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  • BINDING KINETICS OF IMMOBILIZED BIOMOLECULES

    BY MICROCHANNEL FLOW DISPLACEMENT

    SONG YING (M.Sc.), Tianjin Medical University, China

    A THESIS SUBMITTED

    FOR THE DEGREE OF MASTER OF SCIENCE

    GRADUATE PROGRAMME IN BIOENGINEERING

    NATIONAL UNIVERSITY OF SINGAPORE

    2007

  • Acknowledgements First of all, I would like to express my sincere appreciation to my supervisor Dr.

    Partha Roy for introducing me to the magic world of microfluidics, and offering me the

    opportunity to expand my understanding step by step both theoretically and

    experimentally. Also thank for his time and energy devoted to guiding my research

    throughout this project, as well as for exposing me to a broad spectrum of knowledge that

    greatly benefits me.

    I wish to convey my deep thanks to my lab colleague Mr.Darren Tan for sharing

    his valuable experience and contributing inspirational ideas from time to time. Thank our

    lab officer Mr. Daniel Wong for his continuous support and assistance in procuring

    equipments and reagents, which ensured the project proceeded smoothly.

    I also would like to thank Nanobioengineering Lab and Micro Systems

    Technology Initiative (MSTI) Lab for kindly providing essential lab facilities. Without

    their help, my project would hardly have been accomplished.

    I thank my friends Alberto Corrias, Lim Cheetiong, Cheng Jinting, Li Jun, Sui Yi,

    and Wang Chen for their willingness of hand-on help on either computational modeling

    or experimental work for the development of microfluidic chip.

    Last but not the least, my special gratitude goes to my husband who has been with

    me through this memorable journey in a foreign land, sharing happiness in the good times

    and giving me endless encouragement in the hard times. Also, I owe a special debt of

    gratitude to my parents for their never-ending spiritual support and their belief in me.

    i

  • Table of Contents

    Acknowledgements ............................................................................................................ i

    Summary............................................................................................................................ v

    List of Tables ................................................................................................................... vii

    List of Figures................................................................................................................. viii

    List of symbols................................................................................................................... x

    Chapter 1 Introduction.................................................................................................... 1

    1.1 Biomolecular binding and kinetics .......................................................................... 1

    1.2 Significance of measuring binding kinetics............................................................. 3

    1.3 Fluorescence technique............................................................................................ 5

    1.4 Motivation................................................................................................................ 6

    1.5 Objectives ................................................................................................................ 8

    1.6 Organization............................................................................................................. 9

    Chapter 2 Literature Review ........................................................................................ 10

    2.1 Label - dependent methods for kinetic analysis..................................................... 10

    2.2 BIAcore – Label free biosensor technique............................................................. 13

    2.2.1 Introduction..................................................................................................... 13

    2.2.2 Kinetic experiment procedures ....................................................................... 15

    2.2.3 Applications .................................................................................................... 17

    2.2.4 Comparison between BIAcore and fluorescence-based microdevice............. 18

    2.3 Exploration of binding analysis with microfluidic device..................................... 19

    2.4 Protein immobilization methods on PDMS surface............................................... 20

    2.5 Application of avidin-biotin capture system in protein immobilization................ 23

    Chapter 3 Theoretical Modeling................................................................................... 25

    3.1 Modeling of flow-based displacement assay in microchannel .............................. 25

    3.2 Analytical solution to the model (constant u) ........................................................ 28

    3.3 Analytical and numerical solution comparison...................................................... 32

    ii

  • 3.4 Simulation of displacement curve for experimental design................................... 33

    Chapter 4 Experiment Materials and Methods .......................................................... 37

    4.1 Preparation of PDMS microchannel by soft lithography....................................... 37

    4.2 Protein Immobilization in microchannel ............................................................... 39

    4.2.1 Immobilization principle and key reagents..................................................... 39

    4.2.2 Verification of avidin coating in microchannel .............................................. 42

    4.2.3 Immobilization test using fibrinogen as example protein............................... 43

    4.3 Example biomolecular binding systems for measuring dissociation kinetics in

    microchannel................................................................................................................. 43

    4.3.1 Monoclonal antibody-antigen system: insulin & anti-insulin IgG ................ 43

    4.3.2 Enzyme-inhibitor system: aprotinin & trypsinogen....................................... 44

    4.3.3 Preparation of proteins for immobilization and displacement assay .............. 45

    4.3.3.1 Labeling of ligand protein with fluorescein............................................. 45

    4.3.3.2 Labeling of receptor protein with biotin .................................................. 46

    4.4 Flow-injection analysis (FIA) system setup .......................................................... 47

    Chapter 5 Experiment Results and Discussion ........................................................... 49

    5.1 Protein Immobilization in microchannel ............................................................... 49

    5.1.1 Verification of avidin coating in microchannel .............................................. 49

    5.1.2 Immobilization of fibrinogen as test protein................................................... 51

    5.1.2.1 Immobilization of fibrinogen................................................................... 51

    5.1.2.2 Quantification of immobilized fibrinogen ............................................... 53

    5.1.3 Immobilization of insulin - antibody system .................................................. 54

    5.2 Flow-based displacement assay in microchannel .................................................. 56

    5.2.1 Displacement assay with insulin-antibody system ......................................... 56

    Chapter 6 Conclusion and Recommendation.............................................................. 61

    6.1 Conclusions............................................................................................................ 61

    6.2 Directions for further study.................................................................................... 63

    Bibliography .................................................................................................................... 67

    iii

  • Appendix 1 Microfluidic device.................................................................................... 74

    Appendix 2 Simulation of the time trace of displaced labeled ligand for longer

    channel design ................................................................................................................. 77

    Appendix 3 Immobilization test using fibrinogen as example protein ..................... 79

    Appendix 4 Quantification of protein immobilized to PDMS microchannel wall ... 81

    Appendix 5 Binding system selection criteria ............................................................. 84

    Appendix 6 Flow-injection analysis (FIA) system setup ............................................ 85

    Appendix 7 Detection performance of the flow-injection analysis (FIA) system..... 88

    Appendix 8 Flow-based displacement assay in microchannel ................................... 92

    Appendix 9 Matlab codes for simulation of eluting time trace.................................. 94

    Appendix 10 HPLC Profiles.......................................................................................... 97

    Appendix 11 Solution phase b