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New Fabricating Methods Of Active Nano-interface As Applied To Electrochemical Biosensor Design

Posted on:2006-10-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:C X LeiFull Text:PDF
GTID:1118360152970085Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
Electrochemical biosensors combining the specific recognition of biomaterials with the magnification function of electrochemical determination have the advantages of high-sensitivity, nice-selectivity as well as easy miniaturization and automation. They can be applied to a wide range of analytical tasks, such as clinical diagnosis, bioassay, environmental monitoring and industrial analysis. In the design and fabrication of electrochemical biosensors, the development of a simple and effective strategy for the construction of sensitive membrane on or into the electrode is a crucial step. This research is aimed to develop new immobilization strategies of biomaterials for the purpose of improving the performance and long-term stability of biosensors. The detailed materials are summarized as follows:1. Three kinds of novel strategies for forming active nano-Au interface on the surface of electrodes have been developed and successfully applied to immobilize enzymes or immunoreagents fabricating electrochemical biosensors.(1) The chitosan-entrapped carbon paste electrode (CCPE) was obtained by using chitosan-modified graphite powder as conductive material and paraffin as adhesive. Chitosan molecules embedded in CCPE contain lots of amino groups, which have high affinity for nano-Au. So the stable nano-Au monolayer could be formed on the surface of CCPE resulting from strongly electrostatic interaction between nano-Au and -NH2. In chapter 2, a H2O2 biosensor based on immobilization of HRP to a nano-Au monolayer supported by CCPE was described. The research results demonstrated that the negatively charged nano-Au could effectively immobilize positively charged HRP and retain the catalytic activities of enzyme to a large extent. The HRP electrode provided a linear response to H2O2 over a concentration range of 1.22×10-5 to 2.43 × 10-3 mol/L with a sensitivity of 0.013 A-L/mol-cm2. The biosensor was found to be stable over three weeks. Taking account of the gold or nano-Au being a suitable matrix for the immobilization of antibody molecules, which contain -SH, in chapter 7, we utilized the nano-Au monolayer formed on the surface of CCPE as a platform where the antibodies were loaded and the competitive immunoreaction took place.The sequential competitive immunoassay included following steps: Schistosoma japonicum antibody was firstly immobilized to the nano-Au monolayer, subsequently the remaining active sites of nano-Au monolayer was blocked with BSA, then the analyte Schistosoma japonicum antigen (SjAg) and SjAg labeled with HRP was combined to the surface of biosensor and the electrochemical detection was conducted. The investigation indicated that the nano-Au monolayer is an appropriate interface for immunoassay and can be used to sensitive detection of different samples. This methodology has several attractive advantages, such as high stability of nano-Au monolayer formed, easy adsorptive immobilization of antibody on nano-Au monolayer, efficient activity retention of loading immunoreactants as well as simple operation for the formation of nano-Au monolayer. The dynamic concentration range for SjAg assay is 0.11 to 22.4 g/mL with a detection limit of 0.06 g/mL. The regeneration of nano-Au monolayer for reuse after each measurement was realized by treating biosensor with a 0.5 mol/L NaOH-NaCl solution (pH 12).(2) Considering chitosan's excellent film-forming ability and good adhesion, we used it to form a pre-coated film on the surface of the glassy carbon electrode (GCE). The plenty amounts of amino groups of chitosan film on the surface of GCE facilitated the formation of nano-Au film on its surface through strong electrostatic interaction. By immobilizing HRP on the surface of nano-Au film, a H2O2 biosensor could be fabricated. The linear range of detection for H2O2 is from 6.1 10-6 to 1.8 x10-3 mol/1, and the lifetime of the biosensor was over 1 month. By further immobilizing glucose oxidase (Gox) onto the HRP layer, a Gox/HRP bienzyme biosensor can be applied to the determination of glucose with the linear range...
Keywords/Search Tags:Electrochemical biosensor, Active nano-Au interface, Biomaterials immobilization, Electrostatic interaction based on polyions, Rapid electrochemical immunoassay, Inorganic-organic hybrid film
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