Fabrication Of Glucose Biosensors Based On Nanomaterials And Glucose Oxidase Mono/multilayered Films

Rapid, selective and accurate determination of glucose concentration is very important in clinical, biological and chemical samples, as well as food processing and fermentation. Numerous methods such as fluorescence, electrochemistry, and flow injection have been reported for glucose analysis. Among these techniques, amperometric enzyme-based electrodes, which combine the inherent selectivity of enzymatic reactions with the highly efficient electrochemical signal transduction, have attracted considerable interest due to their advantages in terms of simplicity, high sensitivity, and excellent selectivity by substrate specificity.For the construction of amperometric glucose biosensor, how to firmly assemble glucose oxidase on the electrode is very important. Self-assembly is a simple technique for modifying surfaces at the molecular level, which does not require unusual equipment or special synthetic procedures. Compared with self-assembly monolayer film, layer-by-layer (LBL) film contains 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 for constructing 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 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.Nanomaterials, with their size in the range of 1-100 nm, are currently under intense investigation owing to their special properties. Due to their small size, these materials exhibit quanta-size effect, small-size effect, surface effect and tunneling effect that differ from both bulk material and the individual atoms from which the comprised. With these unique properties, they are widely used in the fields of catalysis, optical absorption, medicine, magnetic medium, new materials synthesis and particularly attractive in biological applications. Introduction of nanomaterials to the system of glucose biosensor is of considerable interest, since nanoparticles can play an important role in improving the biosensor performance due to their large specific surface area and excellent biocompatibility.In chapter 2 section 1, a series of amine-functionalized silica nanoparticles (ASNPs) with varying particle size were prepared according to the St?ber method, and we utilized them for the first time to construct a stable enzymatic film by covalent attachment of periodate oxidized GOx (IO4--oxidized GOx) and ASNPs on an aminated Au electrode. From the analysis of cyclic voltammetry of biosensors using different sizes of ASNPs under the same condition, the surface concentration of electrically wired enzyme (ΓET) was estimated, which was increasing with the decrease of ASNPs size. Therefore, in chapter 2 section 2, we utilized 30 nm ASNPs as a model to construct stable glucose sensors. Through the layer–by-layer method, ASNPs and GOx were deposited alternately on the gold electrode using a glutaraldehyde as a covalent attachment cross-linker. The covalent attachment processes were followed and confirmed by electrochemical impedance spectroscopy (EIS), which demonstrated that the ASNPs/GOx multilayer films are formed in a progressive and uniform manner. The gold electrodes modified with the ASNPs/GOx multilayer films were studied by cyclic voltammetry (CV) and showed 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 (ГE) on the electrode was estimated, which showed a linear relationship with the number of ASNPs/GOx bilayers. This suggests that the analytical performance such as sensitivity, detection limit is tunable by controlling the number of attached bilayers. Furthermore, the comparison inГE between the ASNPs/GOx multilayer films and GOx films does however highlight the importance of silica nanoparticles. Firstly, the ASNPs have large specific surface area and produced a three-dimensional assembly of GOx, which can immobilize more enzymes compared to that on the two-dimensional substrate. Secondly, the biocompatible ASNPs provide a favorable microenvironment for the active immobilization of GOx and avoid the direct contact between the enzyme and the electrode, which appears to prevent the denaturation of the enzyme.Due to their high surface area, excellent electrical conductivity and unique chemical properties, carbon nanotubes have found great applications in many fields. Recently, the combination of MWNTs and GOx is receiving considerable interest. In chapter 3, MWNTs functionalized with amino groups were prepared via silane treatment using 3-aminopropyltrimethoxysilane (APS) as a silane coupling agent. The resulting amino terminated MWNTs (AMWNTs) exhibited an improvement of dispersion in water. Then, they were applied to construct glucose biosensors with IO4--oxidized glucose oxidase (IO4--oxidized GOx) through the layer-by-layer (LBL) covalent self-assembly method without any cross-linker. Scanning electron microscopy (SEM) and EIS were used for characterization of multilayer films, and indicated that the assembled AMWNTs were almost in a form of small bundles or single nanotubes, and the surface density increased uniformly with the number of bilayers. From the analysis of voltammetric signals, a linear increment of the coverage of GOx per bilayer was estimated, demonstrating that the multilayer was constructed in a spatially ordered manner. The marked decrease in the overvoltage for the reduction of dissolved oxygen facilitated convenient low-potential stable detection of the glucose. The enzyme electrode exhibited good electrocatalytic response toward the glucose and that the response increased with the number of GOx/AMWNTs bilayers, suggesting that the analytical performance such as sensitivity and detection limit of the glucose biosensors could be tuned to the desired level by adjusting the number of deposited GOx/AMWNTs bilayers. Because of relative low applied potential, the interference from other electro-oxidizable compounds (such as uric acid and ascorbic acid) was minimized, which improved the selectivity of the biosensors.The first-generation glucose oxidase-based biosensors usually involve oxidation of hydrogen peroxide (H2O2) resulting from the enzyme's reaction with its natural cofactor, O2. In this type of biosensors, a relatively high working potential (ca. 0.6 V vs. SCE) must be applied to achieve a sufficiently high sensitivity. At such a high potential, many compounds commonly coexisting in biological samples (such as uric acid, ascorbic acid and acetaminophen) can also be electrochemically oxidized, giving electrochemical signals overlapping on the one for glucose, which certainly influence the selective and quantitative detection of glucose. A promising method to suppress the interference has been developed by the construction of bienzymatic amperometric biosensors coupling horseradish peroxidase (HRP) with glucose oxidase (GOx). In such configurations, produced H2O2 is subsequently reduced to water by HRP. The detection principle of H2O2 switches from an electrochemical oxidation to a reduction process that happens at much lower potentials, and therefore, avoids the interference of coexisting electroactive species. In chapter 4, we constructed a reagentless bienzymatic sensor for the detection of glucose in the low working potentials without interference. Firstly, the SCGNPs/TH multilayer films were fabricated on the gold electrode using LBL technique via the electrostatic and covalent interactions. On the basis of studies of UV-vis spectroscopy (UV), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), we demonstrated that the SCGNPs/TH multilayers were formed in a progressive and uniform manner. Such SCGNPs/TH multilayer films exhibited tridimensional conductivity and porosity, and TH molecules in each bilayer had well electroactive performance. Secondly, IO4--oxidized GOx and HRP were covalently attached to the multilayer film-modified electrode in a homogeneous array. The resulting bienzymatic sensor exhibited high electrocatalytic response and sensitivity toward glucose, and due to the low working potentials, the interference of coexisting electroactive species such as uric acid, ascorbic acid and acetaminophen were avoided. Furthermore, the catalytic capability improved gradually with the increase in the number of TH. Consequently, the sensitivity of the bienzymatic sensor could be controlled by the number of SCGNPs/TH bilayers assembled on the electrode.