Measurement of Force Dependence of Receptor-Ligand Bonding Using a Novel Forced Unbinding System

3 Acknowledgments. 5 List of Tables . 11 List of Figures . 12 Chapter 1: Introduction . 16 1.1 Cell Adhesion Molecules . 16 1.1.1 Cell adhesion . 16 1.1.2 Types of adhesion molecule. 16 1.1.3 Antibody and antigen . 18 1.2 Adhesion Bond Models . 20 1.2.1 Receptor-ligand bonding . 20 1.2.2 Bell model . 22 1.2.3 Catch-slip bond model . 24 1.3 Methods for Applying Force to Bonds . 25 1.3.1 Atomic force microscopy (AFM) . 25 1.3.2 Optical tweezers (OT) . 26 7 1.3.3 Biomembrane force probe . 27 1.3.4 Hydrodynamic flow . 28 1.3.5 Microcantilever . 29 1.4 Elasticity Theory of Cantilever Stiffness . 31 1.5 Thermal Fluctuation Method . 32 Chapter 2: Spring Constant Measurement Results for Fiber Microcantilevers . 36 2.1 Materials and Methods . 36 2.1.1 Fabrication of glass micropipettes . 36 2.1.2 Fabrication of microcantilever . 38 2.1.3 Design of viewing chambers . 40 2.1.3.1 Flow viewing chamber . 40 2.1.3.2 Thin viewing chamber . 42 2.1.4 Optical microscopy . 44 2.2 Calculation of Spring Constants from Elasticity Theory . 45 2.2.1 SEM measurements of glass fiber diameter. 47 2.2.2 Light microscopy measurements of glass fibers . 48 2.3 Thermal Fluctuation Measurement of Spring Constant . 51 2.3.1 Image analysis . 51 2.4 Spring Constant Measurement Results . 54 8 2.4.1 Experimental methods . 54 2.4.2 Monte Carlo simulation for moving average filter . 58 2.4.3 Comparison of theoretical and experimental spring constant estimates . 66 2.5 Results and Discussion . 72 Chapter 3: Hydrodynamic Force on Microcantilever Tip . 77 3.1 Experimental Method . 77 3.1.1 Viewing chamber . 77 3.1.2 Experimental procedure . 79 3.2 Fiber Tip Displacement with Applied Pressure . 80 3.3 Effect on Fiber Tip Displacement Due To Different Relative Positions . 84 3.3.1 Variation with relative vertical positions . 84 3.3.2 Varying the fiber tip-micropipette initial relative position . 88 3.4 Effects of Micropipette Size on Flow Force . 93 3.5 Discussion . 95 Chapter 4: Adhesion Experiments . 97 4.1 Materials and Methods . 97 4.1.1 Human IgG and protein A. 97 4.1.2 Viewing chamber . 98 4.1.3 Mounting viewing chamber on stage . 99 9 4.1.4 Bonding experiment setup . 101 4.1.5 Image capture . 104 4.2 Nonspecific Adhesion Experiments . 105 4.3 Specific Adhesion Experiments . 109 4.3.1 Bonding test using fluorescence microscopy . 109 4.3.2 Bonding experiments of human IgG and protein A . 112 4.3.3 Catch-slip transition fitting . 116 4.3.4 Catch-slip transition fitting method . 118 4.3.5 Bell model fitting . 121 4.4 Results and Discussion . 124 Chapter 5: Conclusion and Suggestions for Future Work . 127 5.1 Concluding Remarks . 127 5.2 Suggestions for Future Studies . 128 References . 130 Appendix 1 LabVIEW Code for Program “Vertical Average X Pixels Value.vi” . 148 Appendix 2 LabVIEW Code for Program “TwominD.vi” . 149 Appendix 3 LabVIEW Code for Program “Peak.vi” . 150 Appendix 4 The LabVIEW Code for “Twomax.vi” . 151 Appendix 5 The LabVIEW Code for “Twomin.vi” . 152 10 Appendix 6 The MATLAB Code for “Movingaveragefilter.m” . 153 Appendix 7 The MATLAB Code for “Read_FFT. m” . 155 Appendix 8 The MATLAB Code for “Method_Verify. m” . 156 Appendix 9 The LabVIEW Code for “AVI Decomposer.vi” . 158 Appendix 10 The MATLAB Code for “Read_Process. m” . 159 Appendix 11 The LabVIEW Code for “Pressure Logger Ultimate.vi” . 161 Appendix 12 LabVIEW Code for Program “Fast Pressure Logger with Valve.vi” . 162 Appendix 13 The MATLAB Code for “Curvefitting_main. m” . 163 Appendix 14 The MATLAB Code for “Curve_fit. m” . 164 Appendix 15 The MATLAB Code for “Fit_equation. m” . 165 Appendix 16 The MATLAB Code for “Probability_catchslip. m” . 166 Appendix 17 The MATLAB Code for “Curvefitting_main_ks. m” . 167 Appendix 18 The MATLAB Code for “Curve_fit_ks. m” . 168 Appendix 19 The MATLAB Code for “Fit_equation_ks. m” . 169 Appendix 20 The MATLAB Code for “Probability_Bell. m” . 170 11 LIST OF TABLES Page Table 2.1 Magnitude of Fourier transformed fiber position vs. discrete frequencies, P is the number of data points. (P=2425 for the 4 mm length fiber) . 62 Table 2.2 kspring with different number of points in moving average filter for a simulated 4 mm fiber . 65 Table 2.3 kspring with different number of points in mov