Preparation And Application Of Bioelectrochemical Sensors

The 21^st century is the century for life science.The intermolecular electron transfer in organism is an important chemical reaction which plays fundamental role in biological function.From the view point of chemistry,many biomaterials are charged particles or molecules,and life are always associated with the movement of charge,such as various redox reactions in organism(respiratory chain,photosynthetic chain),the separation and transfer of substance and charge on membranes,reaction mechanism and biocatalysis. Accordingly,the essence of life is reflected in all kinds of electrochemical phenomena. Studies on the charge transfer process of biomolecules(especially for redox protein and enzyme) not only uncover the information about thermodynamics and dynamics,but also reveal the electron transfer mechanism,energy transfer,material metabolism,as well as understanding structure,physicochemical properties and relationship between different functions of biomolecules.Therefore,the biosensors based on electron transfer mechanism, such as electrochemical enzyme sensor,nanoelectrochemical sensor,DNA sensor and sensor for in vivo detection,are all attracting more and more attentions.Based on electrochemistry,this thesis investigated the electrochemical behavior of varieties of biomolecules and realized in vivo detection of plant using electrochemical sensor.The main research content and results are shown as below:(1) The interaction between dsDNA and CdTe QDs was investigated by an indirect electrochemical method with the assistance of electroactive dsDNA indicator Co(phen)₃^3+/2(phen=1,10-phenanthroline).Influence of CdTe QDs to the system of dsDNA/Co(phen)₃^3+/2+ was studied.The results indicated that CdTe QDs had an obvious effect on Co(phen)₃^3+/2+/dsDNA system.Dissociation method was employed for eliminating the influence of CdTe QDs on the signal from Co(phen)₃^3+/2+.It was found that Co(phen)₃^3+/2+ was more easily dissociated from dsDNA modified gold electrode (dsDNA/Au) in the presence of CdTe QDs.In relatively low ionic strength Tris buffer(pH 7.0,5 mmol/L NaCl),the dissociation coefficient constant of Co(phen)₃^3+/2+ in the presence of CdTe QDs was 3.1 times larger than that in the absence of CdTe QDs,while it was 1.32 times in relatively high ionic strength Tris buffer(pH 7.0,50 mmol/L NaCl), indicating that the binding site of CdTe QDs on dsDNA was probably at major groove of dsDNA.This demonstration offers a new approach to illustrate the QDs cytotoxicity mechanism.(2) Biosensor for hydrogen peroxide was fabricated by co-immobilizing CdTe nanoparticles,chitosan and hemoglobin(Hb) matrix.There was a pair of nearly reversible redox peaks around -0.360V,and the electrochemical behavior of Hb was a surface-controlled process,with electron transfer rate constant of 1.36s(-1) and surface coverage of 2.62×10^-10 mol cm^-2.Fourier transform infrared spectra and UV-Vis spectra indicated that Hb sustained its natural conformation.It was demonstrated that Hb in the matrix kept bioactivity,and exhibited catalytic ability toward H₂O₂,with a response ranging from 7.44×10^-6 to 6.95×10^-4 mol/L,and a detection limit of 2.23×10^-6 mol/L.(3) Superoxide dismutase(SOD) was immobilized on Au electrode by Nation film. Direct electrochemical behavior of SOD at Nation film covered Au electrode was investigated by cycle voltammetry(CV).A pair of quasi-redox peak appeared near +0.16 V at the SOD-Nation modified Au electrode.SOD maintained its bioactivity in Nation film at Au electrode and showed a surface-controlled process with electron transfer rate 0.65 s^-1 at the modified electrode.Further study showed that in Nation film,SOD exhibited catalytic activity toward superoxide anion(O₂^·-) and could detect the concentration of O₂^·-.Moreover,amperometric response of SOD-Nation modified Au electrode was proportional to concentration of O₂^·-in the range of 0.34～3.82 mmol/L, with detection limit of 0.083 mmol/L.(4) Poly-o-phenylenediamine and Pt microparticle modified Pt electrode (POPD/Pt-MP/Pt) as microbiosensor was developed to monitor in vivo oxidative burst induced by Cd²⁺ stress and ultraviolet A(UV-A) and ultraviolet C(UV-C) radiation stress in oilseed rape(Brassica napus L.).After oilseed rape exposure to 84.9 mmol/L CdCl₂ stress,three oxidative bursts were observed in oilseed rape by amperometry at 3.3 h,8.4 h and 13.2 h,respectively.Meanwhile,atomic absorption spectrometry(AAS) verified the existence of Cd²⁺ contained in the leafstalk after Cd²⁺ stress.Under UV stress,twice oxidative bursts were detected,appearing at 2 h and 25 h,respectively.According to the peak area of amperometry,the amount of H₂O₂ induced by UV stress was estimated to be 6.2×10^-10 and 3.1×10^-10 mol for such two oxidative bursts in detected leafstalk, respectively.This novel microbiosensor provides an effective tool for studying the defense reaction of the plant in the situation of UV stress and probing the antioxidative mechanism of the antioxidative enzyme.