Fabrication Of Functional Biosensor Interfaces And Their Applications In Bioanalysis

Cell toxicity detection, antigen-antibody reaction, enzyme catalysis and carbohydrate-biomacromoleculer binding are popular research domains in the bioananlysis field. Biosensors have been widely used in bioanalysis field as a powful analysis tool, which allows detectiong analyte with high sensitivity and excellent selectivity. A crucial step in biosensor design is the sensor interface fabrication-how biomolecules could be efficiently immobilized on kinds of sensing surfaces with high activiey and stability. This thesis focus on fabricating cell, antibody, enzyme and carbohydrate sensor interfaces base on different immobilization methods and materials, and investigate their applications in bioanalysis field.Many processes in living cells have electrochemical characteristics that are suitable for measurement by potentiometric biosensors. The present work fabricates a potentiometric sensor array to real-time, high throughout monitore the cytotoxicity of hydroquinone to cultured mammalian V79 cells. Various electrode substrates (Au, PPy-HQ and PPy-PS) used for cell growth were designed and characterized. The controllable release of hydroquinone from PPy substrates was studied. Our results showed that hydroquinone exposure affected cell proliferation and delayed cell growth and attachment in a dose-dependent manner. Additionally, we have shown that exposure of V79 cells to hydroquinone at low doses (5μM) for more than 15 hours allows V79 cells to gain enhanced adaptability to survive exposure to high toxic HQ doses afterwards. Compared with traditional methods, the potentiometric biosensor not only provides non-invasive and real time monitoring of the cellular reactions but also is more sensitive for in vitro cytotoxicity study.The lipoic carbohydrate were used as blocking reagent on recombinant antibody-scFv modified immune sensor interface and studied as protein and cell resistant biomaterials. Six types of carbohydrates were examined for their abilities to reduce nonspecific adsorption of human serum and Hela cell. Our data suggested that the structures of carbohydrates play an important role in resisting nonspecific binding. Specifically, the resistance was found to increase in the order of:lipoic fucose< lipoic mannose< lipoic N-acetyl glucosamine< lipoic glucose< lipoic sialic acid< lipoic galactose, where lipoic galactose derivative resisted most nonspecific adsorption. Furthermore, the combination of lipoic galactose and BSA was the most effective in reducing the adsorption of even undiluted human serum and the attachment of Hela cells while allowing specific binding. Several control experiments have demonstrated that the resistant-ability of mixed lipoic galactose and BSA was comparable to the best known system for decreasing nonspecific adsorption.Poly (diallyldimethylammonium chloride) (PDDA) was chosen to disperse single-walled carbon nanotubes (SWCNTs). Then, the positively charged PDDA-SWCNTs composite and negatively charged glucose oxidase (GOD) were employed to fabricate multilayer films on platinum (Pt) electrodes by layer-by-layer self-assembly technique. Results demonstrated that SWCNTs were evenly dispersed within the PDDA films and efficiently improved the conductivity of the resulting films. It showed wide liner range of 0.05-12 mM, high sensitivity of 63.84 uA/mM cm', low detection limit of about 4 uM and small value of the apparent Michaelis-Menten constant,8.46 mM.A boronic acid-containing polymer (boropolymer) as multivalent carbohydrate receptor was synthesized and oriented immobilized on the cysteamine coated electrode. Carbohydrates were conjugated to AuNPs to generate a multivalent carbohydrate moiety to amplify the response signal. Thus, the binding of the carbohydrate conjugated AuNPs to the boropolymer surface are multivalent which could simultaneously increase the sensititiy and specificity.We systematically study the binding between five saccharides conjugated AuNPs with the boropolymer. Our studies show that different carbohydrate gives different binding constant. The associate constant (Ka) was in the order of lactose< fucose< glucose< mannose< galactose< maltose. Furthermore, the multivalent binding between carbohydrates and boronic acids are reversible and allow the regeneration of boropolymer surface using 1 M acetic acid to sequentially capturing and releasing the carbohydrate analytes.Furthermore, the nature biomaterial-hydrophobin immobilizing biomolecular for fabricating biosensor interface was studied. We used the class I hydrophobin (HGFI) to disperse multi-walled carbon nanotubes (MWCNTs) in water. MWCNTs could be effectively dispersed by 30 min sonication in a 0.1 mg/ml HGFI solution. Optical absorption and transmission electron microscopy provide evidence for individually stable dispersed MWCNTs. X-ray photoelectron, Fourier transform infrared, and Raman spectroscopies suggest that HGFI can non-covalently bind to MWCNTs through hydrophobic interaction. A quartz crystal microbalance and electrochemical methods were used to demonstrate that the HGFI-coated MWCNTs can be used to immobilize human immunoglobulin G and glucose oxidase in solution.