Ultrasensitive Multiplexed Electrochemical Immunoassay Using Funtionalized Nanomaterials For Signal Amplification

Electrochemical immunosensor, which combines highly specific antigen-antibody interaction with electrochemical sensing technique, has been applied extensively to clinical diagnostics, pharmaceutical analyses, and quality control in environment and food field because of its unique advantages such as excellent selectivity, high sensitivity, real time detection, convenient operation, low cost and easy miniaturation. Compared with single-analyte immunoassay method, electrode array-based multiplexed immunosensor has higher analytical efficiency and throughput. By combining the multiplexed immunosensor with signal amplification by functionalized nanomaterials, an ultrasensitive multiplexed electrochemical immunoassay method can be developed, which indicates a promising potential in early cancer screening and diagnosis. Focusing on the preparation of immunosensor arrays and functionalized nanoprobes, and the construction of the novel ultrasensitive multiplexed electrochemical immunoassay methods based on nano signal amplification strategy, this thesis carried out some researches as follows.1. Disposable immunosensor array for ultrasensitive detection of tumor markers using glucose oxidase-functionalized silica nanosphere tagsAn 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 a-fetoprotein as model analytes, this method showed wide linear ranges with the detection limits down to 3.2 and 4.0 pg/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 showed potential application for multianalyte determination in clinical diagnostics.2. Dual signal amplification of glucose oxidase-functionalized nanocomposites as trace label for ultrasensitive simultaneous multiplexed electrochemical detection of tumor markersA 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 sandwich-type immunoassay. Using carcinoembryonic antigen and a-fetoprotein as model analytes, the simultaneous multiplexed immunoassay method using the immunosensor array and the designed tracer showed linear ranges of three 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.3. 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 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 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 four 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 electrochemical immunoassay, and thus provided a promising potential in clinical applications.4. Electrochemical stripping analysis of nanogold label-induced silver deposition for ultrasensitive multiplexed detection of tumor markersA multiplexed electrochemical immunoassay method was developed for simultaneous ultrasensitive measurement of tumor markers based on electrochemical stripping analysis of silver nanoparticles (Ag NPs) catalytically deposited by nanogold labels on a disposable immunosensor array. 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, and further induced a subsequent silver deposition from the silver enhancer solution. The deposited Ag NPs were then measured directly by anodic stripping analysis in KCl solution. Both the silver deposition and the well-defined stripping sharp peak in KCl solution amplified the detection signal, and the positive stripping potential range excluded the interference of dissolved oxygen. Using carcinoembryonic antigen and a-fetoprotein as model analytes, this 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, respectively. The localized deposition and stripping processes of this method eliminated completely electrochemical cross talk between adjacent immunosensors. The immunosensor array showed acceptable reproducibility, stability and accuracy, indicating a promising potential in multianalyte determination for clinical application.5. Streptavidin-functionalized silver-nanoparticles-enriched carbon nanotube tag for ultrasensitive multiplexed detection of tumor markersA streptavidin functionalized silver-nanoparticles-enriched carbon nanotube (CNT/Ag NP) was designed as trace tag for ultrasensitive multiplexed measurement of tumor markers using a disposable immunosensor array. The CNT/Ag NP nanohybrid was prepared through one-pot in situ deposition of Ag NPs on carboxylated CNT. The nanohybrid was functionalized with streptavidin via the inherent interaction between protein and Ag NPs for further linkage of biotinylated signal antibodies to obtain tagged antibodies. The functionalization process greatly improved the dispersibility and stability of the nanohybrid in water. The immunosensor array was 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 were captured to every single immunocomplex, which were further amplified by a subsequent Ag NPs-promoted deposition of silver from the silver enhancer solution to obtain the sensitive electrochemically stripping signal of Ag NPs. Using carcinoembryonic antigen and a-fetoprotein as model analytes, this proposed multiplexed immunoassay method showed acceptable precision and wide linear ranges over four orders of magnitude with the detection limits down to 0.093 and 0.061 pg/mL, respectively. The assay results of serum samples with the proposed method were in acceptable agreement with the reference values. The newly designed strategy and the functionalized tag avoided cross talk and the requirement of deoxygenation for electrochemical immunoassay, and thus provides a promising potential in clinical application.