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Surface Modifications for Enhnaced Immobilization of Biomolecules: Applications in Biocatalysis and Immuno-Biosensor

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Surface Modifications for Enhnaced Immobilization of Biomolecules: Applications in Biocatalysis and Immuno-Biosensor SURFACE MODIFICATIONS FOR ENHNACED IMMOBILIZATION OF BIOMOLECULES: APPLICATIONS IN BIOCATALYSIS AND IMMUNO-BIOSENSOR DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yunling Bai, M.S. ***** The Ohio State University 2006 Dissertation Committee: Approved by Professor S.T. Yang, Advisor Professor James F. Rathman _______________________________ Professor Hua Wang Advisor Graduate Program in Chemical Engineering ABSTRACT The goal of this study is to investigate the application of immobilization technology into various systems: immobilized cell/enzyme bioreactor, affinity chromatography, and BioMEM surface modification. All these three application areas were investigated to solve a particular application problem. R-2-hydroxy-4-phenylbutryic acid (R-HPBA) is an important intermediate in the manufacture of angiotensin converting enzyme inhibitors. In this work, a recombinant D- lactate dehydrogenase (LDH) was used to transform 2-oxo-4-phenylbutyric acid (OPBA) to R-HPBA, with concomitant oxidation of β-nitotinamide adenine dinucleotide (NADH) to NAD+. The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii, which were pre-treated with toluene to make them permeable. The whole cells used in the process were more stable and easier to prepare as compared with the isolated FDH from the cells. Kinetic study showed that the reaction rate was dependent on the concentration of cofactor, NAD+, and that both R- HPBA and OPBA inhibited the reaction. A novel method for co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using poly (ethyleneimine) (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with high loading (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield (>95%). A ii fibrous bed bioreactor with co-immobilized cells and enzyme on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity of 9 g/L⋅h and product yield of 0.95 mol/mol OPBA when the concentrations of OPBA and R-HPBA were less than 10 g/L. The production of FDH in a 40-liter bioreactor was carried out with methanol as the inducer to reach the final intracellular FDH activity of 35 U/g cell. Among different permeabilization methods studied, treatment with toluene at a relatively small amount resulted in the cells with the highest FDH activity. Crude FDH cell extract obtained by ultrasonically breaking down the cell wall was treated with polyethyleneimine (PEI) to separate FDH from other proteins. By adding PEI at a low concentration of 0.04 mg/mL to the cell extract, ~50% of the proteins formed aggregates with PEI and precipitated; however, FDH was not in these aggregates and remained in the solution. After the PEI treatment, the specific FDH activity increased by 1.6-fold. SDS-PAGE analysis showed that PEI precipitation removed some impurity protein molecules that cannot be separated by affinity chromatography with Sepharose-Procion Blue HERB as the separation ligand, and thus improved the separation efficiency. The adsorbed FDH in the affinity column was eluted with KCl solution. Adding 5 mM NAD+ in 0.2 M KCl improved the FDH elution and increased the specific FDH activity by 1.38-fold as compared to elution with 1 M KCl. Overall, the PEI precipitation and dye affinity chromatographic process obtained a high recovery yield of 56% with a 5.5-fold increase in the specific FDH activity from the crude cell extract. iii A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylene diamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140 × 125 µm) of the microchannel, the time required for antibody diffusion and adsorption onto the microfluidic surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 ng/mL to 1000 ng/mL, as compared to that of the microtiter plate (from 2 ng/mL to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate. The surface modification method was further simplified to enhance polymer-based microchannel ELISA for E. coli O157:H7 detection. By applying an amine-bearing polymer, poly (ethyleneimine) (PEI), onto poly (methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups iv were introduced to PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that the alkali condition is preferable for PEI attachment onto the PMMA surface. The amine groups on the PMMA surface were then functionalized with glutaraldehyde, whose aldehyde groups served as the active sites for binding the antibody by forming covalent bonds with the amine groups of the protein molecules. This surface modification greatly improved antibody binding efficiency and the microchannel ELISA for E. coli O157:H7 detection. Compared with untreated PMMA microchannels, ~45 times higher signal and 3 times higher signal/noise ratio were achieved with the PEI surface treatment, which also shortened the time required for cells to bind to the microchannel surface to ~2 minutes, much less than that usually required for the same ELISA carried out in 96-well plates. The detection in the microchannel ELISA only required 5 to 8 cells per sample, which is also better than 15 to 30 cells required in multi-well plates. With the high sensitivity, short assay time, and small reagent consumption, the microchannel ELISA can be economically used for fast detection of E. coli O157:H7. v Dedicated to my family vi ACKNOWLEDGMENTS First I wish to thank my advisor, Professor S. T. Yang, for his intellectual support, invaluable guidance, discussions, and encouragements throughout my four years stay at The Ohio State University. Thanks him for his help, understanding and support for my study and life here. I would also like to acknowledge Professor L. James Lee and his group for their insightful and encouraging advice on my research in microfabrication and microfluidic device development. Without their support, I would not have the chance to learn the cutting-edge techniques for the microfluidic biosensor development. Thanks to Dr. James F. Rathman and Hua Wang for serving on my dissertation committee. Thanks also go to my collaborators, Dr. Wei-cho Huang, Dr. Roger Juang, Chee Guan Koh, and Chunmeng Lu on the CD-ELISA project. I would also like to acknowledge our former and current group members for their technical support and friendship. I cherish every moments we shared in the past four years! I am grateful for the Allumini Grants for Graduate Research and Scholarship. Finally, I would like to thank my parents for their love and dedications for raising, supporting, and educating me. Great appreciations to my husband, Mr. Qinghua Qin, for his love, patience, support, accompany and understanding through all these years. vii VITA Feberary, 14, 1974. Born – Xuanhua, China July, 1995. B.S. Fermentation Engineering Tianjin University of Commerce Tianjin, China December, 1997. M.S. Fermentation Engineering Wuxi University of Light Industry Wuxi, China 1998 – 2001. .. Assistant Professor East China University of Science and Technology Shanghai, China 2001 – present. Graduate Research Associate, The Ohio State University PUBLICATIONS 1. Bai, Yunling; Yang , Shang-Tian. 2005. Synthesis of R-2-hydroxy-4-phenylbutyric Acid Using Coimmobilized D-lactate Dehydrogenase and Candida boidinii Cells in a Fibrous Bed Bioreactor. Biotechnology and Bioengineering, 92(2), 137-146. 2. Bai, Yunling; Xu, Zhenghong; Sun, Wei; Tao, Wenyi. 2000. Study on fermentation conditions of a bacterial xylanase producer. Weishengwuxue Tongbao 27(4), 278- 280. 3. Xu, Zhenghong; Bai, Yunling; Sun, Wei; Tao, Wenyi. 2000. Screening and fermentation conditions for Pseudomonas strain over-producing xylanase. Weishengwu Xuebao 40(4), 440-443. 4. Xu, Zhenghong; Bai, Yunling; Sun, Wei; Xu, Xia; Tao, Wenyi. 2000. Extraction and viii characteristics of bacterial xylanase. Wuxi Qinggong Daxue Xuebao 19(1), 35-37. 5. Tian, Lei; Bai, Yunling; Zhong, Jianjiang. 2000. Developments in degradation of toxic organic pollutants by microorganisms. Gongye Weishengwu 30(2), 46-50. FIELDS OF STUDY Major Field: Chemical Engineering Minor Field: Biochemical Engineering ix TABLE OF CONTENT Page Abstract …………………………………………………………………………….........ii
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