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  • Strategies for Detecting DNA Hybridisation Using Surface Plasmon Fluorescence Spectroscopy

    Thomas Neumann

  • Strategies for Detecting DNA Hybridisation Using

    Surface Plasmon Fluorescence Spectroscopy

    Dissertation zur Erlangung des Grades

    Doktor der Naturwissenschaft

    am Fachbereich Chemie und Pharmazie

    der Johannes Gutenberg-Universitt in Mainz

    vorgelegt von Thomas Neumann

    aus Witten an der Ruhr

    Mainz, Juni 2001

  • CONTENTS

    1 INTRODUCTION ..............................................................................................1

    2 THEORY...........................................................................................................7 2.1 Fluorescence.............................................................................................................. 7

    2.1.1 Principle of Fluorescence .................................................................................................7

    2.1.2 Quenching of Fluorescence .......................................................................................... 10

    2.1.3 Resonant Energy Transfer............................................................................................. 12

    2.2 Langmuir Adsorption.............................................................................................. 15 2.3 Surface Plasmons .................................................................................................... 18

    2.3.1 Electromagnetic Fields................................................................................................... 18

    2.3.2 Reflection and Transmission of Polarised Light ........................................................ 19

    2.3.3 Surface Plasmons in a Two Layer System................................................................... 23

    2.3.4 Excitation of Surface Plasmons.................................................................................... 25

    2.3.5 Sensitivity of Surface Plasmons to Optical Changes ................................................. 29

    2.3.6 Transfer Matrix Method ................................................................................................ 30

    2.3.7 Field Distributions.......................................................................................................... 32

    3 EXPERIMENTAL METHODS.........................................................................35 3.1 Optical Methods...................................................................................................... 35

    3.1.1 Surface Plasmon Spectroscopy (SPS) .......................................................................... 35

    3.1.2 Surface Plasmon Fluorescence Spectroscopy (SPFS)................................................ 37

    3.1.3 Surface Plasmon Microscopy (SPM)............................................................................ 43

    3.2 Surface Modification Techniques ........................................................................... 47 3.2.1 Cleaning of Glass Samples ............................................................................................ 47

    3.2.2 Thermal Evaporation of Metal and Silicon Oxide..................................................... 48

    3.2.3 Self Assembled Monolayers (SAM) on Gold ............................................................. 48

    3.2.4 Spin Coating .................................................................................................................... 49

    3.2.5 Sol-gel Procedure for the Deposition of Silica Film.................................................. 50

    3.2.6 Preparation of Silane Layers on Silicon Oxide Films................................................ 50

    3.2.7 Labelling of Surfaces with Fluorescence Labels......................................................... 52

    3.3 UV Spectroscopy of DNA ....................................................................................... 54

  • ii

    4 FLUORESCENCE ENHANCEMENT AND EMISSION NEAR METALIC SURFACES.................................................................................................... 57

    4.1 Surface Plasmon Field Enhanced Fluorescence..................................................... 58 4.1.1 Theoretical Considerations ............................................................................................58

    4.1.2 Experimental Verification of the Surface Plasmon Field Enhancement ................58

    4.2 Energy Transitions for Fluorescence near Metal Surfaces..................................... 60 4.2.1 Theoretical Consideration of the Major Decay Channels .........................................61

    4.2.2 Experimental Set-up........................................................................................................62

    4.2.3 Experimental Verification of the Decay Channels.....................................................63

    4.3 Distance Dependence of Decay Channels.............................................................. 67 4.3.1 Theoretical Consideration of the Quenching Profile.................................................67

    4.3.2 Experimental Set-up and Sample Preparation ............................................................72

    4.3.3 Experimental Conformation of the Simulated Quenching Profile ..........................74

    4.4 Conclusion............................................................................................................... 76

    5 FLUORECENCE ON BLAZED GRATINGS .................................................. 77 5.1 Coupled Resonances on Blazed Gratings............................................................... 77

    5.1.1 Grating Coupling.............................................................................................................77

    5.1.2 Coupled Resonances and Localised Electrical Fields.................................................79

    5.2 Excitation and Emission of Fluorescence .............................................................. 83 5.2.1 Simulation of the Excitation and Emission Process ..................................................84

    5.3 Results and Discussion ........................................................................................... 85 5.3.1 Experimental ....................................................................................................................86

    5.3.2 Reflectivity Measurements .............................................................................................87

    5.3.3 Fluorescence Measurements in the Plane of Incidence.............................................89

    5.3.4 Fluorescence Emission Outside the Plane of Incidence ...........................................92

    5.3.5 Bleaching by the Field of Coupled Resonances..........................................................93

    5.4 Conclusion............................................................................................................... 97

  • 6 DNA HYBRIDISIERUNG ON THE SENSOR SURFACE ...............................99 6.1 Nucleic Acid Structure ............................................................................................ 99 6.2 DNA Sequencing and Detection ...........................................................................103 6.3 DNA Immobilisation Architecture ........................................................................105

    6.3.1 Biotin-Streptavidin........................................................................................................ 105

    6.3.2 DNA Probe and Targets ............................................................................................. 108

    6.3.3 Experimental Results.................................................................................................... 115

    6.4 DNA Hybridisation and Mismatch Discrimination..............................................119 6.4.1 Hybridisation with Hairpin Probes and Targets ...................................................... 120

    6.4.2 Hybridisation with Hairpin Probes and Targets ...................................................... 122

    6.4.3 Discussion...................................................................................................................... 125

    6.5 Conclusion..............................................................................................................129

    7 A NOVEL DETECTION SCHEME FOR UNLABELLED TARGETS BASED ON STRUCTURAL CHANGES IN THE PROBE..........................................131

    7.1 Introduction ...........................................................................................................131 7.2 Probe Sequences ....................................................................................................133

    7.2.1 Probe Design................................................................................................................. 133

    7.2.2 Photobleaching.............................................................................................................. 135

    7.2.3 Secondary Structure of Probes and Interaction with the Matrix ........................... 138

    7.3 Detection of Unlabelled Targets............................................................................140 7.3.1 Hybridisation on the Surface ...................................................................................... 140

    7.3.2 Hybridisation in Solution............................................................................................. 140

    7.3.3 Mismatch Discrimination

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