Studies On Several Amperometric Enzyme Electrodes Based On Novel Materials And Methods For Enzyme Immobilization

Combining the recognition ability of bioactive species (e.g. enzyme, antigen/antibody, aptamer) and the physical/chemical transducing technology, biosensing analysis has become one of the most important detection methods of modern analytical chemistry. Biosensor is well acknowledged as a device that detects the target with operation facility, time-effectiveness, ease for real-time monitoring and automation, low-cost, and high selectivity/sensitivity, being competent for working in clinical analysis, food security and environment monitoring. The immobilization of biomolecules is the key step in constructing a biosensor. Immobilization of biomolecules with improved methods and materials is essential to improve the biosensing performance, which has become the frontier and focus of the biosensing area. Polyelectrolyte is always the hot researching area of electrochemistry, and it also receives high and current attention in the biosensing area. In this thesis, we briefly review the biosensor, especially of electrochemical biosensor, and the polyelectrolyte, and conduct several biosensing studies based on enzyme immobilization using sodium alginate (AlgNa), poly(diallyldimethylammonium chloride)(PDDA), metal iron, ferrocyanide (K3Fe(CN)6), and Prussian blue composite for high performance glucose biosensing and lactate biosensing. The main contents are as follows:1. An interpenetrating polymer composite was prepared via electrostatic interaction and mixing AlgNa and PDDA, and it was then cast on an Au electrode surface, followed by incorporation of metal ions (e.g. Fe2+or Ca2+, to form AlgFe or AlgCa hydrogel) and glucose oxidase (GOx)(or lactate oxidase (LOx)), to prepare amperometric enzyme electrodes. The interactions of PDDA, Alg, and Fe3+are studied by visual inspection as well as microscopic and electrochemical methods. Under optimized conditions, the PDDA-AlgFe-enzyme/Au and PDDA-AlgCa-enzyme/Au electrodes can give good analytical performance (e.g. nM-scale limit of detection of glucose or lactate, and sensitivities>50μA cm-2mM-1) in the first generation biosensing mode, which are better than the reported analogues using typical polysaccharide biopolymers as enzyme-immobilization matrices. The enzyme electrodes also worked well in the second generation biosensing mode in the coexistence of p-benzoquione or ferrocene monocarboxylic acid artificial mediator. Biofuel cells (BFCs) with the enzyme electrodes as the bioanodes and glucose (or lactate) as the biofuel were also fabricated with satisfactory results. The proposed protocols for preparation of high performance Alg-based biocomposites may find wide applications in bioanalysis.2. Interaction/reaction of PDDA with K3Fe(CN)6yields a precipitate that cannot be robustly immobilized on an electrode surface. In contrast, Interaction/reaction of PDDA-Alg interpenetrating polymer composite with K3Fe(CN)6yields a precipitate that shows high electroactivity and can robustly immobilized on an electrode surface. Immobilization of LOx using the Fe(CN)63--PDDA-Alg composite led to high-performance lactate biosensors. Various interactions/reactions were studied by visual inspection and electrochemical methods. Under optimized conditions, the such-prepared lactate biosensor gives an LDR of2.0μM-1.61mM, a sensitivity of83.7μA cm-2mM-1and a detection limit of77nM (S/N=3), and its stability is good. The K3Fe(CN)6-PDDA-Alg/Au electrode can efficiently electrocatalyze the reduction of nitrite and give a linear nitrite-detection range of5.0μM-9.7mM and a sensitivity of83.7μA cm-2mM-1under optimized conditions, which is amongst the best results reported for nitrite sensing. The electrode was also used for nitrite electroanalysis in real sample matrices with satisfactory results.3. We found that reaction of HAuCl4with K4Fe(CN)6can yield a new Prussian blue composite (PBc), as characterized by visual inspection and UV-Vis spectrophotometry. In a mixing solution of K3Fe(CN)6+HAuCl4+K2SO4, we prepared an Au electrode-supported film of this Au-PBc/Au by cyclic voltammetry, and the prepared electrode shows a high electrocatalytic activity for H2O2oxidation and a high performance for H2O2electroanalysis (detection potential of-0.05V, linear detection range of0.2-20mM, and sensitivity of104.5μA mM-1cm-2). In addition, a high-performance glucose biosensor was constructed based on the composite of PDDA-Alg-GOx and the Au-PBc/Au electrode.