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Surface-Selective Biochip for the Chemical Analysis of Single Cells by MALDI-TOF MS

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Surface-Selective Biochip for the Chemical Analysis of Single Cells by MALDI-TOF MS Surface-Selective Biochip for the Chemical Analysis of Single Cells by MALDI-TOF MS Liang Jiang A thesis in fulfilment of the requirements for the degree of Masters by Research Supervisor: Dr William A. Donald School of Chemistry Faculty of Science University of New South Wales October 2019 Surname/Family Name : Jiang Given Name/s : Liang Abbreviation for degree as give in the : MSc (Research) University calendar Faculty : Faculty of Science School : School of chemistry Surface-Selective Biochip for the Chemical Analysis of Thesis Title : Single Cells by MALDI-TOF MS Abstract 350 words maximum: (PLEASE TYPE) Single-cell analysis is used to study cell-to-cell variation in large cell populations of multi-cellular organisms, tissues, and cell cultures. Matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) presents a promising platform for single cell analysis owing to its ability to rapidly detect 10s to 100s of molecules nearly simultaneously from a single cell. Current single-cell MALDI MS techniques require all cells in a mixture to be diluted and dispersed onto a target plate. Thus, it is challenging to analyse ‘rare’ but important cells (e.g. circulating tumour cells) that are present at exceedingly low concentrations (e.g. one in a billion). Here, the surface of a transparent indium-tin oxide (ITO) coated borosilicate microscope slide is modified with an antifouling layer, and ‘decorated’ with surface-immobilised anti-EpCAM antibodies. In this way, model circulating tumor cells (MCF-7 cells) that overexpress EpCAM at the cell surface can be immuno-selectively captured from a complex sample mixture and then directly analysed by both microscopy and MALDI-TOF MS owing to the transparency and conductivity of the ITO substrate. The use of such a modified ITO surface can be used to capture MCF-7 cells from a mixture of blood at a ratio as low as 1 MCF-7 cell in 10 million total cells. The subsequent analysis by MALDI-MS imaging resulted in the detection of 10 phosphatidylcholine lipids. This new method will reduce the sample preparation steps required to perform MALDI- TOF MS on rare single cells and provide a platform for examining the molecular heterogeneity between single cells in a sub-population of diseased cells. This method can also be potentially used to analyse an individual single cell using both microscopy and mass spectrometry to obtain morphological and chemical information, which could be used to study the molecular origins of the heterogenous uptake of drugs and nanoparticles within a diseased population of cells. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………………… ……………………………………..……… …………14/10/2019……… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date ………1…4/…10…/2…0…19……………………….............. COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date …14…/1…0…/2…01…9……………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... 14/10/2019 Date ……………………………………………........................... iii INCLUSION OF PUBLICATIONS STATEMENT UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The student contributed greater than 50% of the content in the publication and is the “primary author”, ie. the student was responsible primarily for the planning, execution and preparation of the work for publication • The student has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not. This thesis contains no publications, either published or submitted for ☒ publication (if this box is checked, you may delete all the material on page 2) Some of the work described in this thesis has been published and it has been documented in the relevant Chapters with acknowledgement (if this ☐ box is checked, you may delete all the material on page 2) This thesis has publications (either published or submitted for publication) ☐ incorporated into it in lieu of a chapter and the details are presented below CANDIDATE’S DECLARATION I declare that: • I have complied with the Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Name Signature Date (dd/mm/yy) Liang Jiang 14/10/2019 Postgraduate Coordinator’s Declaration (to be filled in where publications are used in lieu of Chapters) I declare that: • the information below is accurate • where listed publication(s) have been used in lieu of Chapter(s), their use complies with the Thesis Examination Procedure • the minimum requirements for the format of the thesis have been met. PGC’s Name PGC’s Signature Date (dd/mm/yy) iv Acknowledgements First and foremost, I would like to thank my Supervisor, Dr Alex Donald, for his support and guidance throughout my entire research project. I will always appreciate him for the opportunity he provided so that I could work on this project and take this research journey. He always encouraged and pushed me to tackle all the challenges and difficulties I encountered along the way and to achieve better results. During these past two years, what I learnt from him was far more than this master project. Next, I would like to thank my co-Supervisors, Dr Fabio Lisi and Scientia Professor J. Justin Gooding. Fabio helped me with all experimental details and provided me with many compelling ideas. I learned a lot from him, not only the specific scientific topics but also the hands-on way to do research. Justin was an outstanding scientific mentor that kept me on track in the right direction. I gratefully acknowledge all the members in the Donald research group for all the support and company for all these years. Every time when I discuss my project with them, I could always get unexpected suggestions. Special thanks are also given to Diana Zhang and K M Mohibul Kabir for their proofreading of my thesis. I would like to thank Jiaxin Lian and Ying Yang for their help with regards to understanding the cell biology requirements. I would also like to thank Dr. Stephen G Parker and Lachlan Carter for the help with surface chemistry and electrochemistry. I would also like to thank Dr Anjaneyaswamy Ravipati and Ms Sydney Liu Lau from the i BMSF at UNSW for their technical support in operating the Bruker ultrafleXtreme MALDI-ToF/ToF. Finally, I would like to thank my parents and boyfriend. Without their patience and support, I would not be able to deal with all the challenges I experienced alone.
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  • Laurdan Fluorescence Lifetime Discriminates Cholesterol Content from Changes in Fluidity in Living Cell Membranes
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