| The specific attachment of biomolecules onto solid supports, e.g. electrode, is of substantial interest for research in biotechnology. Especially in the fields of biosensors and bioelectronic devices, recent activities have focused on spatially arranged assemblies of biofunctional molecules because of such a molecular level design is key for the development of high-performance biosensors and devices to mimic biological cell membranes.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. Specific biological interactions such as antibody-antigen, streptavidin-(or avidin-)biotin, and lectin-saccharide bindings have proven to be useful forconstructing layered enzyme films. However, in the procedure of construction this kind of multilayer films, the enzyme must be labeled with biotin or other molecules that have biospecific binding ability. It is often time-consuming, especially, that results in some cases in the loss and reduction of the activity of enzymes. Although the layer-by-layer self-assembly technique based on electrostatic interaction of oppositely charged polyelectrolyte and enzymes has proven to be a rapid and experimentally simple way to produce layered enzyme structure, the stability of such assembled films is not always adequate, which limits their development and applications. To date, the quest for the molecularly organized and stable protein thin films still remains a challenge.To overcome the drawbacks associated with above techniques, in chapter two, we developed two simple and convenient methods to construct stable and multilayered enzyme films, (i) Through alternate layer-by-layer deposition of periodate-oxidized glucose oxidase (GOx) and poly(allylamihe) (PAA), covalent coupling of enzyme aldehyde groups and amine groups of polymer via the Schiff base reaction, the multilayer films of GOx/PAA were obtained. X-ray diffraction (XRD) experiments revealed that the films were homogeneous and formed in an ordered manner with a thickness of 2.6 ±0.1 nm per bilayer. Cyclic voltammetry (CV) experiments showed that the gold electrodes modified with the GOx/PAA multilayers had excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator. From the analysis of voltammetric signals, the coverage of active enzyme on the electrode surface was estimated, which had a linear relationship with the number of GOx/PAA bilayer.This suggests that the analytical performance of the sensor such as sensitivity, detection limit, and so on, is tunable by controlling the number of attached bilayer. In addition, the sensor exhibited good reproducibility and stability. In addition, we attached ferrocene to the PAA backbone, using redox polymer PAA-Fc replaced PAA, and alternately deposited with GOx into multilayer films on a gold electrode. Cyclic voltammetry showed one well stable reversible redox peak with a small peak separation (ca. 0.045 V at 0.05 V-s"1) for the multilayer film electrode containing GOx/PAA-Fc; the redox surface concentration was obtained through integration of the ferrocene/ferricinium voltammetric peaks, which increased in step with the number of PAA-Fc layers deposited. Enzyme catalysis for the oxidation of glucose was achieved without additional adding of mediators to the solution, thus realized the construction of reagentless enzyme sensor, (ii) We developed a new way to fabricate covalently attached glucose oxidase multilayer films by exploiting the simplicity of layer-by-layer self-assembly technique combined with the photoreaction of diazonium and carboxylic groups. Diazo-resins (DAR) as polycation and glucose oxidase (GOx) as polyanion were alternately deposited into a multilayer structure using layer-by-layer self-assembly technique based on electrostatic interaction. Upon near UV irradiation, the adjacent interfaces of the multilayer films reacted to form a crosslinking structure, which greatly improved the stability of the enzyme films. The change from the ionic bonds to covalent bonds was monitored and confirmed by UV-vis and IR spectroscopy. Ellipsometric measurements revealed that the enzymes formed sub-molecule layers, and the thickness of the films showed a linear relationship with the number of assembledlayers, demonstrating a spatially well-ordered manner of multilayer structure. The multilayered enzyme film electrode can catalyze oxidation of glucose when ferrocenemethanol was used as a diffusional electron-transferring mediator, and the current response is' rapid and stable for the electrochemical analysis. The sensitivity of the glucose sensor was estimated through the analysis of voltammetric signals, which can be fine turned to the desired level by adjusting the number of attached GOx layers.Introduction of nanometer materials to the system of biosensor is of considerable interest in the fields of biotechnology and bioanalytical chemistry, because nanoparticles can play an important role in improving the biosensor performance due to their large specific surface area and excellent biocompatibility. In chapter three, a nanoparticle biosensor was fabricated by immobilization of GOx on gold nanoparticles, which had self-assembled on Au electrode modified with thiol-containing three-dimensional network of silica gel. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments indicated that these immobilized gold nanoparticles can efficiently improve the electron transfer between GOx and surface of the electrode interface. The sensor exhibited fast amperometric response (3 s) to the mediated electrocatalyzed oxidation of glucose, and the catalytic current is proportional to the concentration of glucose up to 6 mM with a high sensitivity of 8.3 uA-mM^-cm'2 and low detection limit of 23 (iM. hi addition, the sensor has good reproducibility and long-time stability. The surface coverage of electrically wired active GOx of the Au/sol-gel/nanoparticle/cystamine/GOx was determined to be 4.5 * 10"12 mol-cm'2,which is higher than that obtained on Au/dithiol/au/cystamine/GOx (3.2* 10"12 mol-cm"2), in which gold nanoparticles were self-assembled onto gold electrode by two-dimensional dithiol, and much higher than that of 1.0* 10"12 mol-cm'2 for the enzyme electrode, Au/cystamine/GOx, without nanoparticles. From analysis of above data, we can conclude that both the sol-gel technique and gold nanoparticles play important roles in improving the biosensor performance and several advantages of the proposed method for GOx immobilization should be highlighted. First, thiol-containing silica gel can be assembled onto a gold electrode to firm a three-dimensional network, which provides more stereo attaching sites for gold nanoparticles than that based on two-dimensional plane; the porous structure of the silica sol-gel matrix yields a low mass transport barrier and results in a rapid diffusion of substrate and mediator from bulk solution to enzyme. Second, the gold nanoparticles immobilized by silica gel three-dimensional network can act as tiny conducting centers. They were distributed throughout the film and formed a continuous array of gold nanoparticles on the electrode, which can decrease the energy barrier, facilitate electron transfer, and improve biosensor response. The third, the gold nanoparticles have large specific surface area and produced a three-dimensional assembly of GOx, which can immobilize more enzymes as compared to the two-dimensional substrate. Moreover, the gold nanoparticles can provide a favorable microenvironment for the active loading of GOx due to their excellent biocompatibility. Most important of all, self-assembly of sol-gel and gold nanoparticles have little affected on the bulk property of primary gold electrode, such as mass transfer ability, and so on, and thereby, the resulting biosensor have theadvantages of fast response and high sensitivity. The covalent mode of enzyme immobilization ensures that the sensor process good reproducibility and long-time stability.Embedded protein in a polyelectrolyte multilayer matrix is a promising strategy for preparation of ordered enzyme films. In chapter four, we reported on a new approach to construct ordered enzyme films by infiltrating GOx into a basic multilayer films of (PDDA/GN)n/PDDA. The fabrication procedure is simple and convenient and involves only two steps: firstly, layer-by-layer electrostatical self-assembly of PDDA and gold nanoparticles (GN), which forms the basic multilayer films (BMF) needed; secondly, incubation of BMF modified electrode in a GOx solution, which absorbs the GOx into the BMF. The bioelectrocatalytic characteristics of the modified electrodes for glucose were investigated by CV in detail. From the analysis of voltammetric signals, it was found that the coverage of active enzyme on gold electrode showed a linear function to the number of PDDA/GN bilayers. This result confirmed the GOx penetration into the BMF and suggested that the BMF-based enzyme film formed in a uniform manner. Gold nanoparticles in the BMF play an important role not only in adjusting the compactness of the multilayers, thereby adjusting the amount of enzyme absorbed, but also in decreasing the electron transfer resistance (/??) of the sensor, which was verified by electrochemical impedance measurements. So we can proud to say that by using of this simple and convenient method, the GOx could be embedded in the multilayers of (PDDA/GNyPDDA in a uniform manner, and resulting biosensor has the advantages of high enzyme loading, sensitive response and low...
|
PDF Full Text Request