Studies Of Amperometric Biosensors Based On Redox Proteins And Enzyme

Biosensors continues to be a very active area of research in analytical chemistry and technology. The recognition of a biological event such as an antibody-antigen binding reaction, conversion of a substrate by an enzyme or electron transfer reaction of a redox protein using electrochemical methods can lead to the development of novel biosensors or further our understanding of life processes.A range of nanoparticles including nanoparticles, nanotubes and nanowires, prepared from metals, semiconductor, carbon or polymeric species, have been widely investigated for their ability to enhance the response of biosensors. Nanoparticles can be used in a variety of ways, such as modification of electrode surfaces, or to modify biological receptor molecules such as enzymes, antibodies or oligonucleotides. Nanosized particles of noble metals, especially gold nanoparticles (GNPs), have received great interests due to their attractive electronic, optical, and thermal properties as well as catalytic properties and potential applications in the fields of physics, chemistry, biology, medicine, and material science and their different interdisciplinary fields. For electroanalytical chemist, more attention has been paid to GNPs because of their good biological compatibility, excellent conducting capability and high surface-to-volume ratio. The introduction of GNPs onto the electrochemical interfaces has infused new vigor into electrochemistry. GNPs modified electrode surfaces, generating functional electrochemical sensing interfaces, have been reported in great quantity. There are also many reports of the direct electrochemistry of enzymes and proteins at nanoparticle-modified electrodes. The enhanced electrochemistry is due to the ability of the small nanoparticles to reduce the distance between the redox site of a protein and the electrode, since the rate of electron transfer is inversely dependent on the exponential distance between them. In our work, we will report some studies on gold nanoparticles-based electrochemical sensing and electrocatalytic systems.In Chapter II, a novel amperometric sensor of nitrite was developed based on the immobilization of hemoglobin/colloidal gold nanoparticles on a glassy carbon electrode by a titania sol-gel film. UV-Vis spectra suggest that the Hb in the film still retains its original structure close to that of the native state of Hb. The sensor shows a pair of well-defined and nearly reversible cyclic voltammetric peaks for the Hb Fe(III)/Fe(II) with a formal potential (E°') of -0.370 V and the peak-to-peak separation at 100 mV s-1 was 66 mV vs. Ag/AgCl (3.0 M KCl) in a pH 6.9 phosphate buffer solution. The surface coverage of electroactive Hb is 3.52×10-11mol cm-2. The formal potential of the Hb heme Fe(III)/Fe(II) couple shifted linearly with pH with a slope of -50.0 mV pH-1, indicating that an electron transfer accompanies single-proton transportation. Gold Nanoparticles can play the role of an efficient electron-conducting tunnel. It is possible that Hb attached to the surfaces of gold nanoparticles has more spatial freedom in its orientation. Therefore, the presence of colloidal gold nanoparticles facilitates direct electron transfer between the heme site of immobilized Hb and the surface of the electrode. The sensor exhibited excellent electrocatalytic response to the reduction of nitrite. The reduction overpotential was reduced by about 0.45 V compared with that obtained at colloidal gold nanoparticles/TiO2 sol-gel film modified GCE. The linear range for nitrite determination was from 2.0×10^-5 to 3.6×10^-4 mol L^-1(n=19, R=0.999) with a detection limit of 1.2×10^-6 M. The sensitivity is 10.1μA·(mol·L^-1)^-1·cm^-2. The stability, repeatability and selectivity of the sensor were also excellent.In Chapter III, A novel amperometric sensor of hydrogen peroxide was developed based on the immobilization of myoglobin (Mb) and colloidal gold nanoparticles on a glassy carbon electrode (GCE) by a Nafion film. The immobilized Mb displayed a pair of well-defined and nearly reversible cyclic voltammetric peaks with a formal potential of -0.373V in a 0.1M phosphate buffer solution (PBS) of pH 6.9. The formal potential of the Mb heme Fe(III)/Fe(II) couple shifted linearly with pH with a slope of -49.6 mV pH-1, indicating that the electron transfer is accompanied by single-proton transportation. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which an unmediated biosensor for hydrogen peroxide was achieved. The linear range for determination of hydrogen peroxide was from 5×10^-6 to 9.0×10^-5 mol L^-1(R=0.998, n=19) with a detection limit of 5.0×10^-7 mol L^-1 at a signal-to-noise ratio of 3. The sensitivity is 281.3μA (mmol L^-1)^-1 cm^-2. The stability,repeatability and selectivity of the biosensor were also excellent. Layered construction of biofunctional molecules such as enzyme into organized systems has attracted considerable attention in recent years due to its potential application in the areas of biosensors. As compared with that of monolayer film, multilayered assemblies contain more amounts of enzymes, which would be of advantage to further improve the analytic performance of those relative enzyme sensors. In capter IV, A feasible approach to construct multilayer films of glucose oxidase/gold nanoparticles on the Au electrode surface using a cysteamine as a covalent attachment cross-linker is described. The layer-by-layer covalent attachment process was followed and confirmed by electrochemical impedance spectroscopy. When the GNPs were covalent attached to sulphydryl surface, Electron-transfer resistance Ret decreased dramatically. The lower Ret indicated that the GNPs-modified electrode possessed an excellent conductive interface. On the basis of the studies of UV-Vis spectroscopy and cyclic voltammetry, we demonstrated that the GOD/GNPs multilayer film was formed in a progressive and uniform manner. From the calculation of apparent diffusion coefficient of ferrocenemethanol, we know the GOD/GNPs multilayer film has excellent permeability. The CV experiments revealed that the Au electrodes modified with the GOD/GNPs multilayers exhibited an excellent bioelectrocatalytic response to the oxidation of glucose and that the bioelectrocatalytic response was directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized on the Au electrode surface. The biosensor constructed with two, four, six bilayers of GOD/GNPs showed high sensitivity of 2.24μA (mmol L^-1) cm^-2, 4.09μA (mmol L^-1) cm^-2 and 5.72μA (mmol L^-1) cm^-2, respectively. The biosensor shows fast response, as well as good stability and long-term life. The proposed method would be applied to the constructions of thickness and sensitivity controllable biosensing interfaces composed of multienzymes as well as a single enzyme.The attachment of redox mediators to a polymeric material that can be cast on an electrode surface is an attractive route to obtain an efficient electron-transfer system for reagentless biosensors. Until recently, the majority of biosensors were based on one or more enzymes used in conjunction with an electrode. The enzyme reaction could be detected electrochemically by measurement of the loss or formation of a substrate or product, by the use of a small mediator species that shuttles between the enzyme and the electrode. Advances in achieving electron transfer have been made by the modification of an enzyme or electrode surface with a mediator. Redox polymers can be directly attached to the electrode surface and can also electrostatically bind to enzyme, so that the enzyme is said to be'wired', and electron transfer between an enzyme such as glucose oxidase (GOD) and the electrode is possible. In capter V, ferrocene-branched chitosan derivatives (CHIT-Fc) are synthesized by reductive N-alkylation of chitosan with ferrocenecarboxaldehyde. The structures of the products are determined by 1H NMR, UV-vis spectra and FT-IR spectra. CHIT-Fc is used as a functionalized matrix to immobilize GOD on glassy carbon electrodes. We construct electrostatic-based CHIT-Fc/GOD film electrodes and covalent-based CHIT-Fc/GOD-CHO film electrodes and investigate their electrochemistry. CV experiments show that the charge propagate through electron-hopping between neighboring ferrocene redox sites in a concentration gradient. Ferrocenyls in CHIT-Fc exhibit an excellent redox activity and establish efficient electrical communication between GOD and the electrodes for the oxidation of glucose. The development of reagentless glucose biosensors are described. The ferrocenyl functionalization level, i.e., DS of ferrocenyls, has some effects on the electrochemical characteristics of the CHIT-Fc/GOD film electrodes. A higher value of C*D_ct^1/2 is found when the concentration of ferrocene redox centers in the film increases. Also, the CHIT-Fc/GOD film electrode with a higher concentration of ferrocenyls exhibits larger current response to the oxidation of glucose. In addition, an increase in C*D_ct^1/2 for CHIT-Fc/GOD-CHO film electrodes with decreasing film-forming solution pH was observed. The sensitivity of electrostatic-based CHIT-Fc/GOD film electrodes and covalent-based CHIT-Fc/GOD-CHO film electrodes for glucose oxidation are 18.0μA (m mol L^-1)^-1 cm-2 and 73.2μA (mmol L^-1)^-1 cm^-2。The later's stability is better than the former's.