Preparation of Modified DNA Molecules for Multi-Spectroscopy Application
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Preparation of modified DNA molecules for multi-Spectroscopy Application Thesis by Xinyu Zhang In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia November, 2018 2 EXAMINATION COMMITTEE PAGE The thesis of Xinyu Zhang is approved by the examination committee. Committee Chairperson: Prof. Enzo Di Fabrizio Committee Members: Prof. Frederic Laquai; Prof. Antonio Adamo 3 © November, 2018 Xinyu Zhang All Rights Reserved 4 ABSTRACT HENs of modified DNA molecules Xinyu Zhang Hot Electron Nanoscopy and Spectroscopy (HENs) is a current-sensing AFM technique recently developed in our lab, which have proven a new kind of response on conduction at the nanometer scale, casting a new light for the comprehension of electronic states in nanomaterials. Direct imaging of DNA structure has long been investigated, with the development of HENs technology, more structural information about DNA could be revealed by simultaneous measurements of height, phase, Raman signal, and conductivity. With the aim of applying it for the first time on biological molecules, customized double- stranded DNA sequences, including thiol-modified oligonucleotides are designed to create preferential conductive paths through the basis as a benchmark system for the technique on biomolecules. This work aims to a final goal to characterize hot-electron current between gold tip and thiol modified DNA which ideally is covalently bonded to the gold surface and optimized for the application. In this work, high density of DNA absorbed by SERS active gold surface with atomic flat islands has been prepared for HENs application. The samples have been characterized by AFM, SKPM and Raman Spectroscopy, as non- destructive and controlled interactive image analysis. High-resolution images of DNA have been acquired, S-S and Au-S bonding of DNA anchored on SERS active gold substrate are also visible with Surface-enhanced Raman and Tip-enhanced Raman signals. A submolecular feature has also been found in both topographical and electrical results. 5 Herein, we report the synthesis and characterization steps to obtain the optimized operation standard. 6 ACKNOWLEDGEMENTS I would like to thank my thesis advisor: Professor Enzo Di Fabrizio of the Department of Material Science and Engineering. His support is crucial for the completion of this thesis. I would also like to thank all the professional scientists in SMILES lab: Dr. Bruno Torre; Dr. Marco Allione; Dr. Andrea Giugni and Dr. Manola Moretti. They are always helpful and nice whenever I ran into a trouble spot or had a question about my research or writing. They consistently inspired my work but steered me in the right the direction of this work. Especially, many thanks to our former coworkers Dr. Monica Marini, who designs the DNA sequence and helps along the experiments. Also, I am gratefully indebted to Dr. Marini and Dr. Torre’s valuable comments on this work. I would also like to thank the students in our group: Andrea Ricco and Maria Caterina Morello. They are excellent coworkers and friends, without their moral support, this work will not be completed with joy and fun. 7 TABLE OF CONTENTS EXAMINATION COMMITTEE PAGE ................................................................ 2 COPYRIGHT PAGE ................................................................................................ 3 ABSTRACT ............................................................................................................... 4 ACKNOWLEDGEMENTS ..................................................................................... 6 TABLE OF CONTENTS ......................................................................................... 7 LIST OF ABBREVIATIONS ................................................................................ 10 LIST OF ILLUSTRATIONS ................................................................................. 12 LIST OF TABLES .................................................................................................. 17 Chapter 1 Introduction ....................................................................................... 18 Chapter 2 DNA and DNA Characterization ..................................................... 21 2.1 Deoxyribonucleic Acid (DNA): from the 1870s to 1960s. .................... 21 2.2 Structural Characterization of DNA....................................................... 24 2.2.1 Fluorescence Microscopy .................................................................. 25 2.2.2 Scanning Probe Microscopy (SPM) .................................................. 27 2.2.2.1 AFM in biomaterial imaging ........................................................ 28 2.2.2.2 STM .............................................................................................. 33 2.2.3 Electron Microscopy ......................................................................... 35 2.2.3.1 Transmission Electron Microscopy (TEM)/High Resolution TEM (HRTEM) ................................................................................................. 35 2.2.3.2 Cryo-Electron Microscopy ........................................................... 37 Chapter 3 Methodologies .................................................................................... 38 3.1 Electrophoresis ....................................................................................... 38 3.1.1 Introduction ....................................................................................... 38 3.1.2 Mechanism of Nucleic Acid Migration and Separation .................... 41 3.2 Raman Spectroscopy .............................................................................. 44 3.2.1 Introduction of Spectroscopy ............................................................ 44 3.2.2 Basic Raman Theory ......................................................................... 45 3.2.2.1 Scattering ...................................................................................... 46 3.3.2.2 Selection Rule and Molecular Vibrations .................................... 49 3.3.3 Enhancing Raman Scattering ............................................................ 52 3.2.3.1 Resonance Raman Spectroscopy .................................................. 52 3.2.3.2 Surface-Enhanced Raman Scattering ........................................... 53 3.2.3.3 Tip-Enhanced Raman Scattering .................................................. 54 8 3.2.4 IR versus Raman ............................................................................... 54 3.2.5 Raman Instrumentation and Raman Micro-spectroscopy ................. 56 3.3 Atomic Force Microscopy...................................................................... 59 3.3.1 Introduction ....................................................................................... 59 3.3.2 How does Atomic Force Microscope work? ..................................... 64 3.3.4 Imaging Methods in AFM ................................................................. 66 3.3.5 Artifacts: Tip Shape Issues ................................................................ 71 3.4 Electrical Measurements in AFM .......................................................... 73 3.4.1 Scanning Kelvin Probe Microscopy (SKPM) ................................... 74 3.4.1 Conductive AFM (CAFM) ................................................................ 77 3.4.3 Hot Electron Nanoscopy and spectroscopy (HENs) ......................... 80 2.2.4 Raman of DNA.................................................................................. 92 Chapter 4 Materials and Methods ..................................................................... 98 4.1 DNA Constructs Hybridization .............................................................. 98 4.2 Electrophoresis ....................................................................................... 99 4.3 AFM Imaging on DNA ........................................................................ 100 4.3.1 Substrate Preparation....................................................................... 100 4.3.2 Sample Preparation ......................................................................... 101 4.4 Raman Spectroscopy ............................................................................ 102 4.5 SKPM, TERS, and HENs .................................................................... 104 4.6 Additional techniques .......................................................................... 107 4.6.1 SEM ................................................................................................. 107 4.6.2 Optical Microscopy ......................................................................... 107 Chapter 5 Experimental Results and Analysis ................................................ 109 5.1 DNA Modification ............................................................................... 109 5.1.1 DNA Constructs Design .................................................................. 110 5.1.2 List of Sequence of DNA Oligonucleotides .................................... 112 5.2 DNA Constructs Visualization: Agarose Gel Electrophoresis............. 114 5.3 AFM Imaging ....................................................................................... 117 5.3.1 Substrate Morphology ..................................................................... 117 5.3.2 DNA Imaging .................................................................................