Applications Of Enzyme Amplification In Biosensing And Microfluidic Analysis

Biosensors and microfluidic chip have attracted extensive attention. Due to the advantages low reagent consumption, rapid analysis and sensitive detection, the two can variously be constructed as analysis and detection components. Through the combination of biological, materials, computers and other technologies, biosensor and microfluidic are widely used as analytical methods in food safety, exploration of biological metabolic mechanism, environmental testing and other areas. Enzyme, as an important biomacromolecule in participating in all activities of the ecosystem, was introduced in these two fields. In this thesis, we have established new methods on the detection of purine metabolites and sugar. The details are as follows:1. Amperometric detection of hypoxanthine and xanthine by enzymatic amplification using gold nanoparticles/carbon nanohorn hybrid as carrierA novel gold nanoparticles/single-walled carbon nanohorn hybrid (GNPs/SWCNH) was synthesized for construction of amperometric biosensing platform. The GNPs/SWCNH hybrid could be stable in aqueous solution for at least two weeks, and was characterized with scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy. The average diameter of GNPs in situ synthesized on SWCNH was5-8nm, and the good interaction between GNPs and SWCNH was confirmed by ultraviolet-visible absorption spectroscopy. The GNPs/SWCNH immobilized on platinum electrode showed high electrochemical activity toward the oxidation of hydrogen peroxide and uric acid with low applied potentials. Combining with the enzymatic reaction of xanthine oxidase (XOx), a biosensor for hypoxanthine and xanthine was constructed. The XOx/GNPs/SWCNH-based biosensor exhibited good responses to hypoxanthine and xanthine with the linear ranges of1.5to35.4and2.0to37.3μM, and the detection limits of0.61and0.72μM, respectively. The recovery test showed acceptable results. The gold nanoparticles functionalized carbon nanohorns provided a promising way to construct electrochemical platform for sensitive biosensing.2. Enzyme immobilized microfluidic channel as microreactor for amperometric detection of glucoseA simple and efficient method with enzyme immobilized microfluidic channel as microreactor was designed for amperometric detection of glucose. The microreactor was conposed of a polydimethylsilicone/glass hybrid device with three retainers, a cooling cave and a6cm capillary with a sampling fracture as microchannel. The microchannel was modified by thermal polymerization, followed by covalently attaching with glucose oxidase. Through fracture sampling and electrochromatography seperation, the production via enzymatic reaction was deteminated by Pt electrode at the end of capillary. The linear range for the detection of glucose was0.05-7.5mM with detection limit of23μM. The inter-and intra-chip producibility for detemination of2.5mM glucose were98.5%(n=5) and96.0%(n=5). With the advantage of flexible assembly, rapid efficiency, good stablity and low-cost, this microreactor provided a potential platform for establishing a portable enzyme-based chemical detection system in pratical application.