Point-Of-Care Body Fluid Diagnostics in Microliter
Total Page：16
File Type：pdf, Size：1020Kb
POINT-OF-CARE BODY FLUID DIAGNOSTICS IN MICROLITER SAMPLES by LINUS TZU-HSIANG KAO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Adviser: Dr. Miklós Gratzl Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOLA OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Linus Tzu-Hsiang Kao candidate for the Ph.D. degree *. (signed) Miklos Gratzl (chair of the committee) Clive Hamlin Uziel Landau Andrew Rollins Christian Zorman (date) March 19, 2009 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication To my parents and my wife, Yu-Ting Lin, for their love and support Table of Contents Table of Contents 1 List of Tables 7 List of Figures 8 Abstract 10 Chapter 1. Introduction of Point-of-Care Testing, Clinical Enzymology, and Electrochemical Enzyme Assays 12 1.1. Introduction 13 1.2. Point-of-Care Testing 14 1.2.1. Current state-of-the-art 17 1.3. Fundamentals of Enzymology 19 1.3.1. Significance of clinical enzyme analysis 21 1.3.2. Enzyme Kinetics 22 1.3.3. Units for enzyme activity 24 1.3.4. Factors affect enzyme kinetics 25 1.4. Enzyme Assays 26 1.4.1. Continuous assay 28 1.4.2. Discontinuous assay 28 1 1.4.3. Coupled assay 30 1.5. Principles of Developing Electrochemical pH-stat for Point-of-Care Testing 31 1.5.1. Conventional pH-stat versus electrochemical pH-stat 32 1.5.2. Electrochemical pH-stat on Rotating Sample System (RSS) 33 1.5.3. Limitations 35 1.6. Conclusion 36 1.7. References 39 Chapter 2. Ragentless pH-stat for Microliter Fluid Specimens 45 2.1. Abstract 46 2.2. Introduction 47 2.3. Experimental 52 2.3.1. Apparatus 52 2.3.2. Materials 54 2.3.3. Procedures 55 2.4. Results and Discussion 57 2.4.1. System characteristics 57 2.4.2. Current efficiency 59 2.4.3. Determination of AChE activity 60 2 2.5. Conclusions 63 2.6. Acknowledgements 64 2.7. References 65 Chapter 3. Determination of Critical Micelle Concentration with the Rotating Sample System 70 3.1. Abstract 71 3.2. Introduction 72 3.3. Experimental 76 3.3.1. Materials 76 3.3.2. Apparatus 76 3.3.3. Procedures 77 3.4. Results and Discussion 78 3.5. Conclusions 86 3.6. Acknowledgement 86 3.7. References 87 Chapter 4. Serum Cholinesterase Assay Using a Reagent-free Micro pH-stat 90 4.1. Abstract 91 3 4.2. Introduction 92 4.3. Experimental 96 4.3.1. Materials 96 4.3.2. Apparatus 97 4.3.3. Procedures 97 4.4. Results and Discussion 99 4.4.1. Current efficiency of the electrochemical micro pH-stat in fetal bovine serum 99 4.4.2. Effect of sample dilution on current efficiency 100 4.4.3. Determination of ChE enzyme activities in fetal bovine serum with electrochemical pH-stating 102 4.4.4. Determination of ChE activity in human serum with electrochemical pH-stating 105 4.5. Conclusions 105 4.6. Acknowledgements 106 4.7. References 107 Chapter 5. Conclusions and Future Work 110 5.1. Summary 111 4 5.1.1. Proof of concept: electrochemical micro pH-stat 112 5.1.2. Application of electrochemical micro pH-stat in biological samples 113 5.1.3. RSS for determination of Critical Micelle Concentration 114 5.2. Future Work 115 5.2.1. Microfabricated pH-stat chip 115 5.2.2. Measurement of substrate concentration and buffer capacity 117 5.2.3. NADH-based enzyme assay 118 5.3. References 121 Appendix I. Characteristics of the Reagent-Free Micro pH-stat System 125 A-I.1. Schematics of the Reagent-Free Micro pH-stat 125 A-I.2. Current generating circuit and voltage isolation 126 A-I.3. PID control algorithm 128 A-I.4. Chamber for pH-stat on the RSS 130 A-I.5. Elimination of CO2 interference 132 A-I.6. Minimizing the effect of protein adsorption at the liquid-air interface 133 A-I.7. Micro pH electrode 135 A-I.8. Implementation of the advanced PID control 137 A-I.9. Reference 140 5 Appendix II. Possible errors during pH-stating and the trouble-shooting procedures 141 A- II.1. Background pH drift 141 A- II.2. Erratic pH-stating 142 A- II.3. Reference 143 Bibliography 144 6 List of Tables TABLE 1.1. Enzymes of clinical importance that can be analyzed by pH-stating 38 TABLE 2.1. Current efficiency of the micro pH-stat in KNO3 buffer 60 7 List of Figures FIGURE 1.1. Comparison of the clinical testing process in an outpatient situation between the central laboratory and POCT methods 16 FIGURE 1.2. Urea cycle: the primary mechanism for disposal of waste nitrogen resulted from protein turnover and dietary intake 20 FIGURE 1.3. Michaelis-Menten Kinetics 24 FIGURE 1.4. Initial rate measurement of enzyme kinetics 27 FIGURE 1.5. Schematic diagram of the Rotating Sample System 35 FIGURE 2.1. Schematic diagram of the reagentless micro pH-stat based on the Rotating Sample System platform (RSS) 51 FIGURE 2.2. Substrate dependence of AChE activity determined with the micro pH-stat and with spectrophotometry as control 62 FIGURE 2.3. pH dependence of AChE activity 63 FIGURE 3.1. Schematic view of the Rotating Sample System used for CMC detection 74 FIGURE 3.2. Cyclic voltammograms of 25 mM K4[FeCN6] at the CMC of Triton X-100 (140 ppm) 79 FIGURE 3.3. Normalized cyclic voltammetry currents of 25 mM K4[FeCN6] 84 FIGURE 4.1. Schematic of the electrochemical micro pH-stat built on the Rotating Sample System platform 95 8 FIGURE 4.2. Effect of enzyme dilution on the enzyme activity as determined with the micro pH-stat 101 FIGURE 4.3. Comparison of ChE activities in FBS measured with the electrochemical micro pH-stat and with the standard optical assay using Ellman’s reagent 104 FIGURE 5.1. Prototype of microfluidic pH-stat chip 116 FIGURE 5.2. Schematics of lactate dehydrogenase assay using PB modified electrode 120 FIGURE A.1. Schematics of the pH-stat system 125 FIGURE A.2. Voltage controlled variable current source 126 FIGURE A.3. RS-232 optical-isolator 127 FIGURE A.4. The first version of LabVIEW controller 129 FIGURE A.5. Performance of PID controller 130 FIGURE A.6. Acrylic chamber with DI water channels surrounding the RSS cell and air jets housing 131 FIGURE A.7. Sample pH drift due to CO2 influx 133 FIGURE A.8. Protein adsorption in RSS sample 134 FIGURE A.9. Micro pH electrode 136 FIGURE A.10. Typical operation of the pH-stating assay for enzyme kinetics 139 9 Point-of-Care Body Fluid Diagnostics in Microliter Samples Abstract by LINUS TZU-HSIANG KAO Abstract Point-of-care testing (POCT) generally refers to the implementation of laboratory tests near the patient with the goal to minimize turnaround time, reduce medical cost, and improve medical outcome. The aim of this research is to develop, validate, and optimize a preliminary POCT system for body fluid diagnostics using our existing analytical platform, Rotating Sample System (RSS). The RSS is a convective platform for different optical and electrochemical analyses in microliter-sized samples. In this work we have developed a reagent-free electrochemical micro pH-stat for in vitro enzyme assays where a titrant of acid or base is produced by water electrolysis on the RSS platform. As water electrolysis induces no volume change and the current that generates the reagent can be precisely measured even at low levels, very small samples in 10 the 1-20 μL volume range become accessible for pH-stating: a reduction of more than an order of magnitude in specimen size relative to the most conventional methods. More importantly, analysis using untreated biological sample is feasible as turbidity would not influence pH-stat measurement. The RSS micro pH-stat effectively operates as a galvanostat with an output range of ±0.1-100 μA and can withstand sample impedance up to 100 kΩ. The cathodic current efficiency is virtually 100% in both buffer and serum samples. Results of cholinesterase activities in both buffer and serum using the proposed reagent-free pH-stat have been validated with standard optical techniques (r2 ≥ 0.97). This novel technique hence has great potential to become a miniaturized analyzer for point-of-care diagnostics. 11 Chapter 1 Introduction of Point-of-Care Testing, Clinical Enzymology, and Electrochemical Enzyme Assays Linus Tzu-Hsiang Kao Department of Biomedical Engineering Case Western Reserve University, Cleveland, Ohio 44106 12 1.1. Introduction Rudimentary point-of-care testing (POCT) was described as early as 1883 in London (Kiechle 2002). POCT generally refers to the implementation of laboratory tests near the patient’s bedside or at the “point of care" with the goal to minimize turnaround time, reduce medical cost, and improve patient management. Due to its potential to reshape the delivery of medical service, POCT is an emerging modality growing rapidly in the healthcare field. Our long term goal is to develop a portable point-of-care device for an array of body fluid analyses to aid biomedical research and expedite clinical diagnostics. The aim of this thesis is to develop, validate, and optimize a preliminary POCT platform using reagent-free micro pH-stating techniques for universal enzymatic analysis in body fluids. Body fluids such as blood continuously circulate throughout the body, delivering nutrients to cells and transporting products from and toward tissues.