Electron Transfer Of Biomolecules At Bionic Interface And Spectrofluoroelectrochemistry

The electron transfer among the biomolecules is an important reaction in implementing the biofunctions. Electrochemical method is applied widely in life science researches due to its advantages of rapid, simple, lower consume, high sensitivity, real time and real place examination. It can be used to research the electron transfer process among the biomolecules, which not only can obtain the basic thermodynamics and dynamics parameters, but also can show the electron transfer mechanism. This is significant to understand the life course and prepare the electro-biosensors.Ddeoxyribonucleic acid (DNA) is the basic germ plasm of life. The studies of DNA drive the development of life science of 20 century. DNA modified electrodes also can be used to study the interactions between DNA and other molecules. Carbon nanotube as one kind of new nano-materials has attracted widely attention in electrochemical sensors'studies for its perfect electron-transfer capacity and bio-compatibility.However, since information obtained from the normal electrochemical methods has statistical and average characters, it cannot explain the chemical process at a molecular level. Spectrofluorescence method has higher sensitivity and can provide multi-parameter information such as excited and emission spectra, fluorescence intensity and efficiency. The combination of spectrofluorescence method and electrochemical technology realizes in situ study for molecules'electron transfer and promote understanding the structure and function of biomolecules at a molecular level. Multi-photon excitation (MPE) fluorescence overcomes the 1PE's shortcomings such as light damnification and light bleaching by takes advantage of higher penetrability and lower energy at the excitation wavelengths, and furthermore, several kinds of molecules can be excited at a same wavelength. So MPE fluorescence method in conjunction with the electrochemical method has significant meanings and practice values for researching the energy transfer and electron transfer.In this thesis, the method by drying/adsorption was used to prepare DNA and CNTs adsorptively modified electrodes, and adopted electrochemical to research the electrochemical behaviors of molecules, and the main contents and results are summarized as follows:(1) Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) have been used to investigate the interactions of Cytochrome c (Cyt c) with dsDNA and ssDNA at glassy carbon (GC) and gold electrodes, respectively. The results indicate that there are strong interactions between Cyt c and DNA. The binding constant (kds) of Cyt c with dsDNA and ssDNA are (1.69±0.38)×10⁵ and (3.35±0.50)×10⁵ L mol^-1, respectively. The binding sites are achieved to be 3.3 bp per Cyt c molecule with dsDNA and 4.0 nucleotides (ssDNA) binding one Cyt c molecule. This experiment affords a valid method for investigating the interactions between DNA and proteins by electrochemical techniques.(2) The size effect of nano-sized scale materials has been deeply investigated in chemistry, physics etc. Interactions between double-stranded DNA (dsDNA) and [Co(phen)₃]³⁺/²⁺ (Phen=1,10-phenanthroline) at multiwall carbon nanotubes (MWCNTs)-based nanometer-scale size interface and normal interface have been studied by the Cyclic Voltammetry (CV). The results demonstrate that the interactions between dsDNA and [Co(phen)3]3+/2+ increase greatly at MWCNTs-based nano-scale size interface comparing with those at common large size interface. At MWCNTs nano-scale size interface, the binding constant ( k A) of [Co(phen)₃]²⁺ with dsDNA is (2.53±0.22)×10⁴ L mol^-1, which rises evidently comparing with a k Aof (1.68±0.15)×10⁴ L mol^-1 obtained at large size interface. The dissociation rate constant (k) for [Co(phen)₃]²⁺ bound to dsDNA at large size interface is 0.16 min-1, which is also larger remarkably than a k of 0.0025 min-1 obtained at nano-scale size interface. All the researches reveal that the interactions of DNA with small molecules increase remarkably at nano-size interface comparing with those at normal electrodes.(3) The interface behavior and biocatalytic activity of superoxide dismutase (SOD) were studied at carbon nanotube (CNT) surface with Cyclic Voltammetry (CV). The results show that SOD participates in a rapid exchange with CNT and the process is a single-electron, single-proton process. The electron transfer coefficient is calculated to be 0.52, the electron transfer rate constant is 1.4 s^-1, and the average surface coverage is measured to be 6.93×10^-11±4.2×10^-12 mol cm^-2. The bioactive measurements show that SOD keeps its bioactivity at the CNT surface. SOD′s remarkable ability to catalyze the dismutation of the superoxide anion ( O₂^-) has been demonstrated. Spectroelectrochemical methods were adopted to research the electrochemical behaviors of molecules at interface, and the main contents and results are summarized as follows:(4) Electrochemical fluorescence modulation of 5-HT-HCl has been studied here. The results indicate that, when the concentration of 5-HT was higher than 30μmol/L, the electrochemical reaction is approximately a single electron transfer process; when the concentration was less lower than 30μmol L^-1, the number of electrons transferred is about 1.5. These results are consistent with what was obtained by electrochemical method. At the same time, the diffusion coefficient (D0) was calculated as 2.6×10⁽-6( cm² s^-1 by making use of electrochemical modulating fluorescence spectra. This method offers a new approach to research 5-HT's electron transfer.(5) A two-photon spectrofluoroelectrochemical system has been established through combining two-photon-excited fluorescence and electrochemical methods. Ti: Sapphire Laser is used to induce two-photon-excited fluorescence in the researches. The results indicate that, when excited by 740 nm laser photons, riboflavin emits strong two-photon fluorescence, its two-photon adsorption cross section is 35 GM and the quantum yield is 0.26. The formal potential of the electrochemical reaction is -0.56 V and the number of electrons transferred is 1.8.