Disposable Electrochemical Biosensor Based On Au-Chitosan-Modified Indium Tin Oxide Electrode

Electrochemical enzyme biosensors based on the recognition of specific substrateshave the advantages of high-sensitivity, rapid-response, nice-selectivity, no pollution aswell as easy miniaturization and automation. In the design and fabrication of theelectrochemical biosensor the crucial step is how to develop a simple and effectivestrategy for the construction of sensitive membrane. This research is aimed to developnew material for the immobilization of biomolecule, such as enzyme, antigen etc.According to the advantages of excellent film-forming ability and good adhesionproperties, we used chitosan to modify the indium-tin oxide (ITO) electrode, and thenabsorb colloidal gold nanoparticles to immobilize horseradish peroxidase (HRP) andcarcionembryonic antigen (CEA). The detailed materials are summarized as following:1. ITO electrode is used to fabricate a novel disposable biosensor combined withflow injection analysis for the rapid determination of H₂O₂. The biosensor is prepared byentrapping HRP enzyme in colloidal gold nanoparticle-modified chitosan membrane(Au-chitosan) to modify the ITO electrode. The biosensor is characterized by scanningelectron microscope, atomic force microscope, and electrochemical methods. Parametersaffecting the performance of the biosensor, including concentrations ofo-phenylenediamine (OPD) and pH of substrate solution, were optimized. Under theoptimal experimental conditions, H₂O₂ could be determined in the linear calibration rangefrom 0.01 to 0.5 mM with a correlation coefficient of 0.997 (n=8). The detection limitwas 0.005 mM at 3 signal/noise. The amperometric response of the biosensor did notshow an obvious decrease after the substrates were injected continuously 34 times into theflow cell. The prepared biosensor not only is economic and disposable, due to the low-costITO film electrode obtained from industrial mass production, but also is capable with good detection precision, acceptable accuracy, and storage stability for the fabrication inbatch.2. A novel disposable electrochemical immunosensor for the determination of CEA inhuman serum was proposed by using colloidal gold nanoparticles modified Au-chitosan toimmobilize CEA antigen on ITO electrode. A competitive immunoassay format results inbinding of HRP-anti-CEA antibody to the immobilized CEA antigen, theimmunoconjugate was formed by a competitive immunoreaction of HRP-anti-CEAantibody with analyte CEA was retained in the incubation solution, while the conjugateformed on Au-chitosan modified ITO electrode surface was detected by using theOPD-H₂O₂-HRP electrochemical system. Parameters affecting the performance of theimmunoassay system including concentrations of OPD and H₂O₂, pH of substrate solution,dilution of HRP-anti-CEA, incubation temperature and time were optimized. Under theoptimal experimental conditions, the electrocatalytic current decreased linearly with thecompetitive mechanism. CEA could be determined in the linear range from 2.0 to 20ng/ml (correlation coefficient 0.997) with a detection limit of 1.0 ng/ml. The preparedCEA immunosensor is not only economic due to the low-cost ITO film electrode obtainedfrom industrial mass production, but also capable with good stability and reproducibilityfor the fabrication in batch.