The Development of Enhanced Raman Scattering for the Trace Analysis of Biomolecules A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences 2013 David Paul Cowcher Supervised by Professor Royston Goodacre School of Chemistry Contents Contents CONTENTS ....................................................................................................................................... 2 LIST OF FIGURES .......................................................................................................................... 7 LIST OF TABLES .......................................................................................................................... 14 LIST OF EQUATIONS .................................................................................................................. 15 LIST OF SCHEMES ...................................................................................................................... 15 LIST OF ABBREVIATIONS ........................................................................................................ 16 ABSTRACT ..................................................................................................................................... 18 ACKNOWLEDGEMENTS ............................................................................................................ 19 DECLARATION ............................................................................................................................. 20 COPYRIGHT STATEMENT ........................................................................................................ 21 PREFACE ........................................................................................................................................ 22 1. INTRODUCTION ................................................................................................................... 24 1.1. ELECTROMAGNETIC RADIATION AND WAVE-PARTICLE DUALITY ................................... 25 1.2. MOLECULAR ENERGY LEVELS ........................................................................................... 28 1.3. RAMAN SPECTROSCOPY ..................................................................................................... 28 1.3.1. Comparing Raman with Infrared (IR) Spectroscopy .................................................. 32 1.3.2. Fluorescence interference ........................................................................................... 33 1.3.3. Raman Instrumentation ............................................................................................... 33 1.3.4. Raman microscopy ...................................................................................................... 35 1.3.5. Portable Raman and Liquid Sample Analysis ............................................................. 36 1.3.6. A Typical Raman Spectrum ......................................................................................... 37 1.3.7. Enhancing the Raman Signal ...................................................................................... 38 1.3.7.1. Resonance Raman scattering (RRS) ...................................................................... 38 2 Contents 1.3.7.2. Surface-enhanced (resonance) Raman scattering (SER(R)S) ................................ 39 1.4. SURFACE-ENHANCED AND TIP-ENHANCED RAMAN SCATTERING (SERS & TERS) ........ 40 1.4.1. Electromagnetic Theory .............................................................................................. 40 1.4.2. Chemical Enhancement Theory .................................................................................. 41 1.4.3. SERS Surfaces ............................................................................................................. 43 1.4.3.1. Nanoparticles ......................................................................................................... 44 1.4.3.2. Patterned surfaces .................................................................................................. 46 1.4.3.3. Non-metal SERS surfaces ..................................................................................... 47 1.4.4. Tip-enhanced Raman scattering (TERS) ..................................................................... 47 1.5. ANALYSIS OF RAMAN DATA .............................................................................................. 49 1.5.1. Preprocessing .............................................................................................................. 50 1.5.2. Principal Components Analysis (PCA) ....................................................................... 51 1.5.3. Multivariate Regression Analysis ............................................................................... 52 1.6. AIM OF THE THESIS ............................................................................................................ 52 1.7. REFERENCES ....................................................................................................................... 55 2. PORTABLE, QUANTITATIVE DETECTION OF BACILLUS BACTERIAL SPORES USING SURFACE-ENHANCED RAMAN SCATTERING ...................................................... 60 2.1. DECLARATION .................................................................................................................... 61 2.2. ABSTRACT .......................................................................................................................... 62 2.3. INTRODUCTION ................................................................................................................... 63 2.4. EXPERIMENTAL .................................................................................................................. 66 2.4.1. Colloid. ........................................................................................................................ 66 2.4.2. Instrumentation ........................................................................................................... 66 2.4.3. SERS Analysis of Pure DPA. ....................................................................................... 66 2.4.4. Culture of Bacterial Spores ......................................................................................... 67 2.4.5. DPA Extraction and Analysis ...................................................................................... 68 2.4.6. Data Processing .......................................................................................................... 68 3 Contents 2.5. RESULTS AND DISCUSSION ................................................................................................. 69 2.5.1. Univariate Analysis of Pure DPA ............................................................................... 72 2.5.2. Univariate Analysis of DPA Extracted from Bacillus Spores ..................................... 75 2.5.3. Multivariate Data Analysis for Estimating DPA Concentrations and Spore Counts . 75 2.6. CONCLUSION ...................................................................................................................... 78 2.7. REFERENCES ....................................................................................................................... 79 2.8. SUPPLEMENTARY INFORMATION ....................................................................................... 81 3. AN INVESTIGATION OF BORON NITRIDE AS A SUBSTRATE FOR SURFACE- ENHANCED RAMAN SCATTERING AT 244 NM EXCITATION. ....................................... 82 3.1. DECLARATION .................................................................................................................... 83 3.2. ABSTRACT .......................................................................................................................... 84 3.3. INTRODUCTION ................................................................................................................... 85 3.4. EXPERIMENTAL .................................................................................................................. 87 3.4.1. Reagents ...................................................................................................................... 87 3.4.2. Preparation of surfaces and samples: ......................................................................... 88 3.4.3. Instrumentation and measurement: ............................................................................. 88 3.4.4. Data analysis: ............................................................................................................. 89 3.5. RESULTS AND DISCUSSION ................................................................................................. 90 3.6. CONCLUSION ...................................................................................................................... 99 3.7. REFERENCES ..................................................................................................................... 100 4. THE DETECTION OF PROTEIN GLYCOSYLATION USING TERS ........................ 102 4.1. DECLARATION .................................................................................................................. 103 4.2. ABSTRACT ........................................................................................................................ 104 4.3. INTRODUCTION ................................................................................................................