Preparation And Application Of Optical And Electrochemical Immunosensors Base On Nanoparticles For Tumor Marker Detection

Cancer diagnosis, treatment and prognosis are governed by tumor staging. Early diagnosis ensures better prognosis. Stage of tumor can be assessed through the levels of tumor markers in human serum. Therefore, it is very important to quantitatively detect the level of tumor markers in human serum. At present, the quantitative analysis of tumor markers mainly adopts the immunoassay method, and on this basis, a variety of immunosensors have been developed. Compared with other immunosensors, fluorescence immunosensor and electrochemical immunosensor have attracted much attention because of excellent properties. However, with the evolution of the disease, the content of tumor markers is constantly changing and may be very low. As a result, trace detection of tumor markers in human serum give a bigger challenge to the existing immunosensor, which requires building and developing a higher sensitive fluorescence or electrochemical immunosensor. In this paper, we combine the stability of fluorescence immunosensor, the sensitivity of electrochemical immunosensor, the signal amplify effect of nanoparticles and the specificity of immunoassay to construct several optical and electrochemical immunosensors. A series of experimental studies for detecting single, two or triple tumor markers were carried out. Specific works are as follows:1. A sensitive fluorescence immunosensor for detection of prostate specific antigen based on signal amplify strategy of HRP and SiO₂ NSs.In this work, a simple fluorescence immunosensor based on signal amplify strategy has been described. In the design, the primary antibody?Ab1? was first immobilized on the96-well plates. To construct a sensitive immunosensor, silicon dioxide nanospheres?SiO₂NSs?, with large surface area and good biocompatible, were synthesized for the immobilization of horseradish peroxidase?HRP? and the secondary antibody labeled with horseradish peroxidase?HRP-Ab₂?. In the presence of PSA, a sandwich-type immunocomplex from Ab1-Ag-HRP-Ab₂ had been formed. In the L-tyrosine and H₂O₂ system, L-tyrosine was catalyzed oxidation to form the dimmer that can emit fluorescence.Fluorescence technology was employed to obtain the response signal of immunosensor.Infrared spectroscopy, ultraviolet spectrum and scanning electron microscope were used to investigate the characterization of various nanocomposites. Under the optimal conditions,the changes in fluorescence intensity were linear with the logarithm of PSA concentration from 0.05 to 100 ng m L^-1 with a detection limit of 0.01 ng m L^-1.2. Simultaneous fluoroimmunoassay for detection of two tumor makers based on Si/Cd Te QDs and Si/ Au NCs as labels.In this study, a novel fluoroimmunoassay protocol for simultaneous detection of two tumor markers was described. The new approach employed magnetic bead as carrier for the antibody immobilization, while Cd Te quantum dots?Cd Te QDs? and gold nanoclusters?Au NCs? coated-silica nanospheres were used as labels. Carcinoembryonic antigen?CEA? and?-fetoprotein?AFP? were adopted as model proteins. After a typical sandwich-type immunoreaction, the immunocomplex exhibited two distinguishable fluorescence peaks at550 nm and 655 nm corresponding to Cd Te QDs and Au NCs, respectively. Under optimal conditions, fluorescence intensities were linearly increased to the concentrations of CEA and AFP in range of 0.1-400 ng m L^-1. In the meantime, the proposed method was evaluated with human serum, and the potential of immunoprobe in cell labeling and imaging was also investigated.3. A multiplexed electrochemical immunosensor using silver and gold nanoparticles decorated carbon nanospheres as labels.In this work, a sensitive multiplexed immunosensor was reported. In this approach,carbon nanospheres?CNSs? with good monodispersity and uniform structure were employed as the carrier for the immobilization of nanoparticles?Ag NPs or Au NPs?, thionine and secondary antibodies?Ab₂?.Two nanocomposites?CNSs@Ag NPs-Ab₂ and CNSs@Thi-Au NPs-Ab₂? were prepared and used as electrochemical signal probes. CEA and AFP were used model analytes. Two different primary antibodies?Ab1? were immobilized on the surface of glassy carbon electrode modified with Au NPs-reduced graphene oxide?r GO?nanocomposites. In presence of CEA and AFP, two signal probes were attachment onto the surface of the r GO/Au NPs nanocomposites through a sandwich immunoreaction. Two distinguishable peaks, one was at potential of +0.16V?corresponding to Ag NPs? and another was at-0.33V?corresponding to Thi?, were obtained in differential pulse voltmmetry?DPV?. Under optimal conditions, two biomarkers can be assayed in the ranges of 0.01 to 80 ng m L^-1with the detection limits of 2.8 pg m L^-1for CEA and 3.5 pg m L^-1for AFP,respectively.4. A multiplexed electrochemical immunosensor based on metal ion functionalizednanocomposites as immunoprobes.In this study, we described a multiple immunosensor for simultaneous detection of three tumor markers based on metal ion(Pb²⁺, Cd²⁺, and Zn²⁺) functionalized nanocomposites as immunoprobes. The immunosensor was constructed by depositing good nanoparticles?Au NPs? and coating capture antibodies on the surface of glassary carbon electrode.Immunoprobes were prepared by directly assembling metal ion and secondary antibody on gold nanoparticles attached carbon nanospheres?CNSs?. PSA, CEA and AFP were selected as model analytes. In presence of target analytes and immunoprobes, three different sandwich-type immunocomplex were formed from antibody, antigen and immunoprobes. As a result, three distinguishable peaks whose peak potential was at-0.63 V(corresponding to Pb²⁺),-0.85 V(corresponding to Cd²⁺) and-1.12 V(corresponding to Zn²⁺, vs SCE) were obtained in square wave voltammograms?SWV?. The experimental results showed that the immunosensor exhibited good selectivity, high sensitivity and the cross interference was ignored, indicating it can be used in the simutanious determination of multiple tumor makers in a single run.