Preparation And Application Of The Electrochemical Biosensor Based On Nanomatials Modified ITO Electrode

The indium-tin oxide (ITO) film electrodes from industrial mass production havebeen used as the working electrode in our work. Colloidal gold nanoparticle andnanocomposites were used to modify the surface of ITO electrode, and then immobilizethe biomolecules to construct novel biosensors. The paper can be summarized asfollows:1. A disposable pseudo-mediatorless amperometric biosensor has been fabricatedfor the determination of hydrogen peroxide (H₂O₂). In this study, colloidal goldnanoparticles modified chitosan membrane (Au-chitosan) on the surface of an ITOelectrode was used for the coadsorption of horseradish peroxidase (HRP) andtetramethyl benzidine (TMB). The immobilized TMB was used as an electron transfermediator, which displayed a surface-controlled electrode process at the scan rate lessthan 60 mV/s. The biosensor was characterized by scanning electron microscope,photometric and electrochemical measurements. Parameters affecting the performanceof the biosensor including the concentrations of the immobilized TMB and HRP, pHvalue and the reaction temperature were optimized. Under the optimized experimentalconditions, H₂O₂ could be determined in a linear calibration range from 0.1 to 1.15mM with a correlation coefficient of 0.998 (n=10) and a detection limit of 1μM at asignal-to-noise ratio of 3. The stability of the biosensor was assessed by the flowinjection analysis, and the amperometric response of the biosensor did not show obvious decrease after detecting H₂O₂ for successively 17 times. The proposed methodprovides a new alternative to develop low-cost biosensors by using ITO film electrodesfrom industrial mass production. The proposed method provides a new alternative todevelop low-cost biosensors by using ITO film electrodes from industrial massproduction.2. An amperometric quasi-reagentless immunosensor for prostate specific antigen(PSA) has been developed based on co-adsorption of HRP labeled PSA antibody(Anti-PSA) and TMB in Au-chitosan. The immobilized TMB displayed asurface-controlled process at the scan rate less than 45 mV/s, and adiffusion-controlled process at the scan rate higher than 45 mV/s. The immunosensorwith the co-immobilized Anti-PSA and TMB was incubated with sample PSA antigen,and the formed immunoconjugate in the immunosensor was detected by aTMB-H₂O₂-HRP electrochemical system. Under the optimal experimental conditions,PSA could be determined in the linear range from 5.0 to 30 ng/mL with a detectionlimit of 1.0 ng/mL.3. A disposable pseudo-mediatorless amperometric biosensor has beenfabricated for the determination of H₂O₂. ITO electrode was modified with thiolfunctional group by (3-mercaptopropyl) trimethoxy silane. The stable nano-Au-SHmonolayer (AuS) was then prepared through covalent linking of gold nanoparticlesand thiol groups on the surface of the ITO. The HRP and TMB were finallyco-entrapped by the colloidal gold nanoparticles. The immobilized TMB displayed asurface-controlled electrode process at the scan rate less than 50 mV/s. The biosensorwas characterized by photometric and electrochemical measurements. The resultsshowed that the prepared AuS monolayer could not only steadily immobilize HRP butalso efficiently retain HRP bioactivity. Parameters affecting the performance of thebiosensor including the concentrations of the immobilized TMB and HRP, pH valueand the reaction temperature were optimized. Under the optimized experimentalconditions, H₂O₂ could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1μM at aS/N ratio of 3.4. A novel electrochemical biosensor with double enzyme for rapid analysis ofglucose was developed. HRP and glucose oxidase (GOx) were co-immobilized withAg-Carbon nanotubes and Chitosanmembrane (Ag-CNT-Ch) on the surface of the ITOelectrode. The immobilized GOx catalyzed glucose to produce gluconic acid and H₂O₂, then the produced H2O2 was catalyzed the oxygenation by the co-immobilized HRPwithσ-phenylene diamine (OPD). In the presence of GOx and HRP, the intensity of thepeak current is rising with the increased concentration of glucose. The biosensor wascharacterized by electrochemical method. Parameters affecting the performance of thebiosensor including concentration of immobilized GOx and HRP, pH value andconcentrations of OPD were optimized. Under the optimized experimental conditions,glucose could be determined in the linear calibration range from 0.5 to 30μM with acorrelation coefficient of 0.997 (n=7) and a detection limit of 0.1μMat a signal-to-noiseratio of 3.