| The developments of bioengineered surfaces enabling deposition of organized biomacromolecules on the nanoscale continue to attract increasing attention because of their broad biotechnological applications including biosensors and bioprocesses, however,how to realize the direct electron transfer(ET)between the active site of proteins and electrode is the crucial issue.According to Marcus theory,the electron transfer(ET)distance is a crucial factor for direct chemistry of redox protein.The ET distance depends on the overall distance between the redox site within the protein and the electrode surface,the depth of the active site inside the protein and the orientation of the protein on the electrode surface.As a consequence,for an optimally designed electrode configuration one has to make the ET distance between an immobilized redox protein and an electrode surface as short as possible.In this paper,horseradish peroxidase(HRP)was immobilized on three kinds of nanometerials and retains its biological conformation and electrochemical activity.The details are given as follows:Chapter one:PerfaceBasic knowledge on direct electrochemistry of protein is introduced including the history,development and applications on biochemistry and biosensing.Among various kinds of base meterials,nanometerials were demonstrated to be able to offer good bioenvirement for the immobilization of pretains because of their unique properties.Chapter two:Direct electrochemistry of horseradish peroxidase in layer-by-layer nanotubes synthesized on templateA novel method based on electric field directed layer-by-layer assembly(EFDLA)and template synthesisis successfully developed to fabricate horseradish peroxidase(HRP) nanotubes.It provides a rapid and general strategy for fimctional protein nanoarrays. The alternative deposition of poly(diallyldimethylammoniumchloride)(PDDA)and HRP are characterized by SEM,TEM,UV-vis and electrochemical impedance spectroscopy(EIS).Moreover,A pair of redox peaks resulted from the redox reaction of HRP were observed with the formal potential of 0.012V(v.s.SCE)in 0.2 mol.L-1 pH=7.0 phosphate buffer solution(PBS).The number of transference electron was 1.05 and the direct electron transfer(eT)constant(ks)was 0.59-1.The(HRP/PDDA)4 NTs modified electrode displayed an excellent electrocatalytic performance for H2O2 from 1.0×10-6to 0.1 mol.L-1with the formal Michaelis-Menten constant(Kmapp)as 8.7 mmol.L-1.The UV-vis spectrometry,electrochemistry of HRP and biocatalysis to the reduction of H2O2 indicate that immobilization of PDDA/HRP via EFDLA coupled with template synthesis could retain the conformations and bioactivities of HRP.Chapter three:Direct electrochemistry of horseradish peroxidase on TiO2 nanotube arrays via Seeded-growth synthesisHorseradish peroxidase(HRP)was successfully immobilized on vertically oriented TiO2 nanotube arrays(NTAs),which was prepared by a seeded-growth mechanism. The nanotubular structure of TiO2 was characterized by scanning electron microscope (SEM).After encapsulated HRP on TiO2 nanotube arrays,the direct electron transfer of HRP was observed.Owing to the redox reaction of electroactive center of HRP,the HRP/TiO2 NTAs modified electrode exhibited a pair of quasi-reversible peaks with the peak-to-peak separation of 70 mV and the formal potential of-0.122V(vs SCE)in 0.2 mol.L-1phosphate buffer solution(PBS,pH=7.0).The number of transference electron was 0.84 and the direct electron transfer(eT)constant(ks)was 3.82 s-1.The HRP/TiO2 NTAs modified electrode displayed an excellent electrocatalytic performance for H2O2 and the formal Michaelis-Menten constant(Kmapp)was 1.9 mmol L-1.The response currents had a good linear relation with the concentration of H2O2 from 5.0×10-7to 1.0×10-5mol.L-1and 1.0×10-5to 1.0×10-3mol.L-1,respetively.Chapter four:Immobilization of horseradish peroxidase on self-assembled (3-mercaptopropyl)trimethoxysilane film:characterization,direct electrochemistry,redox thermodynamics and biosensing Highly organized(3-mercaptopropyl)trimethoxysilane(3-MPT)films have been prepared via self-assembled coupled with sol-gel linking technology.Horseradish peroxidase(HRP)is successfully immobilized onto the densely packed three-dimensional(3D)3-MPT network and the direct electrochemistry of HRP is achieved without any electron mediators or promoters.Redox thermodynamics of HRP on the 3-MPT films,which is determined from the analysis of the temperature dependence of the reduction potential,suggests that the positive shift of redox potentials of HRP at the interface of 3-MPT originates from the solvent reorganization effects and conformational change of the polypeptide chain of HRP.Based on the direct electrochemistry and electrocatalytic ability of HRP,a sensitive third-generation amperometric H2O2 biosensor is developed with two linear dependence ranges of 5.0×10-7～1.0×10-4and 1.0×10-4～2.0×10-2mol L-1.
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