APPLICATIONS OF OLIGOPEPTIDE-FUNCTIONALIZED SURFACES AND MICROPARTICLES IN LIQUID CRYSTALS- BASED ASSAYS ONG LIAN HAO (B.ENG. (HONS.), NANYANG TECHNOLOGICAL UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2016 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Ong Lian Hao 10 September 2016 i ACKNOWLEDGEMENTS First of all, I am utmost grateful for the opportunity presented by my advisor, Dr. Yang Kun-Lin, to complete this thesis under his guidance. His insightful perspectives and open-door policy provided a conducive environment for me to develop and hone my research skills and ideologies. I am also thankful for the engaging, and more importantly enlightening discussions with our group members, and they include Dr. Liu Fengli, Dr. Chen Chih-Hsin, Dr. Ding Xiaokang, Ms. Liu Yanyang, Ms. Nguyen Le Truc and Dr. Arumugam Kamayan Rajagopalan G. I am also fortunate to receive guidance and advice from Dr. Saif Khan and his group members during our regular intergroup sharing sessions. I would also like to thank the friendly laboratory staff for their assistance in the procurement and operation of equipment, and they include Mr. Boey Kok Hong, Ms. Lee Chai Keng, Mr. Liu Zhicheng, Ms. Lim Kwee Mei, Dr. Yuan Ze Liang and Dr. Yang Liming. Finally, I am grateful to my parents and parents-in-law for their support. I would also like to express my heartfelt gratitude towards my wife, Rylia, for her relentless support and dedicated care for the family over the years. She has always stood by me, and with our beautiful son, Jophiel, they are my motivation to complete this fulfilling life chapter. ii TABLE OF CONTENTS DECLARATION ..................................................................................................... i ACKNOWLEDGEMENTS .................................................................................... ii TABLE OF CONTENTS ....................................................................................... iii SUMMARY ............................................................................................................ x LIST OF TABLES ............................................................................................... xiii LIST OF FIGURES ............................................................................................. xiv LIST OF SCHEMES.......................................................................................... xviii LIST OF SYMBOLS ........................................................................................... xix LIST OF ABBREVIATION ................................................................................. xx CHAPTER 1 INTRODUCTION ............................................................................ 1 1.1 Background ............................................................................................. 2 1.1.1 Importance of Proteases and Protease Assays .................................. 2 1.1.2 Introduction of Liquid Crystals ......................................................... 4 1.1.3 LC-Based Protease Assays ............................................................... 7 1.1.4 Immobilization of Oligopeptide on LC-Compatible Surfaces .......... 8 1.1.5 Signal Enhancement in LC-Based Protease Assays ....................... 10 1.1.6 Oligopeptide-Functionalized Microparticles in LC ........................ 11 1.1.7 Real-Time Protease Activity Monitoring........................................ 12 1.2 Research Objective ..................................................................................... 13 CHAPTER 2 LITERATURE REVIEW ............................................................... 16 2.1 Introduction ........................................................................................... 17 iii 2.2 Homogenous Protease Assays .............................................................. 20 2.2.1 Organic FRET Donors and Acceptors ............................................ 21 2.2.2 Nanomaterials as FRET Acceptors or Donors ................................ 22 2.2.3 QD as FRET Donors ....................................................................... 24 2.2.4 Gold Nanoclusters (AuNC) ............................................................. 26 2.2.5 Nanoparticles in Colorimetric Assays ............................................ 27 2.3 Heterogeneous Protease Assays ............................................................ 29 2.3.1 Electrochemical Assays .................................................................. 30 2.3.2 Label-Free SERS and SPR Assays ................................................. 33 2.3.3 Enzyme-Linked Peptide Protease Assays ....................................... 33 2.3.4 LC-Based Assays ............................................................................ 35 2.4 Outlook ................................................................................................. 40 CHAPTER 3 MECHANISTIC STUDY FOR IMMOBILIZATION OF CYSTEINE-LABELED OLIGOPEPTIDES ON UV-ACTIVATED SURFACES ............................................................................................................................... 42 3.1 Introduction ........................................................................................... 43 3.2 Experimental Section ............................................................................ 46 3.2.1 Materials ......................................................................................... 46 3.2.2 Surface Modifications ..................................................................... 46 3.2.3 Immobilization of Oligopeptides .................................................... 47 3.2.4 X-ray Photoelectron Spectroscopy (XPS) ...................................... 47 iv 3.2.5 Stability Test for Immobilized Oligopeptides ................................. 48 3.2.6 Purpald Test .................................................................................... 48 3.2.7 Streptavidin Binding Assay ............................................................ 49 3.2.8 Oxidation of Thiols ......................................................................... 50 3.2.9 Fluorescence Microscopy ............................................................... 50 3.2.10 Matrix-Assisted-Laser-Desorption-Ionization Time-of-Flight Mass- Spectrometry (MALDI-TOF-MS) .................................................. 51 3.2.11 DTNB Assay ................................................................................... 51 3.3 Results and Discussion ......................................................................... 51 3.3.1 Role of Cysteine in Oligopeptide Immobilization .......................... 51 3.3.2 Effects of Reducing Agent and Buffers .......................................... 54 3.3.3 Effect of UV Irradiation .................................................................. 55 3.3.4 Test for Surface Aldehyde Group ................................................... 57 3.3.5 Role of Cysteine in Immobilization Mechanism ............................ 58 3.3.6 Stability and Orientation of Oligopeptide Immobilization ............. 62 3.4 Conclusion ............................................................................................ 65 CHAPTER 4 LIQUID CRYSTALS-BASED PROTEASE ASSAYS WITH DUAL CYSTEINE-LABELED PEPTIDES FOR IMPROVING PEPTIDE STABILITY ............................................................................................................................... 66 4.1 Introduction ........................................................................................... 67 4.2 Experimental Section ............................................................................ 71 v 4.2.1 Materials ......................................................................................... 71 4.2.2 Surface Modifications ..................................................................... 71 4.2.3 Immobilization of Peptides ............................................................. 72 4.2.4 Fluorescence Protease Assay .......................................................... 73 4.2.5 LC-Based Protease Assay ............................................................... 73 4.3 Results and Discussion ......................................................................... 74 4.3.1 Effect of Dual Cysteine on Peptide Surface Density ...................... 74 4.3.2 Fluorescence Protease Assays ......................................................... 78 4.3.3 Detection of Immobilized Peptides Using LC ................................ 81 4.3.4 LC-Based Protease Assays ............................................................. 82 4.3.5 Limit of Detection (LOD) of LC-Based Protease Assays .............. 85 4.4 Conclusion ............................................................................................ 88 CHAPTER 5 DISTORTION OF LIQUID CRYSTAL PHASE BY MICROPARTICLES FUNCTIONALIZED WITH OCTADECYL- TERMINATED OLIGOPEPTIDES ..................................................................... 90 5.1 Introduction ........................................................................................... 91 5.2 Experimental Section 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