Entwicklung LSPR-basierter optischer Biosensoren zum markierungsfreien Nachweis biomolekularer Wechselwirkungen in hochintegrierten Peptidbibliotheken [Elektronische Ressource] / vorgelegt von Fanny Caroline Liu

De
INAUGURAL-DISSERTATION Zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg vorgelegt von Dipl. –Chem. Fanny Caroline Liu aus Yogyakarta/Indonesien Tag der mündlichen Prüfung: 11. Februar 2011 Entwicklung LSPR-basierter optischer Biosensoren zum markierungsfreien Nachweis biomolekularer Wechselwirkungen in hochintegrierten Peptidbibliotheken Gutachter: Prof. Dr. rer. nat. Reiner Dahint Prof. Dr. rer. nat. Joachim Spatz Development of LSPR-based Optical Biosensors for the Label-Free Detection of Biomolecular Interactions in High-Density Peptide Arrays Referees: Prof. Dr. rer. nat. Reiner Dahint Prof. Dr. rer. nat. Joachim Spatz CONTENTS 1 CONTENTS CONTENTS .............................................................................................................................. 1 ABSTRACT .............................................................................................................................. 5 LIST OF PUBLICATIONS ..................................................................................................... 8 ABBREVIATIONS ..................................................................................................................
Publié le : samedi 1 janvier 2011
Lecture(s) : 119
Source : D-NB.INFO/1011349094/34
Nombre de pages : 154
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INAUGURAL-DISSERTATION


Zur Erlangung der Doktorwürde
der Naturwissenschaftlich-Mathematischen Gesamtfakultät
der
Ruprecht-Karls-Universität
Heidelberg








vorgelegt von
Dipl. –Chem. Fanny Caroline Liu
aus Yogyakarta/Indonesien


Tag der mündlichen Prüfung: 11. Februar 2011




























Entwicklung LSPR-basierter optischer Biosensoren zum
markierungsfreien Nachweis biomolekularer Wechselwirkungen in
hochintegrierten Peptidbibliotheken











Gutachter:
Prof. Dr. rer. nat. Reiner Dahint
Prof. Dr. rer. nat. Joachim Spatz































Development of LSPR-based Optical Biosensors
for the Label-Free Detection of Biomolecular Interactions
in High-Density Peptide Arrays










Referees:
Prof. Dr. rer. nat. Reiner Dahint
Prof. Dr. rer. nat. Joachim Spatz




CONTENTS 1
CONTENTS
CONTENTS .............................................................................................................................. 1
ABSTRACT .............................................................................................................................. 5
LIST OF PUBLICATIONS ..................................................................................................... 8
ABBREVIATIONS .................................................................................................................. 9
1. INTRODUCTION .............................................................................................................. 11
2. THEORY ............................................................................................................................. 14
2.1. In situ peptide synthesis ............................................................................................................................... 14
2.1.1 Photolithographic synthesis ..................................................................................................................... 15
2.1.2 SPOT synthesis ........................................................................................................................................ 16
2.1.3. Particlebased synthesis .......................................................................................................................... 16
2.2. Surface plasmon resonance as a tool in molecular biosensing ................................................................. 19
2.2.1. Surface plasmon resonance ..................................................................................................................... 19
2.2.1.1. Theory of propagating surface plasmon .......................................................................................... 19
2.2.1.2. Theory of localized surface plasmon ............................................................................................... 21
2.2.2. Surface plasmons in optical sensing ....................................................................................................... 21
2.2.2.1. Excitation of surface plasmons ........................................................................................................ 21
2.2.2.2. Quantitative interpretation of SPR response .................................................................................... 23
2.2.2.3. Methods of measurements ............................................................................................................... 25
2.2.2.4. Application of surface plasmons in biosensing ............................................................................... 26
2.3. Protein/resistant films .................................................................................................................................. 26
2.3.1. Poly(ethylene glycol) (PEG) ................................................................................................................... 27
2.3.2. Immobilizing PEG onto surfaces ............................................................................................................ 29
2.3.2.1. Chemisorption ................................................................................................................................. 29
2.3.2.2. Covalent grafting ............................................................................................................................. 29
2.3.2.3. Physisorption ................................................................................................................................... 30
2.3.2.4. Atom Transfer Radical Polymerization ........................................................................................... 31
2.3.2. Zwitterionic polymers ............................................................................................................................. 33
2.4. Surface analysis methods ............................................................................................................................. 34
2.4.1. Excitation and detection of surface plasmons ......................................................................................... 34
2.4.2. Xray Photoelectron Spectroscopy (XPS) ............................................................................................... 35
2.4.3. Scanning Electron Microscopy (SEM) ................................................................................................... 37
2.4.3.1. Basic Principle ................................................................................................................................. 38
2.4.3.2. SampleBeam Interactions ............................................................................................................... 38
3. MATERIALS AND METHODS ....................................................................................... 40
3.1. Fabrication of LSPR biosensors based on optical responsive materials ................................................. 40 CONTENTS 2
3.1.1. Materials ................................................................................................................................................. 40
3.1.2. Deposition of the bottom gold layer ....................................................................................................... 40
3.1.3. Deposition of the dielectric layer ............................................................................................................ 41
3.1.3.1. By simple incubation ....................................................................................................................... 41
3.1.3.2. By spin coating ................................................................................................................................ 41
3.1.3.3. By floating ....................................................................................................................................... 42
3.1.3.4. By thermal evaporation ................................................................................................................... 43
3.1.4. Deposition of the upper gold layer .......................................................................................................... 43
3.1.4.1. By metallization .............................................................................................................................. 43
3.1.4.2. By Physical Vapor Deposition ........................................................................................................ 43
3.2. Synthesis of protein/resistant coatings on the LSPR biosensor via Atom Transfer Radical
Polymerization (ATRP) ...................................................................................................................................... 44
3.2.1. Materials ................................................................................................................................................. 44
3.2.2. Direct synthesis of a proteinresistant coating on the LSPR biosensor ................................................... 45
3.2.2.1. Direct attachment of ATRP initiators on the LSPR biosensor ........................................................ 45
3.2.2.2. Synthesis of a graft polymer film on the LSPR biosensor via ATRP .............................................. 45
3.2.3. Indirect synthesis of a proteinresistant coating on the LSPR biosensor ................................................ 45
3.2.3.1. Synthesis of a silica gel on the LSPR biosensor .............................................................................. 45
3.2.3.2. Synthesis of a graft polymer film on the silica gelcoated LSPR biosensor .................................... 45
3.2.3.2.1. Attachment of ATRP initiators on the silicagel coated LSPR biosensor ................................ 45
3.2.3.2.2. Synthesis of a graft polymer film on the silicagel coated LSPR biosensor via ATRP ............ 46
3.2.3.2.3. Functionalization of the proteinresistant coating on the LSPR biosensor ............................... 47
3.3. Preparation of biomolecule arrays onto the protein resistant LSPR biosensor...................................... 48
3.3.1. Materials ................................................................................................................................................. 48
3.3.2. Fabrication of antibody arrays on the LSPR biosensor by the spotting technique .................................. 48
3.3.3. Fabrication of peptide arrays on the polymer coated LSPR biosensor by the particlebased technique . 49
3.3.3.1. Synthesis of amino acid toner particles for the particlebased synthesis ......................................... 49
3.3.3.2. Partial synthesis of peptide arrays on the polymer coated LSPR biosensor by the particlebased
technique ...................................................................................................................................................... 49
3.3.3.2.1. Liquidphase peptide synthesis ................................................................................................ 49
3.3.3.2.2. Particlebased peptide synthesis ............................................................................................... 49
3.3.3.2.3. Immunostaining with fluorescencelabeled antibodies ............................................................ 50
3.3.3.3. Total synthesis of peptide arrays on the polymer coated LSPR biosensor by particlebased
technique ...................................................................................................................................................... 51
3.3.3.3.1. Particlebased peptide synthesis ............................................................................................... 51
3.3.3.3.2. Immunostaining with infraredlabeled antibodies .................................................................... 51
3.4. Excitation and detection of surface plasmons ............................................................................................ 51
3.5. Analytical measurements ............................................................................................................................. 53
3.5.1. Scanning Electron Microscopy (SEM) ................................................................................................... 53
3.5.2. Xray Photoelectron Spectroscopy (XPS) ............................................................................................... 53
4. RESULTS AND DISCUSSION ......................................................................................... 55
4.1. Overview and introduction .......................................................................................................................... 55
4.2. Study of LSPR based biosensors as a solid support for peptide arrays and a detection tool in the
antibody assay ..................................................................................................................................................... 55
4.2.1. Introduction............................................................................................................................................. 55
4.2.2. Deposition of the intermediate dielectric layer ....................................................................................... 57
4.2.2.1. Silica nanoparticle monolayer as the dielectric layer in the LSPR biosensor .................................. 57 CONTENTS 3
4.2.2.2. Homogeneous polycrystalline silica film as the dielectric layer in the LSPR biosensor ................. 64
4.2.2.3. A composite structure of a polycrystalline silica film and a silica particle monolayer as the
dielectric layer in the LSPR biosensor ......................................................................................................... 64
4.2.3. Deposition of the upper gold layer .......................................................................................................... 65
4.2.3.1. Deposition of the upper gold layer by seeding and plating ............................................................. 65
4.2.3.2. Deposition of the upper gold layer by Physical Vapor Deposition (PVD) ...................................... 66
4.2.4. Localized Surface Plasmon Resonance (LSPR) Spectra ........................................................................ 67
4.2.4.1. Interpretation of the LSPR spectra of the nanoparticlebased biosensor ......................................... 67
4.2.4.2. Plasmon resonance line shape ......................................................................................................... 68
4.2.4.3. Optical homogeneity ....................................................................................................................... 72
4.2.4.4. Optical sensitivity ............................................................................................................................ 78
4.2.4.5. Conclusion ........................................................................................................................................... 82
4.3. Synthesis of protein/resistant coatings on LSPR biosensor via Atom Transfer Radical Polymerization
(ATRP) ................................................................................................................................................................. 85
4.3.1. Direct synthesis of proteinresistant coating on LSPR biosensor ........................................................... 85
4.3.2. Indirect synthesis of proteinresistant coating on the LSPR biosensor ................................................... 86
4.3.2.1. Synthesis of silica gel on the LSPR biosensor ................................................................................. 86
4.3.2.2. Synthesis of a proteinresistant film on the silica gelcoated LSPR biosensor via ATRP ............... 90
4.3.2.2.1. Attachment of ATRP initiators on the silica gelcoated LSPR biosensor ................................ 90
4.3.2.2.2. Surface initiated ATRP on the silica gelcoated LSPR biosensor ............................................ 92
4.3.2.2.3. LSPR response of graft copolymer films with different thicknesses ....................................... 97
4.3.2.2.4. Functionalization of the proteinresistant coating on the LSPR biosensor ............................... 98
4.3.3. Stability of the proteinresistant polymer coated LSPR biosensor in the peptide printing procedure ... 99
4.3.3.1. Blocking of residual hydroxyl groups of the polymer film ............................................................. 99
4.3.3.2. Removal of Fmoc protection groups by piperidine ....................................................................... 100
4.3.3.3. Removal of sidechain protecting groups by trifluoroacetic acid .................................................. 101
4.3.3.4. Treatment of graft polymercoated LSPR biosensor with acetic acid, piperidine, and trifluoroacetic
acid ............................................................................................................................................................. 101
4.3.4. Conclusion ............................................................................................................................................ 102
4.4. Label/free detection of antigen/antibody interactions occurring on a LSPR biosensor coated with a
protein/resistant layer ....................................................................................................................................... 104
4.4.1. LSPR biosensor as labelfree detection tool ......................................................................................... 104
4.4.2. Fabrication of an antibody array on a LSPR biosensor by spotting technique ..................................... 105
4.4.1.1. Qualitative analysis by LSPR imaging .......................................................................................... 106
4.4.1.2. Quantitative analysis by LSPR resonance shift ............................................................................. 109
4.4.2. Fabrication of a peptide array on a LSPR biosensor by particlebased technique for peptide synthesis
........................................................................................................................................................................ 110
4.4.2.1. Generation of a peptide array on a LSPR biosensor by partial particlebased synthesis ............... 110
4.4.2.2. Total synthesis of a peptide array on a LSPR biosensor by particlebased technique ................... 113
4.4.3. Conclusion ............................................................................................................................................ 119
5. OPTICAL BIOSENSORS BASED ON WGM .............................................................. 121
5.1. Introduction ................................................................................................................................................ 121
5.2. Theory ......................................................................................................................................................... 121
5.2.1. Basic equations ..................................................................................................................................... 121
5.2.2. Approximation for q = 1 mode ............................................................................................................. 123
5.2.3. Qfactor ................................................................................................................................................. 123
5.2.4. WGM for biosensing ............................................................................................................................ 124
5.2.5. Excitation of WGM .............................................................................................................................. 124 CONTENTS 4
5.2.6. Determination of the sphere radius ....................................................................................................... 125
5.3. Materials and Methods .............................................................................................................................. 125
5.3.1. Chemicals ............................................................................................................................................. 125
5.3.2. Inking .................................................................................................................................................... 125
5.3.3. Deposition of polyelectrolytes .............................................................................................................. 125
5.3.4. Raman measurement ............................................................................................................................. 125
5.4. Results and Discussion ............................................................................................................................... 126
5.5. Summary and Conclusion ......................................................................................................................... 127
6. SUMMARY AND CONCLUSION ................................................................................. 129
6.1. Optical biosensor based on localized surface plasmon resonance (LSPR) ............................................ 129
6.2. Protein resistant coating on the LSPR biosensor based on poly(ethylene glycol)................................. 131
6.3. Label/free detection of biomolecular interactions on the polymer coated LSPR biosensors .............. 134
6.4. Optical biosensors based on Whispering Gallery Modes (WGM) ......................................................... 137
BIBLIOGRAPHY ................................................................................................................ 138
ACKNOWLEDGEMENT ................................................................................................... 147












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