Design Of Nano-material And Bioconjugate Nano-probe For The Fabrication Of Electrochemical Immunosensor

Electrochemical immunosensor, combined immunologic methods with electrochemical sensing technique, is an analytical method which possessed high sensitivity, good selectivity, fast, the versatility of construction method, simple operation, and so on. Recently, electrochemical immunosensor detection of tumor markers has been received much attention, which achieved by nanomaterials, signal enhancement technique and protein immobilization technology. Compared with single-analyte immunoassay technology, simultaneous multianalyte immunoassays have higher throughput and analytical efficiency. Combining simultaneous multianalyte immunoassays with signal amplification by nanomaterials, an ultrasensitive simultaneous multianalyte immunoassays method can be developed, which indicates a promising potential in early cancer diagnosis. This research focuses on the construction of immunoactive interface sensing interface for immobilization of antibody and the design nano-probes of signal amplification, and was used to prepare amperometric immunosensor. The research contents are described as follows:1. Electrodeposition of gold-platinum alloy nanoparticles on carbon nanotubes as electrochemical sensing interface for sensitive detection of tumor markerA novel electrochemical sensing interface, electrodeposition of gold-platinum alloy nanoparticles (Au-PtNPs) on carbon nanotubes, was proposed and used to fabricate a label-free amperometric immunosensor. On the one hand, the multiwalled carbon nanotubes (MWCNTs) could increase active area of the electrode and enhance the electron transfer ability between the electrode and redox probe; on the other hand, the Au-PtNPs not only could be used to assemble biomolecules with bioactivity kept well, but also could further facilitate the shuttle of electrons. In the meanwhile, horseradish peroxidase (HRP) instead of bovine serum albumin (BSA) was employed to block the possible remaining active sites and avoid the nonspecific adsorption. With the synergetic catalysis effect of Au-PtNPs and HRP towards the reduction of hydrogen peroxide (H2O2), the signal could be amplified and the sensitivity could be enhanced. Using alpha-fetoprotein (AFP) as model analyte, the fabricated immunosensor exhibited two wide linear ranges in the concentration ranges of0.5-20ng-mL-1and20-200ng-mL-1with a detection limit of0.17ng-mL-1at a signal-to-noise of3. Moreover, the immunosensor exhibited good selectivity, stability and reproducibility. The developed protocol could be easily extended to other protein detection and provided a promising potential in clinical diagnosis application.2. Hollow gold nanoparticles attached horseradish peroxidase nanospheres as signal enhancers for ultrasensitive immunoassayA novel signal amplifiation strategy, hollow gold nanoparticles (HGNPs) attached horseradish peroxidase nanospheres (HRPNPs), was designed for highly sensitive detection of tumor marker. Gold colloidal nanoparticles (nano-Au) were absorbed in L-cysteine functionalized chitosan dispersed single-wall carbon nanotubes (CSSH-SWCNTs), which used as platform and increased load primary antibodies. HRPNPs contained a high content of horseradish peroxidase (HRP), which efficiently catalyzed hydrogen peroxide (H2O2) to amplify the voltammetric signal for sandwich-type immunoassay. HGNPs-HRPNPs with the enlarged surface area loaded a large amount of thionine labeled Ab2and improved the sensitivity of the immunosensor. Using a-fetoprotein (AFP) as model analyte, the designed immunosensor showed linear range from0.025to5.0ng-mL-1with the detection limit down to8.3pg-mL-1. The new protocol showed acceptable stability, selectivity and high sensitivity, which could provide promising potential for clinical research and diagnosis of tumor disease.3. Simultaneous electrochemical immunoassay o f three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancersIn this work, we proposed a novel strategy for simultaneous electrochemical determination of three liver cancer biomarkers, in which three electrochemical redox probes (ferrocene (Fc-COOH),2,2’-Bipyridine-4,4’-dicarboxylic acid (Co(bpy)33+) and Thionine (Thi)) yielded distinct voltammetric peaks, were used to label three antibodies as singal tags, respectively. The position and size of voltammetric peaks reflected the identify and level of the corresponding antigen. In the meantime, a signal amplification strategy was introduced based on gold nanoparticles coated carbon nanotubes as the carrier to immobilize redox probes labeled antibodies, which could amplify the response signal effectively and improve the sensitive. Furthermore, the methodology was evaluated for the analysis of clinical samples, receiving a good correlation between the simultaneous electrochemical immunoassay and the enzyme-linked immunosorbent assay (ELISA) as a reference.