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University of Cincinnati UNIVERSITY OF CINCINNATI Date: 8/01/2006 I, Adam Bange , hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy , in: Chemistry , It is entitled: Development and Characterization of Miniaturized Electrochemical Immunosensors This work and its defense approved by: Chair: H. Brian Halsall _ ______ Chair: William R. Heineman ___ Pearl Tsang ________ Carl J. Seliskar _ PhD Dissertation Development and Characterization of Miniaturized Electrochemical Immunosensors Adam Bange A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Chemistry of the Colleges of Arts and Sciences 2007 Committee Chairs: Dr. H. Brian Halsall, Dr. William R. Heineman Abstract The objective of this research was to improve the capabilities of immunosensing devices through miniaturization and using nanoscaled materials. Collaboration between the Chemical Sensors Group and the UC Department of Engineering was established to develop sensors and sensing systems using modern microfabrication technology. One facet of this collaboration included work to miniaturize a microbead-based sandwich immunoassay that was developed by previous group members. A procedure to fabricate poly(dimethylsiloxane) microfluidic devices was developed to control the movement of small amounts of immunoassay reagents and microbeads. The devices were then used to evaluate bead and fluid manipulation, and detection. While the electrochemical and fluorescence measurements required for an immunoassay were demonstrated, we were not able to efficiently manipulate microbeads due to their adsorption to the microchannel walls. Research was then done to improve the detection step of a miniaturized immunoassay. A microbead based sandwich immunoassay was developed using an interdigitated array (IDA) electrode with nanoscale dimensions (220 nm electrode width, 620 nm gap). The IDA was fabricated using an electron beam lithographic lift-off technique. After an antibody-assisted capture of antigen using paramagnetic microbeads, a β-galactosidase labeled secondary antibody was used to convert p-aminophenyl galactopyranoside (PAPG) into the redox active p- aminophenol (PAP). Amperometric detection of PAP with IDA electrodes at +300 and -200 mV vs. a Ag/AgCl reference electrode was used to measure the result, detecting MS2 bacteriophage concentrations as low as 10 ng/mL. iii The third research project focused on making a label-free immunosensor using an array of carbon nanotubes as an electrode and electrochemical impedance spectroscopy (EIS) for detection. Highly aligned multi-walled carbon nanotubes were grown by chemical vapor deposition using a metallic catalyst, Fe/Al2O3/SiO2, on Si wafers. The nanotube towers were removed from the silicon and cast in epoxy, then polished so that one end was exposed for electrical connection and the other used as the electrode array surface. The nanotubes were functionalized electrochemically to form carboxyl groups and then chemically conjugated to antibodies. EIS was used to directly monitor the antibody-antigen binding. iv v Acknowledgements Throughout my graduate career, I have benefited immeasurably from collaborations both within and outside of my research group. From the UC department of engineering, I would particularly like to thank Erik Peterson, Dr. Xiaoshan Zhu, and Dr. Yeoheung Yun for their contributions. Erik and his advisor, Dr. Ian Papautsky, taught me the basics of microfabrication while we were directly working together, then he continued to give me technical, scientific, and professional advice, computer help, and fantastic lunch conversations for my entire graduate career. My collaboration with Xiaoshan and his advisor, Dr. Chong Ahn, was a great learning experience for me, and I sincerely appreciate the long nights of hard work he put in to fabricate the IDA electrodes. Yun and his advisors, Dr. Mark Schulz and Dr. Vesselin Shanov, made the nanotube based biosensor project possible, with their innovative ideas, fast paced work schedule, and patience to pursue very hard problems, even when our experiments weren’t working out as well as we had hoped. I greatly appreciate the support of my research group, and the efforts that everyone made to build a community atmosphere through parties, pot luck lunches, birthday lunches, etc. Also, several group members were important collaborators on my research projects. Irena Nikcevic played a critical role in the microfluidics project, particularly by handling the fluorescence measurements used to characterize microchannels and monitor bead adsorption. Dr. Jian Tu taught me how to efficiently do immunoassays, and was always available to help make measurements or troubleshoot when something was going wrong. I owe a special thanks to Dr. Kevin Schlueter for sharing his insights concerning my research as well as chemistry in general, jobs, real estate, the environment, cars, computers, meteorology, rocket science, pharmaceuticals, the criminal justice system, geopolitics, firearms, botany, nuclear fusion, the energy industry, vi natural disasters, and a few hundred other topics that he knows enough about to make encyclopedias obsolete. None of my graduate school success would have been possible without the support of my research advisors, Dr. William Heineman and Dr. Brian Halsall. While their teaching, advising, and writing styles are very different, I knew I could always count on them for the finest advice concerning all aspects of my life as a graduate student. As a new graduate student, it can be very easy to take exceptional research direction for granted. I admit I didn’t realize how fortunate I was until I began to talk to other graduate students about the way that their labs were run, and saw their jealousy and disbelief when I described how accessible and helpful my advisors were, and how they made their students’ development a priority. I will definitely miss Dr. Heineman’s patience, optimistic attitude, and commitment to his students now that I am leaving UC. He always made sure that I had plenty of opportunities to succeed, by doing things such as introducing me to new collaborators and giving me the opportunity to write review articles, and for this I am very grateful. I am equally grateful for Dr. Halsall’s resolve to make sure that I make the most of my talents and resist settling for mediocrity. He taught me, by example, to constantly strive for improvement and to be critical of myself. I would also like to thank Dr. Carl Seliskar for his generous assistance and advice, as he was like a third advisor to me for much of my graduate career. Finally I would like to thank my parents for teaching me the value of education, and supporting and believing in me for more than twenty years of school. I sincerely appreciate the efforts and sacrifices that they made so that I could have the education and experience to thrive at the graduate level. They, along with my brothers and sisters, cousins, aunts and uncles, friends, and especially my girlfriend Valerie, have been there for me when I was stressed, worn vii out, and needing encouragement. I would especially like to thank my grandmother for her kindness and generosity, particularly during my final year of graduate school. This past year, despite being my busiest and most stressful, has been a pleasure. viii Approval Form Title Page Abstract Copyright Notice Acknowledgements Table of Contents Chapter 1 Introduction to Electrochemical Immunoassay 1 1.1 Chemical Sensors/Biosensors 2 1.2 Immunoassay 4 1.2.1 Antibodies 5 1.2.2 Immunoassay Procedure 7 1.2.3 Labels 9 1.2.4 Bead-based Sandwich Assay 9 1.2.5 Electrochemical Detection 10 1.2.6 Label-Free Detection 14 1.3 Miniaturization 14 Chapter 2 Microfluidic Device for Bead-Based Immunoassay 19 2.1. Introduction to Microfluidics 20 2.1.1 Microfluidic Immunoassay 20 2.1.2 Microfluidic Materials 22 2.1.3 Microfluidics using Microbeads 25 2.1.4 Surface Modification 26 2.2. Fabrication of PDMS Microfluidic Device 28 2.2.1 Creation of the SU-8 master 28 2.2.2 PDMS casting 31 2.2.3 Bonding 32 2.2.4 Connection to Syringe Pumps 35 2.2.5 Proposed On-Chip Immunoassay 37 2.3. Compatibility of Standard Detection Methods with Microfluidic Device 38 2.3.1 Fluorescence Detection 38 2.3.2 Electrochemical Detection 39 2.4. Bead adsorption Study 41 2.4.1 Reagents 42 2.4.2 Equipment 42 ix 2.4.3 Bead Labeling Procedure 43 2.4.4 PDMS Sample Preparation 43 2.4.5 Surface Treatments 43 2.4.6 Data Analysis Procedure 44 2.4.7 Results and Discussion 44 2.5. Conclusions 47 Chapter 3 Electrochemical Immunoassays using Interdigitated Array 54 Microelectrodes 3.1 Introduction 55 3.1.1 Electrochemical Immunoassay Background 55 3.1.2 Miniaturized Electrochemical Immunoassay 56 3.1.3 Analytical advantages of Microelectrodes 57 3.1.4 IDA Electrodes 57 3.2 Characterization of Commercial IDAs 62 3.2.1 Materials 62 3.2.2 Characterization of IDAs 62 3.3 Immunoassay with Nanometer Scaled IDA 68 3.3.1 IDA Fabrication 68 3.3.2 Physical Characteristics of Nanoelectrode 69 3.4 E. coli Immunoassay 73 3.4.1 Materials 73 3.4.2 Buffer preparation 74 3.4.3 Preparation of Biotinylated Antibodies 74 3.4.4 Paramagnetic Bead-Based Immunoassay for E. coli O157:H7 75 3.4.5 Nano IDA Detection 76 3.5 MS2 Immunoassay 77 3.5.1 Materials 77 3.5.2 Preparation of the Immunoassay Buffers 78 3.5.3 Paramagnetic Bead-Based Immunoassay for MS2 78 3.5.4 Nano IDA detection
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