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Development of Carbon Dots As Fluorescent Probes for Fluorescence in Situ Hybridisation (FISH) Application

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Development of Carbon Dots As Fluorescent Probes for Fluorescence in Situ Hybridisation (FISH) Application Development of Carbon Dots as Fluorescent Probes for Fluorescence In Situ Hybridisation (FISH) Application By Phyllis Jacqueline Nishi A thesis submitted to the Faculty of Engineering, Computing and Science Swinburne University of Technology Sarawak In fulfilment of the requirements for the degree of Master of Science by Research 2019 Abstract Fluorescence in situ hybridisation (FISH) is an important bioimaging technique in molecular cytogenetics that utilises fluorescent probes that can bind specifically to a target nucleic acid sequence of a DNA or RNA. It is important that the fluorophores used to fluorescently tag probes are bright, small-sized, non-toxic and come in various colours. Thus, there is a need for new and alternative fluorescent labels to be developed to improve the performance of FISH. This thesis explores on the synthesis and application of carbon dots (CDs) as a class of versatile fluorescent label in FISH. The synthetic method for the production of CDs for use as fluorescent probes in FISH was described. CDs were synthesised by hydrothermal treatment of a carbon source in concentrated phosphoric acid solution. In this work, carboxymethylcellulose (CMC) was selected as the starting precursor and then converted into CDs with carboxylic functional groups. The optical properties and characterisation of the synthesised CDs were carried out. An amine-functionalised oligonucleotide probe was designed to detect and localise glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in human adult low calcium high temperature keratinocytes (HaCaT) cell line. CDs were neutralised, isolated and dried before conjugated with the GAPDH oligonucleotide probe via carbodiimide crosslinker chemistry. The conjugated CD-GAPDH probe were applied for in situ hybridisation in fixed HaCaT cell line. The performance of the probe was evaluated via fluorescence microscopy and discussed in details in this study. i Acknowledgements First, I would like to express my utmost gratitude to my mentor and coordinating supervisor, Associate Professor Ng Sing Muk for all the support, opportunities and invaluable guidance he has provided for me in my postgraduate journey. Thank you for your constructive advices and for being very patient with me. You have set a very good example of a leader and I am very proud to be under your wings. I would also like to thank Dr. Chua Hong Siang for helping out in my research work with his expertise in electronics and Dr. Hwang Siaw San for allowing me to carry out and learn cell culturing while using her utilities. My seniors, Dr. Jessica Fong and Ng Yann Huey for sharing their experiences and knowledge to me. I also dedicate my thanks to Dr. Paul Neilsen and the lecturers of Swinburne University of Technology Sarawak, Dr. Irine Ginjom, Associate Professor Peter Morin Nissom, Associate Professor Moritz Mueller and Dr. Daniel Tan for the advice and suggestions they have provided for my project. My time here in Swinburne would not be possible without the Research and Consultancy Office of Swinburne University of Technology Sarawak and the Tuition Fee Waiver Award granted to me, which I will forever be grateful for. Special thanks to Professor Dr. Zainab binti Ngaini and her students, Arif and Eswaran, from Universiti Malaysia Sarawak (UNIMAS) for providing us with the SEM and FTIR analysis needed for this study and for guiding me throughout the whole process. Likewise, I would like to express my thanks to Sarawak Biodiversity Centre (SBC) for allowing me to use their fluorescence imaging facilities and a very kind thank you to Dr. Kon Nyuk Fong for helping me with using the instrument. Thank you to the science laboratory technicians; Chua Jia Ni, Marciana Jane Anak Richard, Cinderella Anak Sio and Nurul-Arina binti Salleh. They have taken good care of the researchers by making sure all the laboratory safety and technicalities are handled with properly. They also helped me with any problems I faced when dealing with lab equipment and facilities. My thanks also go to my friends and officemates; Andrew Lim, Lee Boon Kiat, Cindy Wee, Diana Choo, Diyana Musa, Edwin Sia, Kong Ee Ling, Fay Fay, Fiona Chung, ii Jasper Sing, Tay Jia Jun, Mertensia Kho, Reagan Entigu, Jong Siu Mei, Vivian Lee and Chan Wen Loong who have been alongside me during my candidature. Many of them provided me with help and advice for my lab work and thesis. I have never felt more at home in the university thanks to you all. Also a very big thank you to Johnny Tang, for being with me and supporting me throughout my candidature. Finally, I would like to thank my parents for the support and care that they have provided for me throughout my life. Without them, getting this far is likely impossible. Last but not least, Phoebe Nishi, for being the sister and best friend that I know I could always lean on. I love you all. iii Declarations I, Phyllis Jacqueline Nishi, hereby declare that this thesis was composed by me and the work presented in it is of my own. Where work was carried out either jointly or wholly by others, due acknowledgement was given to the relative contributors, workers and authors. Any mentions of work and material previously published and written by others were quoted and referenced accordingly. No part of this thesis was previously submitted for any other degree or professional qualification. Signed: Date: 7th June 2019 iv Table of Contents Abstract ......................................................................................................................... i Acknowledgements ....................................................................................................... ii Declarations ................................................................................................................. iv Table of Contents .......................................................................................................... v List of Figures .............................................................................................................. ix List of Tables .............................................................................................................. xv 1 Introduction........................................................................................................... 1 1.1 Brief Overview ............................................................................................... 1 1.2 Problem Statement .......................................................................................... 1 1.3 Thesis Aim ..................................................................................................... 2 1.4 Objectives ....................................................................................................... 2 1.5 Thesis Outline ................................................................................................. 2 1.6 Scope and Delimitation ................................................................................... 4 2 Literature Review .................................................................................................. 5 2.1 Fluorescence Bioimaging ................................................................................ 5 2.1.1 Fluorescence and Fluorophores ................................................................ 7 2.1.1.1 Intrinsic Fluorophores ..................................................................... 10 2.1.1.2 Extrinsic Fluorophores .................................................................... 12 2.1.1.3 Quantum Dots ................................................................................ 15 2.2 Carbon Dots .................................................................................................. 18 2.2.1 Introduction to Carbon Dots ................................................................... 18 2.2.2 Physicochemical Properties ................................................................... 18 2.2.3 Top-down Approaches ........................................................................... 21 2.2.3.1 Arc Discharge Method .................................................................... 21 2.2.3.2 Laser Ablation ................................................................................ 21 2.2.3.3 Electrochemical Methods ................................................................ 22 v 2.2.4 Bottom-up Approaches .......................................................................... 22 2.2.4.1 Thermal Routes .............................................................................. 22 2.2.4.2 Microwave-assisted Methods .......................................................... 22 2.2.4.3 Hydrothermal/Solvothermal Routes ................................................ 23 2.2.5 Surface Functionalisation ....................................................................... 24 2.2.6 Applications .......................................................................................... 25 2.2.6.1 Biosensing ...................................................................................... 25 2.2.6.2 Chemical Sensing ........................................................................... 27 2.2.6.3 Imaging .......................................................................................... 28 2.2.6.4 Catalysis ......................................................................................... 30 2.3 Fluorescence In Situ Hybridisation ................................................................ 32 2.3.1 Mechanism of
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