The Construction Of Several Novel Enzyme Biosensors And Their Applications In Biosensing

Biosensors are constructed with the combination of bioactive materials(enzyme, nucleic acid or antibody/antigen, etc.) and various physical/chemical sensing conversion technologies, and have been widely used in chemical analysis and in the determination of different chemicals. In recent years, the research on amperometric biosensor is still very active, and the investigation of various novel materials to realize the high-effective enzyme-immobilization has received much attention in various areas. In this dissertation, we reviewed the principle, the fabrication, and applications of electrochemical biosensors. Novel enzyme-based biosensors are fabricated by combining the advantages of tyrosinase-catalyzed oxidation and various nanocomposites and utilized in enzyme substrate and enzyme inhibitors sensing. The main contents are as follows:(1) The facile preparation of poly(L-DOPA)-tyrosinase(PDM-Tyr) composite and its application both in substrate(phenol) and inhibitor(atrazine) sensing is reported for the first time. Effective immobilization of enzyme is realized via in-situ entrapping Tyr in poly(L-DOPA)(PDM), which is formed by Tyr catalytic polymerization of L-DOPA. Tyr-catalyzed polymerization of L-DOPA as a new substrate was examined in detail by UV-vis spectroscopy and scanning electron microscopy. The Tyr modified electrode is simply prepared by dipping the PDM-Tyr composite on an Au electrode and then covered by Nafion. The thus-prepared Tyr-immobilized electrode exhibits excellent performance superior to most Tyr-based electrochemical biosensors, the sensitivity to phenol is as high as 5122 ?A mM-1(72.5 mA mM-1 cm-2) in the linear range of 10 nM ~ 1.25 ?M, which increases by several orders of magnitude comparing with most reported values. The apparent Michaelis-Menten constant(KMapp) is determined to be 3.13 ?M, which is obviously lower than the reported values and indicates stronger substrate binding and higher catalytic activity. The biosensor also works well in atrazine biosensing based on the inhibition of Tyr by atrazine, possessing a linear detection range of 50 ppb ~ 30 ppm and a low detection limit of 10 ppb. In addition, the biosensor exhibits excellent stability, precision, high sensitivity and fabrication simplicity, which may find wide applications in diversified fields including biotechnology and surface coating.(2) PDDA-protected(PDDA = poly(diallyl dimethylammonium) chloride) gold nanoparticles(AuNPs) have been successfully synthesized in situ on noncovalently PDDA-functionalized multi-walled carbon nanotubes(MWCNTs) via an effective hydrothermal route in the absence of any seeds and surfactants, in which PDDA, an ordinary and water-soluble polyelectrolyte, acts as both a reducing and a stabilizing agent. The products were characterized with scanning electron microscopy(SEM) and transmission electron microscopy(TEM). Enzyme biosensors were constructed by adsorbing glucose oxidase(GOx) or tyrosinase(Tyr) onto the AuNPs decorated MWCNTs(PDDA-MWCNTs-AuNPs) modified Au electrodes. The resultant biosensors exhibit excellent biosensing performance both in substrate sensing and in inhibitor sensing. The Tyr/PDDA-MWCNTs-AuNPs/Au electrode exhibited a sensitivity high up to 10922 ?A mM-1(154.6 mA mM-1 cm-2) in linear range of 10 ppb ~ 2.35 ppm in phenol sensing and a detection limit of 5 ppb in atrazine sensing, The GOx/ PDDA-MWCNTs-AuNPs /Au electrode shows a sensitivity of 81.8 ?A mM-1 cm-2 to glucose sensing at linear range of 0.05 ?M ~ 5.0 mM and a detection limit of 2.0 nM of Ag+ in inhibitor sensing. which demonstrated the PDDA-MWCNTs-AuNPs nanocomposites reported in this paper may be of great potentials in biosensing applications.(3) Combining the advantages of Co/N-doped porous graphene nanowires(Co-N-GNWs) and metal nanomaterials, Co-N-GNWs-AuNPs nanocomposites are prepared by scattering Co-N-GNWs in DMF solution and then reducing HAuCl4 with NaBH4. Co-N-GNWs-AuNPs/GC electrode was prepared by dipping 5 ?L of 1.0 mg mL-1 Co-N-GNWs-AuNPs on GCE electrode surface. For comparison, Co-N-GNWs/GCE electrode was prepared and immersed in 0.10 M H2SO4 aqueous solution containing 1.0 mM HAuCl4 to realize the further modification of AuNPs by electroplating via multi-potential steps, with Aued/Co-N-GNWs/GCE electrode obtained. 10 ?L 1.0 mg m L-1 GOx solution was dipped on electrode and 2 ?L 0.1wt.% CS was used to reinforce enzyme membrane. The resultant amperometric glucose biosensor is endowed with high sensitivity and excellent selectivity. This method is simple, fast and efficient.