Fabrication Of The Enzyme Interface Dopped With Cytochrome C And Its Application In Biological Sensor

Electrochemical biosensors based on protein interface have valuableapplications in industrial control, environmental monitoring, food inspection andmajor disease diagnosis owing to its excellent selectivity, rapid response, simplicity,low-cost and in vivo test. However, to explore suitable strategy for the fabrication ofprotein interface to implement the signal transition is the most crucial step. A newthinking for protein interface study is employing some materials which is superior inconductibility and biocompatibility to construct the protein interface because it isfavor in keep the activity of the protein and achieve electron transfer, and it makesthat it is possible to realize the quickly electron transition and then make furtherefforts to develop some high-performance biosensors. To follow the thinking, thepaper choice cytochrome c as the model protein, make efforts to search some newimmobilization technique of biosensors and construct a bi-protein interfaceco-immobilized by cytochrome c and horseradish peroxidase to reveal themechanisation of the interreaction of proteins and the communication of them, andthen develop some more sensitive electrochemical biosensor elements. The concretework is divided into the following four aspects:1. Direct electrochemistry of cytochrome c and its biosensing based onPDDA-Gp-Au nanocomposite. The functionalized grapheme nanosheets （Gp-PDDA）with Poly（diallyldimethylammonium chloride）（PDDA） were synthesized and thePDDA facilitated the in situ growth of high dispersed Au nanoparticles on the surfaceof grapheme nanosheets to form PDDA-Gp-Au nanocomposites. And then, make itused to construct a cytochrome c modified electrode. Here, PDDA acts as both areducing agent and a good stabilizer during the formation of grapheme nanosheets.Another important role of PDDA is to facilitate the uniform deposition of Aunanoparticals on grapheme nanaosheets. The synergistic effect of the grapheme andAu nanoparticals makes the prepared nanocomposites superior in conductibility andbiocompatibility. Results showed that direct electron transfer has been establishedbetween cytochrome c and the underlying electrode and the resulted electrode Cytc/RTIL-Au-Gp-PDDA/MUA-MCH/Au exhibited superordinary electrocatalitytowards hydrogen peroxide （H₂O₂）. 2. Direct electrochemistry of cytochrome c and its biosensing based onPFS-DNA3D composite film. DNA is a biological polymer, its stacked base pairscould be considered as a system of connected π electrons to transfer electrons. Itcan be used to enhance the electron transfer of many electroactive species. Anexciting result showed that DNA and PFS could form a3D porous configuration. Inthis part, Cytochrome c （Cyt c） was successfully immobilized onpoly（ferrocenylsilane）–DNA （PFS–DNA） composite film modified gold electrode byelectrostatic adsorption. The direct electron transfer of Cyt c in the composite filmmodified electrode and its application as hydrogen peroxide （H₂O₂） biosensor wereinvestigated. The results suggested that Cyt c could be effectively immobilized in thePFS–DNA composite modified electrode. PFS–DNA composite films showed anobvious promotion for the direct electron transfer between Cyt c and the underlyingelectrode. The immobilized Cyt c also exhibited a good electrocatalytic activitytowards the reduction of H₂O₂. Based on the novel construction, a third-generationbiosensor could be obtained for the determination of H₂O₂.3. The study of electron transfer of Co-immobilized cytochrome c andhorseradish peroxidase in chitosan-graphene oxide modified electrode. In this part,The GO-CHIT nanocomposite as a novel electrochemical platform designed bycombining the biocompatibility of CHIT and the conductivity of GO was used for theimmobilization of the redox enzymes. That is, Cytochrome c （Cyt c） and horseradishperoxidase （HRP） were co-immobilized on graphene oxide （GO）-chitosan （CHIT）modified Au electrode and the electron-transfer between the two proteins wasinvestigated. Results showed that direct electron transfer has been established for thebi-protein system and the average rate constant of electron transfer ks,（Cyt c-HRP）=2.63s^-1was larger than those values at the electrodes modified with single protein(ks,（Cyt c）=1.44s^-1, ks,（HRP）=1.53s^-1). The bi-protein modified electrode exhibitedgood electrocatalytic response to reduction of oxygen （O2） and hydrogen peroxide（H₂O₂）, suggesting that a third-generation biosensor could be obtained for thedetection of O2and H₂O₂.4. In this part, layer-by-layer self-assembly （LBL） technology was used to designa bi-protein multilayer modified electrode. On the basis of the third part, an Auelectrode which covered by a mixed self-assembled monolayer ofmecraptoundecanoic acid （MUA） and6-mercapto^-1-hexanol （MCH） and coated withCyt c was immersed in GO-CHIT nanocomposite and the mix-protein consisted ofCyt c and HRP solution alternately. UV-vis and EIS were used to monitor and confirm the growth process. The influence of the number of the multilayer to themechanisation of the interreaction of proteins and the electron transition wereinvestigated, and then compared the performance of the bi-protein and the signalmultilayer interface to reveal the communication of the proteins further. On the otherhand, the electrocatalytic performance of the electrode based on the bi-proteinmultilayer interface towards O2was investigated. Results suggested that the bi-proteinwas successfully immobilized in the GO-CHIT nanocomposite by LBL technologyand keep their electrobioactivity. The resulted multilayer electrode exhibited excellentelectrocatalytic performance towards O2at a low scan rate.