Fabrication Of Multi-protein Bio-interphase And Its Applicaton In Electrochemical Biosensor

The biosensors based on multi-protein bio-interphase were particularly interestedfor understanding synergistic mechanism of enzymatic reactions in the living systems.The study of the electron transfer process of redox proteins or enzymes in thebio-interphase can provide model system of electronic transmission mechanism in thereal biological system. The sensors based on multi-protein bio-interphase haveexcellent selectivity, simplicity and rapid response, and can be used in vivo onlinedetection. So they have important application prospect in food inspection,environmental monitoring and exploring information transfer in life. To constructbio-interphase which were advantageous to the electron transfer and develop the highperformance of electrochemical biosensor, we adopted the composite materials whichwith good conductivity and biocompatibility, and used the biological molecules whichcan keep the enzyme activity. This study mainly build multi-protein bio-interphaseand committed to the development of new materials for immobilized technology, inorder to improve the biological activity of protein molecules perfect the performanceof the biosensor. In the experiment, we had constructed a series of multi-proteinbio-interface, and the purpose were to reveal interaction mechanism and informationof transfer regular between proteins, and then develop some superior performanceelectrochemical biosensor. The work was divided into the following four aspects:1. The glucose biosensor was constructed by (Glucose Oxidase（GOD）/Concanavalin A（Con A）nmultilayer: Au nanoparticles（AuNPs）-chitosan（CHIT）nanocomposite was electrodeposited on the glassy electrode （GCE） to provide alarge surface-to-volume ratio and good biocompatibility surface for immobilizing ConA. The GOD was immobilized by sugar residues-lectins specific interaction. Repeatthe preparation process of immobilizing Con A and GOD to obtain the multilayer filmstructure. The sugar residues-lectins specific interaction can make the GOD wasorientedly adsorbed on the electrode surface which can be used to study the directelectron transfer. At the same time, the fixing amount of GOD can been increased bylayer-by-layer （LBL） self-assemble technique. The direct electrochemistry and theelectric catalytic properties of GOD were studied in detail in the experiment, and thesensor can be used to detect the glucose concentration.2. A glucose biosensor was developed based on Cytochrome c （Cyt c） and GOD co-entrapped on AuNPs-CHIT nanocomposites which were constructed on GCEby one-step electrodepositing. The AuNPs-CHIT nanocomposites provided a largesurface-to-volume ratio to greatly amplify the surface coverage of GOD-Cyt cmolecules and good conductivity to realize the direct electron transfer of proteins.Simultaneously, the surrounding Cyt c provided biocompatible microenvironmentsaround GOD to keep its biological activity. Furthermore, the generated H₂O₂fromreduction of O2companying by the oxidation of glucose into gluconic acid can reactwith Cyt c to produce O2, leading a wider linear range. The study can provide thebasis for superior performance glucose sensor which was constructed by bi-proteinbio-interface.3. A multilayer bio-interphase composed of DNA and Cyt c-Horseradishperoxidase （HRP） was developed by layer-by-layer assembling DNA and Cytc-HRP on a gold electrode surface modified by a precursor film. The assemblyprocess was monitored by UV-vis spectroscopy and electrochemical technique. The（DNA/Cyt c-HRP）nmultilayer modified electrode showed a pair of well-definedand nearly reversible peak of the protein Fe（III）/Fe（II） redox couples. The（DNA/Cyt c-HRP）nalso exhibited good electrocatalytic response to reduction ofH₂O₂and O2. This study will shed more light on the electron transfer withinmulti-protein arrangements and the development of biosensors.4. A tri-protein bio-interphase composed of Con A, Cyt c-HRP was developed.The assembly uses lectin-sugar biospecific interactions between Con A and HRP. Thefast electron transfer rate at the bio-interphase indicated synergistic interactionsbetween the involved proteins. Moreover, due to the good electrocatalytic responsethe tri-protein bio-interphase can be used as H₂O₂and O2sensor. This study alsoprovides new insights for the electron transfer of multi-proteins in a bio-interphaseand the development of biosensors. The novel tri-protein bio-interphase was formedas a model system to study the mechanism of multienzyme-catalyzed reactions.