Design Of Antigen-amperometric Immunosensor Based On Nan-materials

With the development of nanotechnology, the use of nanomaterials to fabricate electrochemical biosensors has attracted widespread interest, due to their unique mechanical, electrical and optical properties, such as high surface-to-volume ratio, good stability, small dimension effect, good biocompatibility and strong adsorption ability. Particularly attractive for numerous bioanalytical applications are gold nanoparticles, graphene (GR) and carbon nanotubes (CNTs) in applications of biological detection.It is important to develop highly sensitive assay in the immuno-analytical field. Amperometric immunosensor, combined electroanalytical chemistry with immunologic methods, is an important analytical method which possessed good selectivity, high sensitivity, fast, simple operation, the versatility of construction method, and so on. With the rapid development of nano-technique, all kinds of nano-matericals have been used in the field of biosensor due to their unique electricity property, large surface-to-volume area, highly surface free energy, good biocompatible, and so on. This dissertation focuses on the construction of nano-active sensing interface for the immobilization of antibody and the design bioconjuate nano-probes using nano-material as carrier of enzyme and antibody for the analyte recognization and signal amplification, and were further used to prepare amperometric immunosensor. The research details are described as follows:1A label-free amperometric immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticlesIn this work, a novel label-free amperometric immunosensor has been constructed for detecting a-1-Fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) functionalizing carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuC14) to immobilize horseradish peroxidase functionalized carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (electronic transmission force and electrostatic force). Subsequently, AuNPs were doped on the surface of the nanocomposite to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels (HRP-CNTs), which had lager specific surface area and good electronic conductivity compared to carbon nanotubes. Under optimized conditions, the linear ranges were from0.2to200ng-mL-1with a detection limit of0.067ng-mL-1(at an S/N of3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection the recoveries of AFP by standard addition methods in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis.2Sandwich-type electrochemical immunoassay of tumor marker with signal multi-amplification based on ferrocenemonocarboxylic nanospheres as tracer and HRP as enhancerA new bioconjugate probe, based on nanospheres of ferrocenemonocarboxylic (Fc-nanospheres) as tracer, horseradish peroxidase (HRP) as enhancer and gold colloidal nanoparticles (GNPs) as trager, was designed for the fabrication of sandwich-type electrochemical immunosensor with enhanced sensitivity. The presence of the Fc-nanospheres not only provided a large room for the immobilization of antibody molecules (Ab2), but also aggregated more electro-activity substances to enhance the electrochemical signals. The signal intensity of the immunosensor using Fc-nanospheres as tracer labels for carcinoembryonic antigen (CEA) detection was higher than using Fc (bulk) as labels, so we adopted Fc-nanospheres as tracer for amplification electrochemical signals. Moreover, HRP as enhancer and GNPs as trager could further amplify electrochemical signals. The presence of the chitosan enwrapped gold colloidal nanoparticles-graphene composite (AuNPs-CS-GR) was not only for immobilized anti-CEA (Ab,) with bioactivity kept well as matrix, but also improved the area-to-volume ratio of electrode and played a conducting pathway in this immunosensor. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of0.1-200ng-mL-1with a detection limit of0.067ng-mL-1(S/N of3). The proposed immunosenor provided a novel method for the detection of protein in clinical diagnosis.3A doubly-amplification amperometric immunosensor based on Au(core)@Pt(shell) nanospheres and HRP modified glass carbon electrode for AFP detection immunoassayWe modified a layer bioconjugates of carbon nanotubes and graphene on the glassy carbon electrode (GCE), and then the Pt hollow nanospheres was dropped on the pretreated glassy carbon electrode and dried at4℃in the refrigerator by interaction of metal nanoparticles and matrix The Au(core)@P(shell) nanospheres performed good catalytic properties to H2O2. After that, the AFP antibody was fixed on modified electrode by taking advantaged of the good biological compatibility and strongly adsorption effect of the Pt hollow nanospheres. Finally to block remaining active groups of Pt hollow nanospheres and better catalytic reduction of H2O2, HRP solutions was dropped onto the electrode for4h at room temperture. An amplification amperometric immunosensor was constructed based on synergism of Pt hollow nanospheres and HRP to catalytic reduction of H2O2. The Au(core)@Pt(shell) nanospheres have been extensively applicatived for the construction of biosensing devices during the past decade owing to their unique atomic structure, high area-to-volume ratio, and excellent electronic, chemical, thermal, mechanical, optical properties, which could immobilized more of AFP antibody and made the immunosensor more sensitively. Cyclic voltammetry (CV) was the effective and convenient tools to monitor the characteristics of the immunosensor. The experimental parameters, including concentration of H2O2in detection solution, pH of the working buffer, and incubation time, which could affect performance of the immunosensor, were optimized by CV measurement. With the optimization of experimental parameters, the resulted immunosensor of different concentrations of AFP and calibration plots of the cathodic peak current response were displayed. The corresponding calibration plot of peak current versus the logarithm of the concentration of AFP was linear over the range from0.1to200ng-mL-1and the detection limit of0.03ng-mL-1(at an S/N of3). In short, the proposed immunosensor possessed excellent performance for the clinical detection of AFP with simple operation process, low consumption, good selectivity and long-term stability, which was easily to extend other analyzes measured by immunoassay.