybrid is functionalized with streptavidin via the inherent interaction between the protein and Ag NPs for further linkage of biotinylated signal antibodies to obtain tagged antibodies. The functionalization process greatly improves the dispersibility of the nanohybrid in water. The immunosensor array is prepared by covalently immobilizing capture antibodies on chitosan-modified screen-printed carbon electrodes. Through a sandwich-type immunoreaction on the immunosensor array, numerous Ag NPs are captured onto every single immunocomplex and are further amplified by a subsequent Ag NP-promoted deposition of silver from a silver enhancer solution to obtain the sensitive electrochemical-stripping signal of the Ag NPs. Using carcinoembryonic antigen and α-fetoprotein as model analytes, this proposed multiplexed immunoassay method shows acceptable precision and wide linear ranges over four orders of magnitude with detection limits down to 0.093 and 0.061 pg mL−1, respectively. The assay results of serum samples with the proposed method are in acceptable agreement with the reference values. The newly designed strategy and the functionalized tag avoid cross-talk and the requirement of deoxygenation for electrochemical immunoassay, and thus provide a promising potential in clinical application.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: An ultrasensitive multiplexed electrochemical immunoassay method was developed for the detection of tumor markers by combining a newly designed trace tag and a disposable immunosensor array. The array was prepared by immobilizing capture antibodies on gold nanoparticles which were assembled on carbon nanotubes-chitosan modified screen-printed carbon electrodes. The trace tag was prepared by loading signal antibodies and high-content glucose oxidase on amino-functionalized silica nanosphere. With a sandwich-type immunoassay format, ultrahigh sensitivity was achieved by the enzymatic signal amplification with ferrocenecarboxylic acid as electron transfer mediator and the accelerated electron transfer by carbon nanotubes. Using carcinoembryonic antigen and α-fetoprotein as model analytes, this method showed wide linear ranges with the detection limits down to 3.2 and 4.0pg/mL, respectively. The proposed immunosensor array exhibited acceptable stability and reproducibility. The assay results of serum samples were in acceptable agreement with the reference values. This method excluded completely the effect of dissolved oxygen and showed potential application for multianalyte determination in clinical diagnostics.
[Show abstract][Hide abstract] ABSTRACT: This work proposed a simple, sensitive and low-cost multiplexed immunoassay by combining a disposable chip with gold nanoparticle (AuNP) as an electrochemical label. The immunosensors array as the disposable chip was firstly prepared by immobilizing capture antibodies on different screen printed carbon working electrodes by passive adsorption. With a sandwich mode, the analytes were then bound to the corresponding capture antibodies for further capture of the gold nanoparticle labeled antibodies. Gold nanoparticles were finally electrooxidized in 0.1 M HCl to produce AuCl(4)(-) for differential pulse voltammetric detection. Using human IgG and goat IgG as model targets, under optimal conditions this method achieved linear ranges from 5.0 to 500 and 5.0 to 400 ng mL(-1) with limits of detection of 1.1 and 1.6 ng mL(-1), respectively. This method eliminated completely electrochemical crosstalk between adjacent immunosensors due to the strong adsorption of the AuCl(4)(-) on the printed carbon surfaces. The immunosensors array showed acceptable precision, reproducibility and stability, and could be readily applied to multianalyte determination for clinical diagnostics.
[Show abstract][Hide abstract] ABSTRACT: A novel tracer, glucose oxidase-functionalized nanocomposite, was designed to label the signal antibodies for ultrasensitive multiplexed measurement of tumor markers using a disposable immunosensor array. The immunosensor array was constructed by coating layer-by-layer colloidal Prussian blue (PB), gold nanoparticles, and capture antibodies on screen-printed carbon electrodes. The preparation of glucose oxidase-functionalized nanocomposites and the labeling of antibody were performed by one-pot assembly of glucose oxidase and antibody on gold nanoparticles attached carbon nanotubes. The PB immobilized on immunosensor surface acted as a mediator to catalyze the reduction of H2O2 produced in the enzymatic cycle. Both the high-content glucose oxidase and carbon nanotubes in the tracer amplified the detectable signal for the sandwich-type immunoassay. Using carcinoembryonic antigen and alpha-fetoprotein as model analytes, the simultaneous multiplexed immunoassay method using the immunosensor array and the designed tracer showed linear ranges of 3 orders of magnitude with the detection limits down to 1.4 and 2.2 pg/mL, respectively. The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The dual signal amplification of glucose oxidase-functionalized nanocomposites provided a promising ultrasensitive simultaneous multiplexed immunoassay approach for clinical applications.