BODY-FLUID DIAGNOSTICS IN MICROLITER SAMPLES by GAUTAM N. SHETTY Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Miklós Gratzl Co-advisor: Dr. Koji Tohda Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Gautam N. Shetty . candidate for the Ph. D. degree *. (signed) Miklós Gratzl (chair of the committee) Koji Tohda Barry Miller Clive. R. Hamlin Mark D. Pagel (date) 01/26/06 *We also certify that written approval has been obtained for any proprietary material contained within. I grant to Case Western Reserve University the right to use this work, irrespective of any copyright, for the University’s own purposes without cost to the University or to its students, agents and employees. I further agree that the University may reproduce and provide single copies of the work, in any format other than in or from microforms, to the public for the cost of reproduction. Gautam N. Shetty . (sign) To my hardworking parents iv TABLE OF CONTENTS List of figures…………………………………………………………………………….vii List of tables………………………………….……………………………………….......ix Acknowledgements……………………………………………………………………......x List of Abbreviations……………………………………………………………………..xi Abstract…………………………………………………………………………………..xii Introduction: Significance, hypotheses and specific aims………………………………...1 Part I Optimization of RSS system parameters Chapter 1 Hydrodynamic Electrochemistry in 20 μL drops in the Rotating Sample System……………………………………………………………………4 Part II Investigation of RSS performance in biological samples Chapter 2 Rotating Sample System: Hydrodynamic Electrochemistry in Biological Matrices………………………………………………………………28 Chapter 3 Rotating Sample System: A Simple Tool for Rheological Examination of the Air-Solution Interface……………………………………………………..50 Part III Investigation of electrode ‘fouling’ in biological samples Chapter 4 Protein Adsorption on the electrode of the Rotating Sample System…66 Chapter 5 Electrochemical Desorption of Proteins……………………………...82 Part IV Trace Pb analyses Chapter 6 Rotating Sample System: Trace Pb(II) Analyses in Serum and Blood Samples…………………………………………………………………………..96 v Part V Chapter 7 Summary and Future Work………………………………………….114 Appendix A……………………………………………………………………………..122 Appendix B……………………………………………………………………………..129 Appendix C……………………………………………………………………………..137 Bibliography……………………………………………………………………………147 vi List of Figures Figure 1-1 Schematic diagram of home-made Rotating Sample System Figure 1-2 Schematic diagram of microfabricated Rotating Sample System Figure 1-3 Cyclic voltammograms for electrode position close to the axis of rotation at different rotation rates of the sample Figure 1-4 Images of dye injection to visualize bulk flow patterns Figure 1-5 Trace Pb analyses in aqueous (non-biological) samples using the RSS Figure 2-1 Schematic diagram of Rotating Sample System (Top and Front view) Figure 2-2 Cyclic voltammetry in rotated and stationary samples containing different dilutions of fetal bovine serum Figure 2-3 Lipid-protein interplay illustrated by cyclic voltammetry Figure 2-4 Affect of electrode protein adsorption on mass transport properties Figure 3-1 Cyclic voltammetry in rotated sample of different BSA concentration Figure 3-2 Cyclic voltammograms depicting lipid-protein interfacial interactions Figure 3-3 Calculating CMC from plateau currents in RSS Figure 4-1 Using Hydrogen UPD to get electrode active surface area Figure 4-2 Adsorption kinetics Figure 4-3 Adsorption kinetics with and without Nafion coating Figure 4-4 Comparison in voltammograms with and without Polyurethane coating Figure 5-1 Adsorption and Desorption kinetics Figure 5-2 Desorption in fetal bovine serum matrix Figure 6-1 Anodic stripping voltammetry of 2.5 ppm Pb for optimization of CAP vii membrane Figure 6-2 Repeatability of detection of Pb in 10 μL serum samples Figure 6-3 Convection properties in hemoglobin samples Figure 6-4 Pb analysis in human blood Figure 6-5 Trace Pb detection in human blood viii List of Tables Table 1-1 Diffusion layer thickness as a function of the position and the inner diameter of nozzle for a single air jet Table 1-2 Plateau and edge currents at various air flow rates for different positions of the Pt mini-disc electrode, using two anti-parallel air jets for sample rotation Table 4-1 Comparison of different membrane for coating electrode Table A-1 Comparison of electrode area obtained using different techniques Table C-1 Summary of Standards and Regulations for Pb ix Acknowledgements I would like to thank my advisor Prof. Gratzl; I am fortunate to have had the opportunity to learn under him. I am indebted for the education which I am certain will hold me in good stead for the future. I would also like to thank my co-advisor Dr. Tohda, who has always been a great resource (all things except whitewater rafting!). I am grateful to Dr. Barry Miller, Dr. Clive Hamlin and Dr. Marty Pagel for serving on my committee; I would also like to thank for their constant guidance and encouragement. I would like to thank my colleagues at the Laboratory for Biomedical Sensing; my research experience would be incomplete without you all. I would like to sincerely thank for first employers at the Center for Health Promotion Research, Department of Epidemiology and Biostatistics for their support in my initial period here; I would never have made it this far if not for my job there. I would like to thank my friends at Case, Cleveland chapter of Asha for Education, the Cleveland Cricket Club and numerous others in the Cleveland community for enriching my Cleveland experience. I would like to thank my family and friends for their constant support. Coming from a country where one in three children do not have access to primary schooling, I would like to thank all my teachers; I am indebted to them for the gift of education. x List of Abbreviations BSA : Bovine serum albumin CAP : Cellulose acetate hydrogen phthalate CCD : Charge coupled device CV : Cyclic voltammetry CMC : Critical micelle concentration HSA : Human serum albumin ppm : parts-per-million ppb : parts-per-billion ppt : parts-per trillion RDE: Rotating disc electrode RSS : Rotating sample system WE : Working electrode UPD : Under-potential deposition xi Body-fluid Diagnostics in Microliter Samples Abstract by Gautam N. Shetty The Rotating Sample System (RSS) has been conceived in our laboratory for diagnostics of microliter samples. The design of the RSS enables effective convection generation in microliter sized samples. Convection aids in mass-transport and is essential in improving sensitivity in applications such as trace metal diagnostics. In diagnostics applications such as titration and enzyme activity measurements, convection helps in homogenization of the sample. Capability to investigate microliter sized samples is essential to extend diagnostic capability for neonates and small children without having to draw body-fluids (e.g blood) in the order of milliliters for analyses. Also, smaller size of the system would make it portable and attractive for use in point-of-care applications eliminating need for storage and transportation of samples. Small samples also ensure that storage and disposal issues are minimal. Natural physical properties such as surface tension, which are usually ignored for larger sample volumes, become prominent with microliter sample sizes; these can be engineered to develop simple yet robust tools for body fluid diagnostics. Optimization of system parameters for optimal system performance has been undertaken as part of this work. Study of the hydrodynamic performance of the RSS in xii biological matrix was conducted and revealed interplay between proteins and lipids at the liquid-air interface. The RSS is unique in the sense that it imparts convection to the sample via its surface. Hence, utility of the RSS as a tool to probe the interfacial properties of samples containing surface-active molecules has been investigated. The RSS by providing information about both bulk and surface properties of a sample fosters better diagnostics of biological samples. Challenges are posed to electrochemical analysis by non-specific adsorption of proteins onto electrode surfaces; hence methods to protect the electrode by coating with a suitable spacer polymer membrane have been developed. For the first time, a technique to electrochemically effect desorption of proteins is demonstrated. Using the RSS’ favorable convective properties, trace Lead (Pb) analyses in model aqueous samples and detection capability in serum and blood matrices has been demonstrated. A detection limit of 260 ppt for Pb was achieved in aqueous samples. xiii INTRODUCTION: Significance, hypothesis and specific aims Conventional analytical systems and techniques are limited in their ability to address small samples. These systems designed for larger samples cannot be scaled down in size to work with microliter sized samples - a pre-requisite for biomedical applications. Natural properties of liquids such as surface tension, which tend to be ignored for larger volumes, become significant in smaller volumes. This has been engineered to enable us to build a platform for analysis of microliter sized samples. The Rotating Sample System (RSS) was thus conceived in our laboratory. The RSS consists of a microliter sized drop placed atop a hydrophilic substrate such as glass and kept in position by a hydrophobic ring.