Layer-by-layer Assembly Of Protein Multilayer Films And Its Application In Biosenors

Recently, construction of protein multilayer films is being a progressively concerned filed due to its potential in application of fabrication sensitive biosensors, highly catalytic microreactors, and biomedical materials. Layer-by-layer (LbL) assembly is a kind of effective approach to fabricate organic nano-thin films. It is important to study the multilayer protein films of special functions and structures using LbL method.Monolayer and multilayer films of three proteins: horseradish peroxidase(HRP) , hemoglobin(Hb), and lyozyme(Lyz) were fabricated with polyelectrolytes using LbL assembly method in the dissertation. Different kinds of ionizations and different kinds of assembly conditions and methods in the assembly process were systematically investigated. The negatively charged HRP and Hb and the positively charged Hb and Lyz were found successfully in assembling multilayer films with oppositely charged polyelectrolytes through electrostatic LbL(ELbL) method. The amounts of protein and polyelectrolyte in the assembly films were increased linearly with the number of deposited layers. The alternate deposition of positively charged HRP with poly (sodium-p-styrenesulfonate)(PSS) was unsuccessful in assembling PSS/HRP multilayer films. The activities of the assembly films were increased only within two alternate bilayers of (PSS/HRP). Obviously, HRP with different kinds of ionizations have different characters. The activity of the (Con A/HRP) multilayer films was increased linearly with the number of assembly layers when constructed with suger-lectin interactions. We developed a kind of pre-absorbed method to fabricate the mono-charged small organic dye and protein composite films with LbL assembly in the dissertation.The film morphology, deposition mass amount, thickness, and the surface character were studied using atomic force microscopy (AFM), UV-vis spectroscopy, ellipsometry, and static contact angle measurement. The activities of the multilayer films were also discussed. Sub-monolayer films of HRP were obtained by using electrostatic LbL methods and the assembled HRP had almost the same height. The size of HRP molecules on the Con A / HRP films correspond to its X-ray diffraction data, which demonstrated the suger-lectin assembly cancontrol the orientation of deposited HRP.The bioinformatics of HRP, Hb, and Lyz were introduced to investigate the surface charges distributions of the proteins. The negative charges distribution of HRP and Hb, the positive charges distribution of Hb and Lyz were found to be even. The even distribution of surface charges of the protein benefits the multilayer assembly. The positive charges distribution of HRP was found uneven, while the positively charged HRP is difficult to construct multilayer films with PSS by ELbL method. Further analyzing of the other eight proteins assembly systems published in the references, we demonstraed the distribution of surface charges of protein is the key factor to the construction the protein multilayer films.On the surface of functional thiols modified gold electrode, several kinds of HRP multilayer films including suger-lectin method, ELbL method, and pre-mix ELbL method were constructed. The performances and the stabilities of the HRP-modified electrodes were evaluated by the electrochemical methods. It is demonstrated that the HRP modified electrode constructed by LbL assembly method have fast response, high sensitivity, and good stability. The study extended protein multilayer films to the biosensors' application.