Development Of Direct Electrochemical Enzyme Biosensors Using Novel Nanoscaled Hybrid Materials

Recent years witness the increasing interests of developing direct or reagentless electrochemical enzyme sensors, most known as the third generation enzyme sensors. In this regard, the effective immobilization of enzyme onto the electrodes is considered to be one of the key steps to facilitate desirable sensing properties including high loading amount and well retentive bioactivity of immobilized enzymes, and good electron transferring ability and the lifetime of biosensors. However, most of the current enzyme immobilization methods need to be further improved in achieving these purposes. With the rapid development of nanostructured materials and nanotechniques, many nanomaterials with unique physical and chemical properties have been widely explored for electrocatalytic sensing applications. In this thesis, several novel enzyme immobilization plateforms have been successfully developed for direct electrochemical enzyme sensors by synergistically using ZnO nanoparticles, nano-sized gold particles (Nanogold), magnetic Fe3O4 nanoparticles and biomimetic polydopamine, aiming at increased loading amount and high bioactivity of immobilized enzymes towards enhanced detection limit and life time the resulting biosensors. The detailed works are shown as follows:1. A novel immobilization platform has been developed for fabricating enzyme-based biosensors of direct electrochemistry by synergistically using ZnO crystals and Nanogold. ZnO crystals were synthesized with flower-like structure to be casted on the electrode mediated by chitosan so as to provide larger surface area for anchoring horseradish peroxidase (HRP)-labeled Nanogold. The resultant enzyme biosensor was tested for the determination of H2O2 as a model of test system. Experimental results showed that HRP could be immobilized onto the nanocomposite matrix with high loading amount and well-retained bioactivity. Moreover, rapid and direct electron transferring could be achieved between the enzyme's active sites and the electrode surface, thus facilitating the direct electroanalysis of H2O2. The developed enzyme sensor can directly determine H2O2 in the concentration range from 1.5×10-6 to 4.5×10-4 mmol/L, with a detection limit of 7.0×10-7 mmol/L.2. A highly robust enzyme immobilization platform has been fabricated for electrochemical biosensors by synergistically using bio-mimetic dopamine polymer film and Nanogold. Taking HRP as an enzyme model, this presented platform has been successfully exploited to develop a novel electrochemical enzyme sensor for the detection of H2O2. Experimental results show that the nanocomposite matrix can allow HRP to be immobilized with high loading amount and well-retained bioactivity. Moreover, it can achieve rapid electron transferring between the enzyme's active sites and the electrode surface, facilitating quick electroanalysis of H2O2. In addition, compared with other polymer materials (i.e., chitosan), the as-proposed biosensor design using bio-mimetic dopamine polymer can show much better analytical performances in terms of linear concentration range, detection limit and sensitivity, detection reproducibility, and storage stability.3. In order to improve the loading amount and stability of enzyme, a new enzyme immobilization platform has been developed using higly adhesive dopamine, conductive Nanogold and magnetic Fe3O4 nanoparticles. Here, Fe3O4 nanoparticles were first labeled with enzyme and then conjugated with Nanogold mediated by dopamine to be magnetically immobilized onto the piezoelectric electrode. Experimental results show that the synergistical effects of inner polymer conjugation and the exterior magnetic and the electron channel function of Nanogold could lead to greatly increased loading amount and stability of immobilized enzyme (HRP as a model system), and emhanced detection sensitivity and lifetime of H2O2 biosensors showing direct electrochemistry.