The Applications Of Nano-Inorganic And Sol-Gel Materidals In Electrochemical Biosensor

The immobilized protein in suitable matrixes could realize relatively faster direct electron transfer. So, great efforts have been devoted to developing general, stable matrixes for carrying active proteins in direct electrochemistry study and construction of the third generation biosensors.The objective of this dissertation is to explore mesoporous silica, inorganic layered material and ionic liquids based sol-gel material for fabrication the third generation biosensor. More details are summarized below:(1) Intercalation of horseradish peroxidase (HRP) into layered titanate by assembling it with titanate nano-sheets (TNS) was firstly used for fabrication of enzyme electrode (HRP-TNS electrode). XRD result revealed that HRP-TNS film featured ordered layered structures with HRP monolayer intercalated between the titanate layers. UV-Vis spectra indicated the intercalated HRP in TNS film well retained its native structure. The HRP-TNS film was uniform with porous structure which was confirmed by SEM. The immobilized HRP in the TNS film exhibited fast direct electron transfer and showed a good electrocatalytic performance to H₂O₂ with high sensitivity, wide linear range and low detection. The excellent electrochemical performance of the HRP-TNS electrode was attributed to biocompatibility of the titanate sheets, ordered and porous architectures of the HRP-TNS film which retained activity of HRP to large extent, avoided aggregation of HRP, provided better mass transport and allowed more HRP loading per unit area. Thus, the simple method described here provided a novel and effective platform for immobilization of enzyme in realizing direct electrochemistry and had a promising application in fabrication of the third-generation electrochemical biosensors.(2) Layered titanate sheets (TNS) as support matrixes for immobilization of Myoglobin (Mb) were firstly used to fabricate the protein electrode to study the direct electron transfer between redox center of protein and the electrode and establish a model of mediator-free (the third generation) biosensors. The HRTEM and X-ray diffraction pattern revealed that Mb-TNS film featured ordered layered structures with Mb monolayer intercalated between titanate layers. UV-Vis and FTIR spectroscopy showed the immobilized Mb well retained its native-like secondary structure in the film. A pair of well-defined and nearly reversible redox couples with the apparent formal peak potential (E_ρ) of -0.24 V (vs. Ag/AgCl) in pH 5.4 buffer were observed at the Mb-TNS electrode, characteristic of Mb-Fe^III/Fe^II redox couple. The possible reaction mechanism was that a single proton transfer coupled to reversible single electron transfer and H⁺ ions in TNS film might take part in the electron transfer process of Mb. The Mb-TNS electrode exhibited electrocatalytic reduction of hydrogen peroxide in the wide linear range from 2×10^-6 to 1.6×10^-4M, with low detection limit of 0.6μM and low apparent Michaelis-Menton constant (K_m) of 137μM. This excellent catalytic performance of the Mb-TNS electrode attributed from maintaining Mb activity to large extent and porous structure of Mb-TNS film to increase the accessibility toward the substrate. Furthermore, due to compatible micro-environment and the protective effect provided by the layered titanate nano-sheets, the Mb-TNS electrode could work in extreme pH station as low as 2.2 and exhibit enhanced thermal stability that the immobilized Mb retained 92% initial activity after even heating at 85℃for 20 min.(3) Hb modified electrode was successfully fabricated to realize direct electrochemistry by immobilizing of Hb in bimodal mesoporous silica (BMS) and chitosan (CS) inorganic-organic hybrid film. Here, BMS acted as a support to immobilize Hb due to its large pores and chitosan acted as a binder to increase film adherence and stabilizer to prevent the leakage of Hb. The resulting electrode (Hb/BMS/CS) gave a well defined, reversible redox couple for Hb-Fe^III/Fe^II with a formal potential of about -0.32 V (vs. Ag/AgCl) in pH 7.0 phosphate buffer solution. Hb/BMS/CS electrode showed a better electrocatalytial performance to H₂O₂ with wider linear detection range, lower detection limit, and higher sensitivity than that at electrode without BMS. The improved electrocatalytic performance for Hb/BMS/CS electrode was possibly contributed to BMS bimodal structure, whose large pores with 10-40 nm provide favorable conditions for protein immobilization and small pores with 2-3 nm avoid the mass-transfer limitations. In addition, UV-Vis and FTIR spectra indicated that Hb well maintained its native structure in the hybrid film.(4) Ionic liquid of [BMIM][BF4] based sol-gel modified electrode ([BMIM][BF₄]/Gel/Au electrode) was prepared by immobilization of [BMIM][BF₄] into silica sol-gel matrix on the surface of Au electrode via a simple sol-gel method. Taking advantage of ion exchange reaction between [BMIM][BF₄] and K₃Fe(CN)₆, Fe(CN)₆^3- was entrapped into the sol-gel matrix when [BMIM][BF₄]/Gel/Au electrode was immersed in K₃Fe(CN)₆ solution. Following by continuously scans, the cyclic voltammograms of the resulting [BMIM]₃Fe(CN)₆/Gel/Au electrode in K₃Fe(CN)₆ solution behaved "PB (Prussian Blue)-growing pattern". FTIR, elemental analysis and UV-Vis absorbance spectra results manifested the formation of PB in the sol-gel matrix. Furthermore, the formation mechanism, kinetics and stability of the PB/Gel/Au electrode formed by this method were discussed.In addition, sol-gel covalently modified with [PMIM][PF₆] as support for Hb (the resulting composite noted as [PMIM][PF₆]-Gel/Hb) was constructed the third biosensor. UV-Vis spectra indicated that Hb well maintained its native structure in [PMIM][PF₆]-Gel/Hb composite. Immobilized Hb at the resulting electrode gave a well defined, reversible redox couple for Hb-Fe^III/Fe^II with a formal potential of about -0.32 V (vs. Ag/AgCl) in pH 7.0 PBS solution. The [PMIM][PF₆]-Gel/Hb electrode exhibited electrocatalytic reduction of hydrogen peroxide in the linear range from 10×10^-6 to 70×10^-6 M. The [PMIM][PF₆]-Gel composite provides a new platform to construct the third biosensor.