The Fabrication And Application Of Novel Electrochemical Immunosensor As Enhanced Sensitivity Strategy

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, low cost and convenient operation. Research on new electrochemical immunosensors is of great significance. In this Paper, the nanotechnology, bio-sensing technology and electrochemical analytical techniques are combined for immunoassay and detection analysis. Ultrasensitive electrochemical immunoassay methods were developed, which indicates a promising potential in early disease screening and diagnosis. Meanwhile, a new method of Fourier Transformed Sinusoidal Voltammetry was introduced and researched in ultrahigh sensitive and selective detection of target analytes. Therefore, this research focuses on the preparation of multi-functionalized nanomaterials, the construction of the immunoreaetion interface, the development of sensitivity enhancement electrochemical immunosensors and the research on application of Fourier Transformed Sinusoidal Voltammetry. The detail contents are as follows:1) Procalcitonin, as a medium of inflammation, has become a new marker of the identification of severe bacterial infections in recent years. A novel method for the determination of procalcitonin(PCT) was developed based on a sandwich-type electrochemical immunosensor, which combined a simple immunosensor array as well as an effectively designed trace tag. The immunosensor was fabricated by layer-by-layer coating graphene, carbon nanotubes, chitosan, glutaraldehyde composite on the working electrode, which can increase the electronic transfer rate and improve the surface area to capture a large number of primary antibodies(Ab1). The trace tag was prepared by loading high-content signal horseradish peroxidase labeled secondary PCT antibody(HRP-Ab2) with Au NPs, which were coated with mesoporous silica nanoparticles through thionine linking. Incomparison with conventional methods, the proposed immunosensor for PCT provided a better linear response range from 0.01 to 350 ng·m L-1 and a lower limit of detection of 0.5 pg·m L-1 under optimal experimental conditions. In addition, satisfactory results were obtained for the determination of PCT in real human serum samples.2) Compared with the enzyme catalytic amplified signal last experiment, a simple sandwich-type electrochemical immunosensor for the determination of Tcd B was developed based on high content enzyme catalysis technology and enhancement nanotechnique. Greatly enhanced sensitivity was achieved based on fabricating the immunosensor by layer-by-layer coating carbon nanotubes, prussian blue, chitosan, glutaraldehyde composite on the working electrode as well as using graphene oxide as a nanocarrier in a multienzyme amplification strategy. In comparison with conventional methods, the proposed immunoassay exhibited a better linear response range for the detection of Tcd B, from 0.003 to 320 ng·m L-1 and a lower limit of detection of 0.7 pg·m L-1 under optimal experimental conditions. Moreover, satisfactory results were obtained for the determination of Tcd B in real human stool samples, indicating that the developed immunoassay has the potential to find application in clinical detection of Tcd B and other tumor markers as an alternative approach.3) Based on high content enzyme catalysis technology last experiment, a new electrochemical immunosensor for enhanced sensitive detection of human immunodeficiency virus p24(HIV-p24) based on graphene oxide as a nanocarrier linked to horseradish peroxidase- signal antibody and enzyme encapsulated in carbon nanotubes-silica as a matrix in a multienzyme amplification strategy was reported. The increase of response current was proportional to the HIV-p24 concentration in the range of 0.5 pg·m L-1 to 8.5 ng·m L-1 with the detection limit of 0.15 pg·m L-1. The assay results for HIV-p24 spiked in human plasma also show good accuracy. This simple and low-cost immunosensor shows great promise for detection in clinical applications.4) Compared with single-analyte immunoassay method in front of the several immunosensors, multianalyte detection has higher analytical efficiency and throughput. A facile, novel multianalyte electrochemical immunoassay for simultaneous detection of a-fetoprotein(AFP) and human epidermal growth factor receptor type-2(HER-2) was proposed, using metal-containing nanomaterials confined in the ordered mesoporous carbon matrix(OMC-M) as labels. Well-dispersed uniform metallic nanocrystallites incorporated OMC materials were fabricated through a simple, economical, and green preparative strategy toward phenolic resol as a carbon source, metal nitrate as metal sources. Under optimal experimental conditions, the proposed immunoassay exhibited a better linear response range from 0.001 to 150 ng·m L-1 for AFP and for HER-2, with a lower limit of detectionof 0.6 pg·m L-1 and 0.35 pg·m L-1, respectively. The immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility.5) The detection methods of several electrochemical immunosensor in front are adopted by the traditional electrochemical analysis technology, it is of great theoretical importance and application value to develop new analytical methods, Fourier Transformed Sinusoidal Voltammetry. The intention of this study is to carry out the applications of Fourier Transformed Sinusoidal Voltammetry based on electrochemical harmonics: 1. using kinetics difference revealed by higher harmonics of Fourier Transformed Sinusoidal Voltammetry for discrimination and quantitative evaluation of electroactive species, especially thermodynamic similar ones; 2. studies of ions transfer across the liquid/liquid interface based on Fourier Transformed Sinusoidal Voltammetry; 3. studies of electrochemical immunoassay in combination with Fourier Transformed Sinusoidal Voltammetry in ultrahigh sensitive detection of target analytes.