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Cholesterol Oxidase Modified Microelectrodes for Detection of Cholesterol in The CHOLESTEROL OXIDASE MODIFIED MICROELECTRODES FOR DETECTION OF CHOLESTEROL IN THE PLASMA MEMBRANE OF SINGLE CELLS by ANANDO DEVADOSS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. James David Burgess Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedicated to my parents and brother TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................. i LIST OF FIGURES .......................................................................................................... v LIST OF SCHEMES .....................................................................................................xiii LIST OF ABBREVIATIONS ....................................................................................... xiv ACKNOWLEDGEMENTS ........................................................................................... xv ABSTRACT ..................................................................................................................xvi INTRODUCTION............................................................................................................. 1 CHAPTER 1. DETECTION OF CHOLESTEROL THROUGH ELECTRON TRASFER TO CHOLESTEROL OXIDASE IN ELECTRODE- SUPPORTED LIPID BILAYER MEMBRANE.................................... 4 1.1 INTRODUCTION. ........................................................................................................ 4 1.2 EXPERIMENTAL.......................................................................................................... 8 1.3 RESULTS AND DISCUSSION.................................................................................... 11 1.4 CONCLUSIONS.......................................................................................................... 18 APPENDIX 1-1................................................................................................................ 20 APPENDIX 1-2................................................................................................................ 24 APPENDIX 1-3................................................................................................................ 28 APPENDIX 1-4................................................................................................................ 30 APPENDIX 1-5................................................................................................................ 32 1.5 REFERENCES. ........................................................................................................... 33 i CHAPTER 2. STEADY-STATE OXIDATION OF CHOLESTEROL CATALYZED BY CHOLESTEROL OXIDASE IN LIPID BILAYER MEMBRANES ON PLATINUM ELECTRODES.............................. 40 2.1 INTRODUCTION. ...................................................................................................... 40 2.2 EXPERIMENTAL........................................................................................................ 44 2.3 RESULTS AND DISCUSSION.................................................................................... 48 2.4. CONCLUSIONS......................................................................................................... 60 2.5 ACKNOWLEDGEMENTS. ......................................................................................... 61 2.6 REFERENCES. ........................................................................................................... 62 CHAPTER 3.ENZYME MODIFICATION OF PLATINUM MICROELECTRODES FOR DETECTION OF CHOLESTEROL IN VESICLE LIPID BILAYER MEMBRANES ...................................... 66 3.1 INTRODUCTION. ...................................................................................................... 66 3.2 EXPERIMENTAL SECTION. ..................................................................................... 68 3.3 RESULTS AND DISCUSSION.................................................................................... 71 3.4 CONCLUSIONS.......................................................................................................... 86 3.5 ACKNOWLEDGEMENTS. ......................................................................................... 86 3.6 REFERENCES. ........................................................................................................... 87 CHAPTER 4. 89STEADY STATE DETECTION OF CHOLESTEROL CONTAINED IN THE PLASMA MEMBRANE OF SINGLE CELL USING LIPID BILAYER MODIFIED MICROELECTRODES INCORPORATING CHOLESTEROL OXIDASE............................. 89 4.1 INTRODUCTION. ...................................................................................................... 89 ii 4.2 EXPERIMENTAL........................................................................................................ 90 4.3 RESULTS AND DISCUSSION.................................................................................... 91 4.4 CONCLUSIONS.......................................................................................................... 96 4.5 ACKNOWLEDGMENT............................................................................................... 96 APPENDIX 4-1................................................................................................................ 97 4.6 REFERENCES. ......................................................................................................... 102 CHAPTER 5. 103COVALENT MODIFICATION OF CHOLESTEROL OXIDASE TO PLATINUM MICROELECTRODES FOR DETECTION OF CHOLESTEROL IN THE PLASMA MEMBRANE OF SINGLE CELLS ................................................................................................... 103 5.1 INTRODUCTION. .................................................................................................... 103 5.2 EXPERIMENTAL...................................................................................................... 104 5.3 RESULTS AND DISCUSSION.................................................................................. 105 5.4 CONCLUSION.......................................................................................................... 110 5.5 ACKNOWLEDGEMENTS. ....................................................................................... 111 5.6 REFERENCES. ......................................................................................................... 112 CHAPTER 6. SCANNING FORCE MICROSCOPY IMAGES OF CHOLESTEROL OXIDASE IMMOBILIZED IN SUPPORTED LIPID BILAYER MEMBRANE......................................................... 113 6.1 INTRODUCTION. .................................................................................................... 113 6.2 EXPERIMENTAL SECTION. ................................................................................... 115 6.3 RESULTS AND DISCUSSION.................................................................................. 116 6.4 CONCLUSIONS........................................................................................................ 119 iii 6.5 ACKNOWLEDGEMENTS. ....................................................................................... 119 6.6 REFERENCES. ......................................................................................................... 120 BIBLIOGRAPHY......................................................................................................... 122 iv LIST OF FIGURES Figure1-1. Idealized structure of the electrode-supported lipid bilayer membrane containing cholesterol oxidase.................................................................... 6 Figure 1-2. Dual chambered electrochemical dialysis cell. The dimensions of the cell are 1 cm x 5 cm x 5 cm. The electrode is mechanically clamped in the sample chamber and the dialysis membrane is sandwiched between the flow chamber and the sample chamber............................................................... 9 Figure 1-3. A) Atomic force microscopy images of an indium tin oxide surface (contact mode). B) A zoomed portion of the scan from A showing a cross-section. ................................................................................................................... 13 Figure 1-4. Cyclic voltammetry of 5mM potassium ferricyanide at A) bare ITO, B) ITO modified with the thiolipid, and C) ITO after deposition of the outer lipid leaflet......................................................................................................... 15 Figure 1-5. Amperometric responses for exposure of cholesterol to a cholesterol oxidase modified electrode and to a lipid bilayer modified electrode containing no cholesterol oxidase (control). The electrode area is 0.2 cm2. ................................................................................................................... 17 Figure A1-1. Linear sweep voltammograms
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