Preparation Of Immobilized Enzyme Capillary Microreactors And Their Applications

Enzymes, as biomacromolecules with catalytic activity, are closelyrelated to a variety of biological physiological behaviors, dominatingprocesses like biological metabolism, nutrient absorption and energyconversion, etc. Immobilized enzymes are prepared through fixing freeenzymes on solid supporter or keeping them in a certain area, which canenhance reusability and recoverability and reduce cost. Capillary enzymemicroreactors are a class of immobilized enzymes and particularlysuitable for the treatment of precious samples, possessing high enzymestability, low consumption and easy separation from reaction mixtures.Because of these advantages, immobilized enzyme capillarymicroreactors have attracted wide attention during recent years and havepotential applications in the fields of analytical chemistry, drug screeningand clinical medicine. The enhancement of immobilized enzyme concentration promote, enzymolysis efficiency and stability are the mainobjectives in the development of new immobilized techniques.In this thesis, new types of immobilized enzyme capillarymicroreactors have been developed, which combines the remarkableadvantages of polyamidoamine dendrimer, magnetic nanoparticles andimmobilized enzymes. The main research work is listed as follows:1. G3polyamidoamine dendrimer was grafted onto the surface ofsilica by microwave irradiation. A novel enzyme immobilized materialwas developed through immobilizing cellulase onto the prepared G3polyamidoamine dendrimer-grafted silica and applied in hydrolysis ofchitosan. Enzymatic hydrolysis conditions were optimized by singlefactor and orthogonal experiments. The results indicate that thepolyamidoamine dendrimer has a good performance as a carrier, and canbe potentially adapted to support other biomacromolecules in capillarymicroreactors.2. Different generations of polyamidoamine dendrimer wereintroduced onto the inner wall of fused-silica capillaries by microwaveirradiation and a new type of glucose oxidase capillary enzymemicroreactor was developed based on enzyme immobilization in theprepared polyamidoamine dendrimer-grafted fused-silica capillaries. Theoptimum enzymolysis conditions for β-D-glucose in the microreactorwere evaluated. The introduction of polyamidoamine dendrimer enabled larger amounts of glucose oxidase to be immobilized, which markedlyimproves enzymolysis efficiency with increasing PAMAM generation.The experimental results provide the possibility of increasing enzymebinding capacity and foundation for the development and application ofonline immobilized capillary enzyme microreactors.3. Glucose oxidase was immobilized in the inlet end of capillary as aplug and a new type of online immobilized glucose oxidase capillaryenzyme microreactor was developed based on enzymatic redox reactionwith1,4-benzoquinone as an acceptor of electrons. The enzymatichydrolysis, electrophoretic separation and detection could beaccomplished simultaneously via the prepared capillary, which combinedthe advantages of both enzyme microreactor and capillary electrophoresis.In addition, online enzyme inhibition study was performed by the onlineimmobilized glucose oxidase capillary. The suggested experimentalplatform is recommended for wide applications in high-throughputenzyme assays.4. A new type of immobilized alkaline phosphatase was developedbased on enzyme immobilization on the prepared superparamagneticnanoparticles with a clear three-dimensional core-shell architecture andhigh saturation magnetization. It was applied in the enzymolysis andenzyme inhibitive assay with theophylline, L-tryptophan as modelinhibitors. The magnetic nanoparticles coated with immobilized alkaline phosphatase was introduced into capillary used as enzyme microreactorarea by fast oriented aggregated via magnets. An online enzyme capillarymicroreactor was established based on magnetic nanoparticles, which canpotentially realize high-throughput screening of enzyme reactions andenzyme inhibitors.