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

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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.

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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).

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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.

ii Abstract

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.

iii List of Abbreviations

ADP - Adenosine diphosphate

AFM - Atomic force microscopy

AMP - Adenosine monophosphate

ATP - Adenosine triphosphate

CD45 - Protein tyrosine phosphatase, receptor type, C

CE - Capillary electrophoresis

CTCs - Circulating tumor cells

CV - Cyclic voltammetry

DHB - 2,5-Dihydroxybenzoic acid

DMEM - Dulbecco's modified eagle medium

DMF - Dimethylformamide

DMPC - 1,2-Dimyristoyl-sn-glycero-3-phosphocholine

DNA - Deoxyribonucleic acid

DOPC - 1,2-Dioleoyl-sn-glycero-3-phosphocholine

DPBS - Dulbecco's phosphate buffer saline

EDC - 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

EpCAM - Epithelial cell adhesion molecule

ER - Estrogen receptor

ESI - Electrospray ionisation

FACS - Fluorescence activated cell sorting

FBS - Fetal bovine serum

FTIC - Fluorescein isothiocyanate

FWHM - Full width at half maximum

HCCA - α-Cyano-4-hydroxycinnaamic acid

iv HER2-neu - Receptor tyrosine-protein kinase erbB-2

ITO - Indium-tin oxide

LCM - Laser capture microdissection

MALDI - Matrix assisted laser desorption/ionisation

MCF - Michigan Cancer Foundation

MES - 2-(N-morpholino) ethanesulfonic acid mRNA-Seq - Messenger ribonucleic acid sequencing

MS - Mass spectrometry nESI - Nanoelectrospray ionisation

NHS - N-Hydroxysuccinimide

OLED - Organic light emitting diodes

PBMCs - Peripheral blood mononuclear cells

PC2- Physical containment level 2

PCR - Polymerase chain-reaction

PDMS - Poly(dimethylsioxane)

PEG - Polyethylene glycol

16-PHDA - 16-Phosphohexadecanoic acid

PR - Progesterone receptor

PTFT - Polytetrafluoroethylene qPCR - Quantitative polymerase chain-reaction

RNA - Ribonucleic acid

SAMs - Self-assembled monolayers

SIMS - Secondary ion mass spectrometry

ToF - Time of flight

XPS - X-ray photoelectron spectroscopy

v List of Figures

Figure 1.1 A model of metastatic cancer development. Figure 1.2 Cell capture by laser capture microdissection. Figure 1.3 Principle of fluorescence-activated cell sorting (FACS). Figure 1.4 AFM-based imaging of single cell analysis with molecular resolution. Figure 1.5 Mass spectrometry imaging. Figure 1.6 Single cell analysis by nESI mass spectrometry. Figure 1.7 MALDI-TOF mass spectrometry. Figure 1.8 A schematic view of cleaned ITO coated surface composition with hydrolysis, oxyhydroxides and oxygen vacancies. Figure 1.9 Images of ITO coated glass slides for MALDI imaging substrate. Figure 1.10 General diagram for the formation and structure of a self-assembled monolayer. Figure 1.11 Illustration of PEG chain on a substrate which imparts surface resistance to non-specific protein or cell adsorption. Figure 1.12 Schematic of organosilane-based formation of SAMs on an oxide surface. The presence of water influences the surface modification by organotrimethoxysilane. Figure 1.13 Schematic of possible binding modes of an alkylphosphonic acid on an oxide surface. Figure 1.14 A schematic summarising the proposed use of an antibody-conjugated ITO surface to capture circulating tumor cells from a mixture of whole blood for integrated fluorescence microscopy and mass spectrometry analysis of single rare cells. Figure 1.15 A schematic showing how a specific antibody will be modified onto an ITO coated surface to capture circulating tumor cells. Figure 2.1 A model of a typical three electrode cell for electrochemical cyclic voltammetry measurement. Figure 2.2 Contact angle (θ) of a liquid droplet deposited on the surface of a solid. Figure 2.3 Water contact angle measurements of different layers on ITO surfaces. Figure 2.4 Cyclic voltammograms of ITO surfaces before and after modification. Figure 2.5 X-ray photoelectron survey spectra.

vi Figure 2.7 Fluorescence images of antibody conjugation on modified ITO surfaces. Figure 2.8 Fluorescence images of live cells (25,000 cells/mL) plated on different modified surfaces in cell culture media for 30 min. Figure 2.9 Images of a mixture of MCF-7 cells and MDA-MB-231 cells (total cell: 25,000 cells/mL) plated on anti-EpCAM conjugated surfaces for 30 mins. Figure 2.10 Images of a mixture of MCF-7 cells whole blood plated onto anti-EpCAM conjugated surfaces and antifouling surfaces for 30 mins. Figure 3.1 MALDI mass spectrum of calibrant mixture of standard peptides listed in Table 3.2. Figure 3.2 ImagePrep for matrix deposition on an ITO coated glass slide for MALDI imaging. Figure 3.3 Zoom review of an entire ImagePrep deposition encompassing five phases. Figure 3.4 A model of sublimation chamber used to deposit matrix on ITO coated glass slides for MALDI imaging mass spectrometry experiments. Figure 3.5 Brightfield images of matrix crystals deposited onto an ITO surface. Figure 3.6 Mass spectrometric image of 100 μM standard phosphatidylcholine. Figure 3.7 Fluorescence image of MCF-7 cells captured on a modified ITO surface from whole blood (1 MCF-7 cell per 10,000 blood cells). Figure 3.8 Mass spectrum of MCF-7 cell. Figure 3.9 MALDI-ToF MS/MS identification of m/z 782 as PC (34:1) in positive ion mode. Figure 3.10 Optical imaging and fluorescence imaging after MALDI-MS analysis, and the heatmap results of MALDI imaging of captured from whole blood (1 MCF-7 cell per 10,000 blood cells). Figure 4.1 A diagram of a potential future microcontact printing strategy to improve the localisation of single cells.

vii List of Tables

Table 1.1 Selected features of discussed cell isolating technologies. Table 1.2 Information obtained by using different single cell analysis methods. Table 2.1 General chemicals, media, and solutions. Table 2.2 Solvents for EDC/NHS activation and amine compound attachment. Table 3.1 General chemicals. Table 3.2 Mass calibrants for MALDI-ToF mass spectrometry. Table 3.3 Tentative assignments lipids detected from bulk MCF-7 cells by MALDI- TOF/TOF.

List of Schemes

Scheme 2.1 16-phosphohexadecanoic acid SAMs on a cleaned ITO surface. Scheme 2.2 Polyethylene glycol attached onto the 16-PHDA SAMs modified surface. Scheme 2.3 Antibody-conjugation for cell capture. Scheme 2.4 Pre-treatment for a mixture of blood cells. Scheme 2.5 Surface characterisation methods for different surfaces and parameter optimisation. Scheme 2.6 Reaction of EDC/NHS activation for carboxyl groups.

viii Table of Contents

Acknowledgement……………………………………………………………………….i Abstract...……………………………………………………………………………….iii List of Abbreviations………………………………………………………………...….iv List of Figures………………………………………………………………...…………vi List of Tables……………...…………………………………………………………...viii List of Schemes.………………………………………………………………………..viii

Chapter 1…………………………………………………………………………………1 General Introduction……………………………………………………………………..1 1.1 Circulating tumour cells and single cell analysis………………………………….2 1.2 Single-cell isolation methods……………………………………………………...4 1.2.1 Limiting dilution……………………………………………………………...4 1.2.2 Manual cell isolation………………………………………………………….5 1.2.3 Physical property-based methods……………………………………………..6 1.2.4 Immuno-based methods………………………………………………………8 1.3 Single-cell analysis methods……………………………………………………..10 1.3.1 Microscopy analysis…………………………………………………………10 1.3.2 Electrochemical analysis…………………………………………………….12 1.3.3 Polymerase chain-reaction analysis…………………………………………13 1.3.4 Single cell mass spectrometry……………………………………………….14 1.3.4.1 Capillary electrophoresis electrospray ionization mass spectrometry ………………………………………………………………………………….16 1.3.4.2 Nanoelectrospray ionisation……………………………………………16 1.3.4.3 Secondary ion mass spectrometry………………………………………17 1.3.4.4 Matrix-assisted laser desorption ionisation mass spectrometry…………18 1.4 ITO surface modification………………………………………………………...21 1.4.1 ITO surfaces…………………………………………………………………21 1.4.2 Self-assembled monolayers………………………………………………….23 1.4.3 Self-assembly on ITO……………………………………………………….25

ix 1.5 Research aims……………………………………………………………………27 1.6 References………………………………………………………………………..31

Chapter 2………………………………………………………………………………..45 Surface Modification for Capturing CTCs……………………………………………45 2.1 Introduction………………………………………………………………………46 2.2 Materials and experimental methods……………………………………………47 2.2.1 Materials…………………………………………………………………….47 2.2.1.1 Chemicals and substrates……………………………………………….47 2.2.1.2 Antibodies and cells…………………………………………………….49 2.2.1.3 Cleaning protocols……………………………………………………50 2.2.2 Experimental methods……………………………………………………….50 2.2.2.1 Phosphonic acid self-assembled monolayers…………………………...50 2.2.2.2 Attachment of polyethylene glycol……………………………………..51 2.2.2.3 Antibody-conjugation…………………………………………………..53 2.2.2.4 Cell culture protocols…………………………………………………54 2.2.2.5 Cell sample preparation and MCF-7 cells capture protocols……………56 2.2.3 Characterisation……………………………………………………………..57 2.2.3.1 Contact angle goniometry………………………………………………57 2.2.3.2 Electrochemical cyclic voltammetry……………………………………58 2.2.3.3 X-ray photoelectron spectroscopy………………………………………59 2.2.3.4 Fluorescence microscopy……………………………………………….59 2.3 Results and discussion……………………………………………………………60 2.3.1 Contact angle goniometry…………………………………………………...61 2.3.2. Cyclic voltammetry measurements…………………………………………62 2.3.3 X-ray photoelectron spectroscopy…………………………………………64 2.3.4 Fluorescence microscopy……………………………………………………68 2.3.5 Cell capture results…………………………………………………………..70 2.4 Conclusions………………………………………………………………………75 2.5 References………………………………………………………………………77

x Chapter 3………………………………………………………………………………..79 MALDI Mass Spectrometry Analysis…………………………………………………79 3.1 Introduction………………………………………………………………………79 3.2 Materials and experimental methods……………………………………………..81 3.2.1 Materials…………………………………………….………………………81 3.2.1.1 Chemicals and MALDI targets…………………………………………81 3.2.1.2 Cleaning protocols……………………………………………………83 3.2.2 Equipment and methods……………………………………………………..84 3.2.2.1 Sample preparation……………………………………………………84 3.2.2.2 Matrix deposition methods……………………………………………85 3.2.2.3 Surface characterisation…………………………………………….88 3.2.2.4 MALDI-TOF standard operating conditions……………………………89 3.3 Results and discussion……………………………………………………………90 3.3.1 Optimisation of the matrix deposition methods…………………………..90 3.3.2 MALDI-MS for MCF-7 cell analysis…………..……………………………93 3.3.2 Imaging analysis for MCF-7 cells…………………………………………98 3.4 Conclusions……………………………………………………………………101 3.5 References……………………………………………………………………..103

Chapter 4………………………………………………………………………………106 Summary, Conclusions and Future Work……………………………………………106 4.1 Summary………………………………………………………………………107 4.2 Conclusions……………………………………………………………………107 4.3 Future directions………………………………………………………………..109 4.3.1 Improving capture efficiencies……………………………………………110 4.3.2 Improving the cell localisation approach…………………………………110 4.3.3 Improving the information obtained………………………………………113 4.4 References………………………………………………………………………113

xi Chapter 1

General Introduction

In this dissertation, an indium-tin oxide (ITO) coated glass slide was modified with an antifouling layer and anti-EpCAM antibody to capture MCF-7 cells for microscopy and single cell mass spectrometry analysis. This project is motivated by the importance of single circulating tumour cell analysis in cancer progression and early tumour detection1–4. This presents a significant challenge, as the concentration of circulating tumour cells is extremely low in the bloodstream compare with normal blood cells5–9. In this study, an immuno-based method was used to capture circulating tumour cells in pre-concentrated blood mixtures. Analysis of single cells was then conducted using both fluorescence microscopy and MALDI-TOF mass spectrometry, eliminating time-consuming processes and providing sensitive detection. To have a better understanding of this project, the introductory part will focus on the existing single cell isolation and analysis methods, as well as the applications of single cell mass spectrometry analysis and the fundamental theory of surface chemistry for ITO modification.

1 1.1 Circulating tumour cells and single cell analysis

In a typical cancer tumour, there can be millions or billions of cells and not all of them necessarily remain in the same location during their lifecycles. Some of these cells can be released from the tumour and enter the bloodstream and/or lymphatic system.

These so-called circulating tumour cells (CTCs) can struggle to survive in circulation, cluster together as they travel, or lodge themselves in new tissues (Figure 1.1). Such cells can result in the growth of satellite tumours remote from the precursor tumour site and this metastatic cancer is responsible for 90% of cancer-related deaths10,11. Since the CTCs were first detected in a cancer patient in 1869, a number of researchers have demonstrated that CTCs can be used as a biomarker for predicting disease progression in early stage cancer patients. Thus, studying circulating tumour cells is important for our understanding of metastasis processes12,13, cancer characterization14,15, survival prediction16–18 and treatment responses19–21.

Primary tumour Cancer cells shed into bloodstream

Red blood cell Circulating tumour cells (CTCs) Secondary White blood cell tumour

Figure 1.1 A model of metastatic cancer development22,23. Including tumour cells intravasation into blood, circulating around the blood stream, and extravasation to a new site. For 1 ml of blood from a typical cancer patient, there are approximately one to ten single CTCs, one CTC cluster, millions of white blood cells and billions of red blood cells.

2 Breast cancer is the cancer that is most frequently diagnosed and the leading cause of female cancer death, which is responsible to 23% of total cancer cases and 14% of cancer related death24. The use of in vitro cell lines are widely used in cancer research, and can be used as models for developing methods for molecular diagnosis of breast cancers25. MCF-7 is the cell line that is most commonly used in breast cancer studies and widely used as a model of circulating tumor cells26–28. The MCF-7 line was first isolated from a 69-year-old patient with metastatic disease in 1973 at the Michigan Cancer

Foundation (MCF)29. It has been widely used in studies of estrogen receptor (ER)-positive cancer and progesterone receptor (PR)-positive cancer30. In addition, the MCF-7 cell is weakly aggressive and non-invasive cell line which was considered as having a low metastatic potential30 and thus, it has been used as a model cell line in breast cancer analysis31.

With the development of analytical methods that can probe at the level of a single cell, recent research has revealed that within a given tissue or even in the same cell line, each cell has a distinct function and state owing to the specific genome, epigenome, environment and history of the cell32,33. For example, advanced tumors may be comprised of different cancer cells that can differ genetically and phenotypically, resulting in different histopathology and different responses to the same therapy20. In this case, single cell analysis has attracted significant interest because by reducing biological noise, rare events can be observed without being masked by the bulk cell population.

3 1.2 Single-cell isolation methods

Before initiating a single-cell analysis, isolating and identifying single cells is necessary. For this purpose, there are several approaches to capture single cells that differ in terms of throughput, target cell purity and cell capture efficiency. General classes of approaches for capturing single cells include: (i) limiting dilution; (ii) manual cell capture;

(iii) size-based physical property methods; and (iv) the methods based on cellular biological characteristics.

1.2.1 Limiting dilution

Like normal pre-treatments of high concentration samples of cells, limiting dilution is one of the simplest single cell isolation methods. Either hand-pipettes or pipetting robots are used to dilute a cell suspension until it is observed using a microscope that a single cell or a limited number of cells are dispensed per aliquot. This method has been used for decades in cell quantification34, stem cell research35 and now is widely used in monoclonal cell culture36,37. It is simple, reproducible and to a certain extent cost- efficient because the degree of automation can be very high. However, since the number of the cells per aliquot is described by Poisson distribution37, this technique is limited by its statistical nature, which means not every aliquot will contain a single cell. On average, only about 30% of aliquots will actually contain a single cell38 and a separate process such as obtaining a microscope image is required to confirm the specific number of the cells in each aliquot.

4 1.2.2 Manual cell isolation

Manual cell picking is often used by an operator-controlled micromanipulator which typically consists of an inverted microscope to observe the cells and an ultrathin glass micro-capillary with a tip diameter that is close to the size of a single cell and a finely mechanised stage. By controlling the manipulator, the tip of a capillary pipette can move in 3 dimensions near the position of the target cells in a sample. A negative pressure connected to the capillary can then remove the single cell from its environment and within or on the tip of the pipette. The cells can then be transferred to a collection vessel or deposited elsewhere for analysis. With this method, specific functional single cells can be captured from a suspension39–41.

Laser capture microdissection (LCM) is also a manual isolation method by which individual cells can be harvested from mostly solid tissue samples with a laser beam in a one step process. Under direct microscopic visualisation, a tissue section of interest can be observed, and the target cells can be identified and removed from unwanted cells, thus obtaining histologically pure and enriched cell populations. The individual cells are then embedded in a thermal polymer film and transferred to sample containers (Figure 1.2)42,43.

However, the need for an experienced operator-based system strongly limits the throughput of this approach.

5 Transfer film

Selected cell Laser pulse

Transfer of selected cell

Vacancy following selective procurement

Figure 1.2 Cell capture by laser capture microdissection44. With the laser pulse, the transfer film was activated and became adhesive to target cells.

1.2.3 Physical property-based methods

Physical property-based methods are approaches to distinguish specific cells by their physical properties such as size45,46, deformability47–49, electric charging50 and density. For example, with a certain size of wells, single cells of a specific size can be trapped within a microwell. Well structures use a physical boundary to mechanically separate individual cells. In this method, single cells are trapped by creating wells that are similar in size to the cells of interest. For different applications, a microwell device can be designed in numerous different ways by selecting different parameters such as shape, size, material, and the number of wells. For example, in a smaller cell-scale microwell, due to the shorter diffusion distances within the microwell, the effect of an induced disease on a particular cell can be examined faster (in several hours) than in a

6 cell culture51. The number of wells in a given chip is dependent on the size of the microwells, in which higher numbers of wells are often used for rapid and accurate data interpretation. By integrating bright-field or fluorescence microscopy imaging, high- throughput screening of live-cells responding in real time to stimuli is possible52,53.

To improve the throughput of single cell isolation, fluid dynamics can be integrated with micro-environments. Microfluidics, which refers to fluidic behaviour and the control of it within channels of micrometre dimensions, is a field of study closely related to single cell chemical cytometry analysis54,55. With the recent development of multilayer soft lithography fabrication, the control of fluids and cells in microfluidic devices has improved considerably56. In single cell analysis, with a poly(dimethylsiloxane) (PDMS) valve, single cells can be moved to a desired location on the chip and isolated in a chamber for further treatment. Microfluidic systems provide numerous advantages for single-cell analysis such as economies of scale, parallelisation and automation, and increased sensitivity and precision that comes from small reaction volumes57.

With these physical property-based methods, single cells can be isolated with high throughput. However, since the size of the circulating tumour cells is similar with the leukocytes in blood (average diameter of about 20 μm), it is hard to completely separate these cells58. Thus, even more advanced methods are needed to target these specific cells.

7 1.2.4 Immuno-based methods

To selectively capture circulating tumour cells from a mixture of contaminated cells or even in whole blood, it is important to find a unique biomarker that is only expressed on these cells. On the surface of tumour cells, there are some surface antigens that are absent on normal cells like leukocytes. These markers include epithelial cell surface markers, such as the epithelial cell adhesion molecule (EpCAM); cytokeratin; and more cancer-specific markers, such as HER2-neu and mucin 1 for breast carcinoma59–62.

By using these antibodies, circulating tumour cells can be either labelled with certain fluorescence dyes or directly captured on the analyser chips or magnetic nanoparticles for further analysis. In addition, the removal of unwanted blood cells is also an alternative approach to isolating single circulating tumour cells by using negative surface markers such as using leukocyte antigen CD45 to remove leukocytes5,63.

To isolate single circulating tumour cells with the label of a specific fluorescent dye, fluorescence activated cell sorting (FACS) can be used. As shown in Figure 1.3, in flow cytometers, the instrument can detect cells using parameters including cell size, morphology, and protein expression, and then sort target cells for further study64,65. By staining the rare target cells of interest and the unwanted subpopulation of cells with different fluorescence colours, FACS has the ability to detect and sort the specific stained cells at a frequency of 1 in 107 peripheral blood mononuclear cells (PBMCs)66. In this system, cell suspensions are pressure driven through a flow cell. The cell flow rapidly passes through a laser beam for optical excitation, where optical detectors are then used downstream to identify cells of interest for capture. In this way, specific cells can be sorted in addition to size analysis and counting.

8 Cell suspension

Laser Detector Current

Charged plates

Figure 1.3 Principle of fluorescence-activated cell sorting (FACS)67.

As another example, the CellSearch system (Veridex LLC) can be applied for the enrichment and enumeration of circulating tumour cells before and during treatment as approved by US Food and Drug Administration for clinical use68,69. In this system, ferrofluid nanoparticles with anti-EpCAM antibodies are used to capture the specific circulating tumour cells containing the epithelial cell adhesion molecule on the surface.

Subsequently, the captured cells can be preconcentrated or isolated by the magnetic beads for further analysis. With these immuno-based methods, specific circulating tumour cells with specific surface antigens can be captured in a mixture of other cells. Thus, the detection of CTCs in blood was finally achieved. After cell capture, a method which is able to conduct single cell analysis directly after cell isolation is necessary.

9 1.3 Single-cell analysis methods

Once isolated, different methods were applied for single cell analysis. However, since the amounts of analytes present in a cell are extremely low (about 7 pg of ribonucleic acid, RNA; and 700 pg of protein in a typical mammalian cell)70,71, performing analysis of single cells is considered challenging. Recently, a number of chemical and physical sensing methods have been applied in the field of single cell analysis, such as microscopy, electrochemical analysis, polymerase chain reaction-based analysis (nucleic acid content) and mass spectrometric analysis (proteins and metabolites).

1.3.1 Microscopy analysis

Imaging-based techniques such as fluorescence microscopy can be used to visualise the cells or to dynamically monitor cell growth and morphological changes72,73.

Fluorescence imaging relies on the illumination of fluorescently labelled proteins or other intracellular molecules with a defined wavelength of light, ideally near the peak of the fluorophore excitation spectrum, and detection of light emitted at a longer wavelengths74.

Cellular fluorescence can be detected and quantified with high sensitivity and precision using a fluorescence microscope75. Advanced techniques based on fluorescence microscopy have been developed to analyse and track individual molecules, allowing images to be acquired at a resolution below 10 nm76,77.

Another type of microscopy, atomic force microscopy, can also be applied to study single cell membranes and cell adhesion. As shown in Figure 1.4, an atomic force microscope that equipped with a detector tip is able to analyse cell surfaces at a high

10 resolution. In this case, even membrane proteins can be detected on the surfaces of cells78,79. Cell force spectroscopy is another utilisation of atomic force microscopy. It is used to study single cell adhesion, which is commonly defined as the binding of a cell to a substrate e.g. another cell, a surface or an organic matrix80. It is an effective tool for studying membrane and sub-membrane cell structures in investigation of cytoskeleton characteristics and dynamics such as the study of migrating cells. In addition, it can also be used to study the structural state (cytoskeleton or membrane lipid phase) of living cells in order to diagnose different human diseases78,81,82.

Scanning direction

Probe

Protein

Figure 1.4 AFM-based imaging of single cell analysis with molecular resolution83.

Microscopy is a powerful method for imaging-based analysis at a very high resolution. With the labels of different fluorescence dyes, the study of protein interactions84–86 and drug delivery87,88 can be achieved at the single-cell or subcellular level. However, since the use of microscopy can only be used to obtain limited information regarding cell morphology or the location of specific chemicals,

11 electrochemical analysis is sometimes conducted in combination with microscopy-based methods.

1.3.2 Electrochemical analysis

Electrochemical-based methods involve the use of electrical stimulation to analyse the chemical components of a system, usually in highly targeted analyses. Since the electrodes for the signal acquisition can be miniaturised, the analysis can be integrated on a micro-sized platform, which is an ideal size for single cell analysis. One of the most widely used electrochemical methods is cyclic voltammetry (CV)89. This approach has been used in the electrical measurements of neurotransmitter secretion to characterise intercellular communication, which occurs through the release of biochemical messengers from an emitting cell to a target cell90–92. In this approach, high sensitivity towards electrochemically active molecules is critical for the analysis of neuronal cells, which makes it possible to investigate the influence of external parameters such as drugs on exocytosis93,94.

Another electrochemical analysis method is impedance spectroscopy, which is a technique used to characterise tissues or cells based on the knowledge of their electrical properties in the frequency spectrum. It is a label-free tool that can provide electrochemical information on the response of cells to chemical or biological stimuli.

This can then aid study of cellular processes, such as cell spreading, adhesion, invasion and toxicology95–98. One development of this method to obtain spatial information for studying cellular heterogeneity is electrochemical impedance microscopy. In this new

12 method, the optical information rather than electrochemical current is measured, thus allowing for fast and non-invasive imaging of impedance99,100.

Electrochemistry can be used to study the stimulation of the cells and can also be integrated with single-cell microscopy imaging. However, this approach yields limited information about many of the thousands of molecules that are contained in single cells, including key biomarkers of circulating tumour cells. To obtain more genetic and molecular information at the single cell level, polymerase chain-reaction and mass spectrometry are used in single cell analysis.

1.3.3 Polymerase chain-reaction analysis

Polymerase chain-reaction (PCR) based techniques are used to amplify small copy numbers of DNA for genetic sequencing101. Single cell genomics is a relatively mature technology, and several commercial devices are already available with the capability of measuring DNA and RNA at the single cell level102. Due to genetic heterogeneity, different gene expressions can be detected within a tissue or a cell population103. In 2011, a commercially available high-throughput single cell quantitative

PCR (qPCR) instrument was reported to enable automated studies of large sets of single cells, such as the identification and clonal analysis of cancer stem cells104. Recently, further improvement in the breadth of single cell mRNA analysis has been achieved using mRNA sequencing (mRNA-Seq)105–107 or qPCR57,108. However, this method is disadvantaged by background contamination during the amplification of nucleic acids from single cells. Moreover, information about proteins and metabolites are more

13 representative of function than DNA and RNA. Thus, alternative methods are required to obtain chemical information.

1.3.4 Single cell mass spectrometry

Mass spectrometry (MS) is now widely used in chemical and biological analysis109,110. MS can be used to obtain more chemical information by measuring the mass-to-charge ratio (m/z) of ions to identify and quantify molecules in simple and complex mixtures. Owing to the ability of MS to rapidly and sensitively differentiate hundreds to thousands of proteins in complex cellular mixtures without any pre-labelling,

MS instruments have also been applied in the analysis of single cells111–113. However, the number of metabolites that can be measured in single cells by MS are limited to 10s to low 100s114–117. Thus, new breakthroughs in sample manipulation, preparation, ionisation and data analysis are required to perform single cell MS with deep proteome and metabolome coverage.

The metabolome is the full complement of small-molecule metabolites (usually less than 2 kDa) in a specific cell, organ, or organism that provides a ‘fingerprint’ of on- going biological processes114,118. It includes endogenous and exogenous small molecules

(e.g., pyruvate, lactate, sugars, adenosine monophosphate (AMP), adenosine diphosphate

(ADP), adenosine triphosphate (ATP), etc.), drugs and their metabolites, lipids, and peptides114. Because metabolites cannot be amplified like nucleic acids, the deep characterisation of whole metabolomes is considered highly challenging.

14 As shown in Figure 1.5, by use of imaging mass spectrometry, the spatial profiling of different molecules can be performed. For example, proof-of-principle single cell analysis has been used to characterize peptides and lipids by use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry to detect membrane-based lipid molecules119–123, while electrospray ionisation (ESI) mass spectrometry are usually used to detect other small molecules usually from intercellular matrices. Capillary electrophoresis has been couple with ESI-MS (CE-ESI-MS)10,111,124–126 to achieve high reproducibility and separation efficiency. Secondary ion mass spectrometry (SIMS) has been used to perform high resolution MS imaging127–133. These methods are discussed in detail below.

(x1, y1) Laser spots

(xn, yn) Intensity Intensity

(x1, y1) m/z a m/z b m/z c m/z (xn, yn)

m/z a m/z b m/z c

m/z a +m/z b +m/z c

Figure 1.5 Mass spectrometry imaging. Single cells are placed onto the target and mass spectra are acquired in a raster pattern. The spatial distribution of a single m/z can be represented as an ion density map122.

15 1.3.4.1 Capillary electrophoresis electrospray ionization mass spectrometry

Capillary electrophoresis (CE) separates analytes based on their electrophoretic mobility. In CE, a voltage is applied across a capillary containing the analyte mixture flowing through a buffered electrolyte solution. In CE-ESI-MS, the fractionated analyte solution is then introduced to the mass spectrometer via online electrospray ionisation.

This method has been used in single cell analysis to identify metabolites134,135 and proteins136 from single frog (Xenopus laevis) blastomeres (approx. volume of 1 μL71). An advantage of CE-ESI-MS is nanoscale sample consumption and analyte separation prior to MS analysis which greatly reduces the problem of ion suppression, i.e. analyte molecules compete for charge during ionisation137. This is a major challenge for mass spectrometry-based analysis, as certain analytes do not readily ionise and require extensive separation and derivatisation in order to be delivered as an ion to the mass analyser. However, CE-ESI-MS samples take time (at best 1 cell/minute) to elute from the electrophoresis capillary, and CE parameters require extensive optimisation for each target analyte and sample mixture.

1.3.4.2 Nanoelectrospray ionisation

The concept of nanoelectrospray ionisation (nESI) was first reported in 1994138.

In standard ESI mass spectrometry, an electrolyte solution containing analyte(s) of interest is pumped at flow rates of a few μL/min through a metal capillary needle biased to a high (positive or negative) potential relative to a counter electrode. In nESI, ion emitters are formed from glass capillaries that are tapered at one end by use of a glass capillary puller to give orifices of around 1 μm139,140 or even less than 300 nm139,141. nESI

16 has become an invaluable tool for biological research due to its high sensitivity and straightforward online coupling with liquid-phase separations.

Recently, nESI mass spectrometry has been used to analyse single cell samples142–

145. As shown in Figure 1.6, a single live cell can be ‘stabbed’ by a nESI emitter with the assistance of a micromanipulator, and then directly electrosprayed into a mass spectrometer. With this method, 10s to 100s metabolites can detected in both plant143 and animal cells142,144,145. As cell contents can be delivered directly to the mass analyser, the losses relating to sample preparation can be reduced, thus potentially increasing sensitivity.

nESI emitter Metal coating nESI emitter MS inlet

Taylor cone

5 mm

Sample Nano droplet

Single cells

Figure 1.6 Single cell analysis by nESI mass spectrometry. A nESI emitter can be used to ‘stab’ the single cell and applied the voltage for nanosprayer to feed the cell content directly to mass spectrometer146.

1.3.4.3 Secondary ion mass spectrometry

Secondary ion mass spectrometry, including nanoscale secondary ion mass spectrometry (NanoSIMS) and time-of-flight secondary ion mass spectrometry (TOF-

SIMS), has emerged as a powerful tool for biological imaging, especially for single cell

17 imaging. In a SIMS experiment, a focused primary ion beam with high energy bombards on the sample surface. The energy of ion impact can be transferred from primary ions to induce the fragmentation of molecular ions as they are transferred from the sample surface to the mass analyser. The ionisation efficiency of these process is relatively low with only ~1% of the material that is ejected from the surface being ionised and detected147.

Usually ToF–SIMS is capable of detecting biological molecules under 1000 Da

(i.e. lipids, metabolites and small fragments), and can achieve a high spatial resolution at about 100 nm148. Thus, SIMS imaging can provide information on the subcellular distribution of endogenous and exogenous chemicals, including metallodrugs, from membranes, cytoplasm and nuclei with high spatial resolution and chemical specificity without the need for labelling149–151.

1.3.4.4 Matrix-assisted laser desorption ionisation mass spectrometry

Matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was first reported in 1988152. In this approach, a laser is used to irradiate a solid surface sample comprised of a relatively high concentration of ‘matrix’ molecules that absorb the laser energy and a lower concentration of analyte molecules to form ions of small and large molecules from the surface for detection by MS (Figure 1.7). The analyte and matrix molecules are typically co-crystallised together on a target plate. The matrix and analytes are irradiated by a laser under vacuum to form ions. Compared with

SIMS, MALDI is a ‘softer’ ionisation approach, which means less energy is deposited into the analyte ions and thus typically MALDI results in the formation of intact ions.

That is, the precursor ions of molecules can be directly detected by MALDI-TOF which

18 is important for identifying molecules particularly when combined with tandem mass spectrometry ion fragmentation methods. Mass analysis is achieved based on the flight time for a particular ion, which varies according to its mass-to-charge ratio. Owing to the ability of MALDI-TOF to accurately and rapidly analyse large molecules, it can be used to obtain the molecular weights of proteins and peptides in complex samples (Figure 1.7).

MALDI-TOF has become an important tool in proteomics and single cell analysis121,153–

155.

Laser beam Vacuum system Ion-molecule

reaction Lineardetector Reflector MALDI target MALDI Ion source

Reflection detector

Analyte

Matrix Acceleration region Field-free drift zone

Figure 1.7 MALDI-TOF mass spectrometry. A schematic diagram showing sample ionisation and detection using MALDI-TOF. Here, the sample is prepared on the plate target and embedded in a surplus of matrix molecules. The matrix is then excited and protonated into the gas phase by a pulsed UV-laser together with the analyte molecules. The ions are then accelerated by an electrical field into the field-free flight tube of the mass spectrometer.

MALDI MS imaging is a method in which the spatial distribution of compounds can be measured in tissues including at the single cell level. The tissue or cells of interest are first placed on an indium tin oxide (ITO) coated glass slide to ensure that the surface of the slide is conductive, and the entire slide is transparent. Matrix molecules are then

19 applied to the analytes, cells and/or tissues of interest, and mass spectra are acquired as a function of different positions on the surface. The spatial distribution of a single m/z can be presented as a 2-dimensional ion density map119,122,123,127,133 (Figure 1.5). Single cell

MALDI MS is capable of relatively high throughput compared to other single cell mass spectrometric techniques. However, there are still several major challenges for single cell

MALDI MS, such as mass range limitations and matrix deposition procedures for reproducible analyte detection.

One of the major challenges for MALDI MS imaging is the spatial resolution, which can make it somewhat challenging to apply MALDI MS imaging for analysis at the cellular level and subcellular levels129,156. The two major effects that can significantly influence spatial resolution is the laser spot size and the matrix crystal size. With a given laser spot size, a relatively high spatial resolution can be achieved by using a homogeneous layer of matrix with a very small matrix crystal size. In addition, ‘gaps’ between matrix crystals can also have a significant effect in single cell MALDI MS imaging. Since the diameter of human cancer cells is around 15-25 μm, if the gaps between matrix crystals are larger than the size of the cells, some cells may not be covered by the matrix and therefore cannot be detected. Thus, the choice of matrix and matrix deposition method are critical factors that can impact spatial resolution. Several different matrix deposition methods have been developed including manual and automatic methods such as the use of a commercial sprayer (e.g. ImagePrep form Bruker Pty.

Ltd)157–160, ‘dried droplet’ (which directly spots the saturated matrix solution on the analyte with micropipette)161, inkjet printing162,163, electrospraying164 and sublimation165–

169.

20 Another challenge in MALDI imaging is the limitation of the detectable mass range. Although suitable for analysis of a wide range of biomolecules, MALDI is often not ideal for the detection of small molecules and metabolites as they can often have similar masses as the organic matrix ions170,171. In addition, different matrices can be suitable for analyte molecules of different mass ranges. For example, HCCA has been widely used in the analysis of peptides and proteins while DHB is often the matrix used for phospholipids, peptides and small proteins. Recently, some studies have used nanoparticles of various materials such as iron oxide172,173, silver174–176, titanium177, diamond177,178 and silica179,180 as an alternative material for the standard organic matrix.

With the use of the surface modification of these inorganic or metallic nanoparticles, such matrices can be used for the analysis of low mass range molecules and higher performance for spatial resolution.

In previous studies involving single cell MALDI MS181,182, single cells were often obtained by plating such cells onto the MALDI target. A method to capture circulating tumor cells from blood and for MALDI MS single cell analysis has not been reported in the literature.

1.4 ITO surface modification

1.4.1 ITO surfaces

As shown in Figure 1.8, indium tin oxide (ITO) is a solid mixture of indium (III) oxide (In2O3) and tin (IV) oxide (SnO2), typically 90% In2O3, 10% SnO2 by weight, which for MALDI MS target slides, is normally deposited on a transparent glass or plastic

21 substrate by physical vapor deposition183. ITO is known for having a high electrical conductivity and being optically transparent. Thus it has been used in many different technological applications, such as organic light emitting diodes (OLED)184,185, sensors186–188, electrodes184,189, DNA detection190, solar cells191,192, as well as a substrate for MALDI MS imaging.

OH OH HO In(OH)3 OH In In OH HO In OH HO OH OH OH OH ‘InOOH’ O O OH In In In Sn In In O O O ITO bulk In Sn In In In In

Oxygen vacancy site

In2O3/SnOx + defectst In Sn O e 2-y y 3 y

Figure 1.8 A schematic view of cleaned ITO coated surface composition with hydrolysis, oxyhydroxides and oxygen vacancies193.

An advantage of using an ITO coated glass slides as a MALDI MS imaging target plate is that it is transparent (Figure 1.9). This means that the same sample can be studied under a light microscope, before being coated with matrix and placed into a mass spectrometer. In this case, an individual cell can be studied by both microscopy and mass spectrometry. This method can potentially provide a useful tool to gain information about

22 the correlation between molecular expression and drug-resistance to develop strategies to design better therapeutics.

Figure 1.9 Images of ITO coated glass slides for MALDI MS imaging substrate. Glass substrates remain transparent with a thin layer of indium tin oxide coating (from Bruker, AU).

To enable further interfacial structures and functions, a number of studies have been reported for the surface modification of ITO190,194,195. Among these methods, self- assembled monolayers (SAMs) based surface modification methods are becoming increasingly popular to form well controlled molecular layers on ITO surfaces196.

1.4.2 Self-assembled monolayers

Self-assembled monolayers are molecular assembles with high degrees of structural order. They are formed from the adsorption of active molecules with a specific affinity to its head-group on a corresponding solid surface197. With these organic materials, the free energy of the interface on the surface is lowered compared to in the absence of such a layer. Thus, these materials will tend to be spontaneously adsorbed by the bare metal or metal oxide surfaces in the ambient environment198. The formation of

23 the self-assembled monolayers is shown in Figure 1.10. Although SAMs are typically extremely thin (usually less than 2 nm199), the surface properties can be completely modified by using various molecules with tailorable head and tail groups.

Solution of surface-active Substrate Immersion time material seconds to hours

Group-specific interactions Air-monolayer (H-bonding, dipolar) interface group

Alkyl groups Intermolecular interactions Surface-active Chemisorption head-group at the surface Substrate

Closely packed ordered SAMs

Figure 1.10 General diagram for the formation and structure of a self-assembled monolayer197.

For example, with a polyethylene glycol (PEG) based surface modification, the non-specific adsorption of proteins or cells can be resisted by surfaces; i.e. PEG-based surface modification is used to form antifouling coatings on surfaces200,201. The resistance of the hydrophilic PEG polymer to the absorption of proteins and other biomolecules is attributed to the strong interactions between PEG and water (Figure 1.11). In addition, owing to the charge neutrality and the absence of hydrogen bond donors, the efficiency of the surface resistance increases as the length and density of the PEG chains

24 increases202–204. Due to the property of antifouling, PEGylated surfaces are widely used in biomaterial applications205–208.

Proteins or cells

PEG layer

Substrates

Figure 1.11 Illustration of PEG chain on a substrate which imparts surface resistance to non-specific protein or cell adsorption.

1.4.3 Self-assembly on ITO

The preparation and performance of SAMs on inorganic oxide surfaces is considered to dramatically depend on the type of molecules used to form monolayers, including both the functional groups and molecular shapes of the head and tail molecules.

For ITO surfaces, several different classes of molecules can be used to form a covalent chemical bond with the reactive sites on the surface, involving either terminal hydroxide groups or exposed metal atoms. These modification molecules include organophosphonates209–213, organosilanes214,215 and alkanethiols216,217.

The thiol (-SH) group in alkanethiols is a typical headgroup which has a high affinity for metal surfaces such as gold, silver, platinum, and indium218. However, the stability and surface coverage of alkanethiol-based SAMs are reported to be relatively low. Compared with thiol groups, organosilane-based SAMs on ITO are of higher stability196,214,219. However, organosilanes tend to form multilayers and thus are difficult to control. For example, as shown in Figure 1.12, even with a tiny amount of water,

25 organosilanes can cross-react with each other, making the formation of silane SAMs more complex and difficult to reproduce220,221. Therefore, the organophosphonic acid SAMs are emerging as the most widely used surface modification approach for ITO due to its well-ordered formation, high stability and surface coverage (Figure 1.13). In addition, because of the high reactivity of the carboxyl acid group in the terminus of organophosphonic acid, further modification can be performed on such SAMs.

OMe OMe R OMe Si R HO R R Si OMe Excess O R Si O Si O of H2O R R OMe Si O O Si + Si O O O OH OH OH OH OH OH O OH OH O M M M M M M M M M M M M

Multilayer Anhydrous Optimum amount of H O conditions 2

R R R R R R OMe Si Si Si O Si O Si O Si O O O O O O OH O O OH O O M M M M M M M M M M M M Partial monolayer Dense monolayer

Figure 1.12 Schematic of organosilane-based formation of SAMs on an oxide surface. The presence of water influences the surface modification by organotrimethoxysilane220.

26 R O P OH OH +

Oxide surface R O P OH R P O O O O R R O P (a) P O O O O (d) H O

(b) (c)

Figure 1.13 Schematic of possible binding modes of an alkylphosphonic acid on an oxide surface222. In the phosphonic acid moiety, up to three oxygen atoms can form chemical bonds to the surface.

1.5 Research aims

In this chapter, a general introduction to circulation tumor cells and strategies for single cell isolation, analysis and surface modification on ITO surface has been provided.

In summary, the study of circulating tumor cells is important in understanding the biology of metastasis and cancer research. To analyse single circulating tumor cells, several methods have been applied. The feature of cell isolating methods and single cell analysis methods are shown in Table 1.1 and Table 1.2 respectively. A method to selectively capture circulating tumor cells from whole blood and then directly perform chemical analysis is necessary.

27 Table 1.1 Selected features of discussed cell isolating technologies.

Technology Feature Manual or automatic Limiting diluting Limited single cell aliquot High selectivity Manual cell isolation Limited throughput Physical property-based methods High throughput (microfluidics) Limited selectivity Immuno-based methods High selectivity

Table 1.2 Information obtained by using different single cell analysis methods.

Technology Information Microscopy Cell morphology analysis Location of specific chemicals Electrochemical Cell response to stimulation analysis Cellular process Polymerase chain- Gene expression reaction analysis Mass spectrometry Chemical information such as analysis proteins or metabolites

The overarching aim of this project was to develop a tool that could be used to capture rare tumor cells from blood and obtain more molecular-level information at the single cell level than is currently possible using more conventional methods. Such an approach could ultimately lead to the development of improved methods for early cancer detection. As shown in Figure 1.14 an antibody modified ITO slide was used to capture circulating tumor cells in a mixture of blood, observed by fluorescence microscope and analysed using mass spectrometry. It is anticipated that this approach will be powerful for improving the single cell chemical analysis of rare cells that are selectively captured from complex mixtures.

28 Blood cells

Rare cells

Analysis

1) Microscopy 2) Mass spectrometry

Intens.

200 400 600 800 m/z

Figure 1.14 A schematic summarising the proposed use of an antibody-conjugated ITO surface to capture circulating tumor cells from a mixture of whole blood for integrated fluorescence microscopy and mass spectrometry analysis of single rare cells.

Specifically, the first objective is to modify an ITO coated glass slide to capture

MCF-7 cells in a mixture of whole blood. As shown in Figure 1.15, an ITO slide, which is normally used as an MALDI MS imaging target plate, will be modified with 16- phosphohexadecanoic acid (16-PHDA) then amino polyethylene glycol acid is

29 immobilised on the surface as an antifouling layer to prevent the adsorption of non- specific proteins or cells. After that, an anti-EpCAM antibody will be connected to the surface to capture MCF-7 cells (breast cancer cell line) from a mixture of whole blood and the captured cancer cells will be analysed by fluorescence microscopy. The surface characterisation of each modified surface and the cell capture performance of such antibody-conjugated surfaces will be discussed in Chapter 2.

NH HO O O O

4 4 O O

NH NH HO O O O O O 13 13 13

O O O P P P O O O O O O

ITO ITO ITO ITO

Figure 1.15 A schematic showing how a specific antibody will be modified onto an ITO coated surface to capture circulating tumor cells.

Once specific single MCF-7 cells can be captured, the next objective is to chemically analyse captured single cells by MALDI imaging MS. Different matrix deposition methods and the chemical analysis of a cell suspension and of single cells will be reported in Chapter 3.

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44 Chapter 2

Surface Modification for Capturing CTCs

Surface functionalised materials for the antibody-based capture of CTCs are a powerful way to isolate rare cells. The preparation of these materials requires extensive surface modification and characterisation to selectively capture CTCs while preventing the remaining cells from sticking onto the surface. In this chapter, the preparation of antibody-functionalised ITO surfaces and their ability to capture CTCs will be investigated and discussed. For this purpose, the ITO coated glass slides were modified with polyethylene glycol, as an antifouling layer to prevent the adsorption of non-specific cells or proteins, and specific antibodies to capture certain cells. Each surface was characterised to confirm the success of modification. To investigate the selectivity of such surfaces, lab cultured MCF-7 cells were used as a model cell line of circulating tumor cells. Given that EpCAM is overexpressed in MCF-7 cells, the corresponding antibody anti-EpCAM was used to selectively capture circulating tumor cells in a complex mixture.

As a negative control, a non-specific antibody was used. The performance of capturing specific cells were tested by a cell suspension of MCF-7 cells in cell culture media, a mixture MCF-7 cells and another cell line containing less EpCAM on the membrane surface as the contaminated cells and a mixture of MCF-7 cells in whole blood. Finally, a microscopy imaging of fluorescently labelled MCF-7 imaging was taken after cell capture, demonstrating the ability of such a surface to capture specific circulating tumor cells in a complex mixture.

2.1 Introduction

As discussed in Chapter 1.1, the detection and characterisation of extremely rare1,2 circulating tumor cells plays an important role in cancer research. CTCs have been used as a cancer biomarker in over 400 clinical studies3. The study of circulating tumor cells may help with the understanding of metastasis development in cancer patients4–6 and the real-time response to treatment7,8. Due to the low concentration of circulating tumor cells (which is about 1-10 circulating tumor cell in 1 mL of blood or ~10 billion red blood cells2), a number of technologies have been used to enrich and detect the circulating tumor cells2,9,10. Owing to the significant heterogeneity of the tumor mass being a major cause of failure in cancer prognosis and prediction11, even within the circulating tumor cells,

12 the highly heterogeneous nature should not be ignored . To achieve this, the study of circulating tumor cells at the single cell level is necessary. Although many innovative methods have been developed to capture circulating tumor cells in whole blood10,13–15, the technologies with the ability of capturing and then directly performing the chemical analysis on single circulating tumor cells are limited.

Chemical analysis at the level of a single cell can be performed using MALDI MS.

The MALDI target substrate is often ITO coated glass slides because such a substrate is transparent and conductive. Owing to the successful demonstration that well-defined monolayers can be prepared by reacting organophophosphates with ITO surfaces16–18, it should be possible to prepare an antifouling layer and immobilise an antibody on ITO surfaces to capture specific circulating tumor cells. Moreover, because ITO coated glass slides are transparent, cells captured onto the ITO surfaces can be characterised by fluorescence microscopy prior to performing mass spectrometry analysis. Thus, single

46 cell microscopy could potentially be directly integrated with MALDI MS imaging to connect fluorescence-based information to a specific chemical profile of a single cell measured by MALDI MS imaging mass spectrometry.

In this chapter, an ITO coated glass slide was modified with an antifouling layer and the anti-EpCAM antibody to capture MCF-7 cells (as a model of circulating tumor cells). The performance of the surface selectivity was tested with cultured MCF-7 cells that were fluorescently labelled in a mixture of other cells or whole blood and then observed using an optical microscope.

2.2 Materials and experimental methods

2.2.1 Materials

2.2.1.1 Chemicals and substrates

Chemicals

A list of the chemicals used in the experimental procedures of this chapter is shown in Table 2.1. All chemicals were used as received, unless otherwise specified. In addition, ultrapure water (18 MΩ·cm) was collected through a MilliporeTM water purification system and was used to prepare solutions and buffers.

Table 2.1 General chemicals, media, and solutions

Item Formula Supplier Specification or Grade

Acetonitrile CH3CN Chem-supply, AU HPLC

Quanta Amino-dPEG® -acid C H NO > 90% 4 11 23 6 BioDesign, US Quanta Amino-dPEG® -acid C H NO > 90% 8 19 39 10 BioDesign, US Dimethylformamide Sigma-Aldrich, HCON(CH ) ≥ 99% (DMF) 3 2 AU Dulbecco's Modified Gibco, US Eagle Medium (DMEM) Dulbecco's Phosphate BioPerformance Gibco, US Buffer Saline (DPBS) Certified, pH 7.4

Ethanol CH3CH2OH Chem-supply, AU Undenatured 100% 1-Ethyl-3-(3- Sigma-Aldrich, dimethylaminopropyl) C H N · HCl ≥ 97.0% 8 17 3 AU carbodiimide (EDC) Fetal bovine serum Sigma-Aldrich, GMP (FBS) AU Density 1.077 g/mL Ficoll-PaqueTM Plus Sigma-Aldrich, (20°C) density gradient media AU Lymphocyte isolation Sigma-Aldrich, 30%, semiconductor Hydrogen peroxide H O 2 2 AU grade Enrichment of Human CD 45 depletion RosetteSep, AU circulating epithelial cocktail tumor cells

Isopropyl alcohol (CH3)2CHOH Chem-supply, AU 99.50% Calcein AM LIVE/EDAD® Fisher Scientific, Ex/Em (nm): 485/530 Viability/Cytotoxicity US Ethidium Homodimer Kit Ex/Em (nm): 530/620

MES C6H13NO4S Chem-supply, AU ≥ 99%

Methanol CH3OH VWR, AU HPLC Sigma-Aldrich, N-Hydroxysuccinimide C H NO 98% 4 5 3 AU 16- Sigma-Aldrich, Phosphohexadecanoic C H O P 97% 16 33 5 AU acid (16-PHDA)

Potassium carbonate K2CO3 Chem-supply, AU 99%

Potassium ferricyanide C6FeK3N6 Chem-supply, AU ≥ 99 % C FeK N · Potassium ferrocyanide 6 4 6 Chem-supply, AU ≥ 99% 3H2O Sigma-Aldrich, Sulfuric acid H SO 98% 2 4 AU Tris(hydroxymethyl)ami Sigma-Aldrich, NH C(CH OH) ≥ 99.8% nomethane 2 2 3 AU Sigma-Aldrich, Trypan Blue solution 0.40% AU Trypsin Invitrogen, US 2.5%, 10 ×

Substrates

Indium tin oxide (ITO) coated glass slides (75 × 25 mm, 0.9 mm thickness, part

No. #237001) were obtained from Bruker, AU. All ITO surfaces were characterised to have low crystallinity and low surface roughness, which is relevant to forming high- quality monolayers by self-assembly of organophosphonic acids on surfaces.

2.2.1.2 Antibodies and cells

Fluorescently labelled Cy3 AffiniPure Donkey Anti-mouse IgG (H + L) were used as the negative control for cell capture and were purchased from Jackson

ImmunoResearch (West Grove, PA, USA). The primary antibodies, Anti-mouse [4G10]

Anti-EpCAM IgM used to capture MCF-7 cells were purchased from Abcam (Cambridge,

MA, USA). A line of cancerous cells, MCF-7, to test the surface was purchased from

CellBank Australia (Westmead, NSW, Australia). An MDA-MB-231 cell line, which expresses less EpCAM antigens on the cell surface, was used to produce ‘contaminant’ cells and was purchased from Abcam (Cambridge, MA, USA). A whole blood sample was obtained from Australian Red Cross Blood Service.

2.2.1.3 Cleaning protocols

Glassware cleaning

All glassware used in the experiment such as beakers, petri dishes, and sample tubes were cleaned by piranha cleaning for 30 min, rinsed by copious amounts of ultrapure water, dried and stored in oven at 120°C before use.

ITO substrate cleaning

ITO-coated glass slides were cleaned by ultrasonication (Unisonics, Ultrasonic cleaning, AU) in methanol for 10 min and then in 3:1 methanol:water with 0.5 M potassium carbonate for 30 mins to remove any residual organic contaminants18. Copious amounts of ultrapure water and isopropyl alcohol were then used to remove any remaining salts. Slides were dried with nitrogen and cleaned using an oxygen plasma cleaner (Plasma cleaner PDC-002, Harric plasma, US) for 10 min. All surfaces were used immediately after cleaning.

2.2.2 Experimental methods

2.2.2.1 Phosphonic acid self-assembled monolayers

After plasma cleaning, the hydrophilic ITO surfaces were ready to modify with

SAMs using the organophosphonic acid, 16-phosphohexadecanoic acid. The process for cleaning and functionalisation of the ITO surface is shown in Scheme 2.1.

Surface 2: Cleaned Surface 1: Bare ITO Surface 3: 16- (before cleaning) PHDA SAMs ITO HO O Ultrasonicate, 40 mins 16-PHDA, MeOH, 24 h 13 Plasma treatment, 10 mins 120°C, vacuum, 24 h O P OH OH O O

ITO ITO ITO

Scheme 2.1 16-phosphohexadecanoic acid SAMs on a cleaned ITO surface.

A solution of 1 mM 16-phosphohexadecanoic acid in methanol was adsorbed onto clean ITO surfaces for 24 h to form SAMs. Surfaces were rinsed with copious amounts of ultrapure water to remove unreacted chemicals, dried with nitrogen gas, and annealed in a vacuum oven (DZ-1BC II Vaacuum drying oven, Laboratory equipment, AU) at

120°C for 24 h to promote the formation of stable covalent bonding. The annealed SAMs modified surfaces were then rinsed with copious amounts of ultrapure water to remove possible multilayers and weakly bonded molecules. Subsequently, the surfaces were dried with nitrogen gas. All surfaces were prepared immediately before use.

2.2.2.2 Attachment of polyethylene glycol

After the first SAMs was formed, a polyethylene glycol layer was then attached

(Scheme 2.2) to form an antifouling layer that prevented the adsorption of non-specific proteins or cells to the surface.

Layer 4: PEG HO attachment O

n Layer 3: 16-PHDA O SAMs O O N NH HO O O O O O 13 200 mM EDC, 400 mM NHS 13 0.1 mM NH2-PEG-COOH 13 solvent, 1 h solvent, 16 h O O O P P P O O O O O O

ITO ITO ITO

Scheme 2.2 Polyethylene glycol attached onto the 16-PHDA SAMs modified surface.

Activation of terminal acid group

To attach the PEG to the 16-phosphohexadecanoic acid modified surface via a stable covalent bond, the carboxyl group of the organophosphate monolayer and the amino group were linked together by EDC/NHS coupling. Solutions of 200 mM 1-ethyl-

3-(3-dimethylaminopropyl) carbodiimide (EDC) and 400 mM N-hydroxysuccinimide

(NHS) were used to activate the terminated carboxyl acid on the surface. The whole slide was immersed into the coupling solution and the uncapped beaker was left in the dark at room temperature with frequent shaking to remove air bubbles that formed on the surfaces.

The activated ITO surface was then removed from the reaction mixture when no bubbles were observed on the surface after 20 min (usually about 1 h total) and then quickly washed with copious amounts of ultrapure water before being modified further.

Amine-reaction for PEG attachment

To avoid two or more PEG sequentially bonding on one NHS linker, the activation of carboxyl groups and the attachment of PEG was performed in multiple steps. After the

EDC/NHS coupling reaction, the activated surface was immediately cleaned and added into a solution of 0.1 mM amino polyethylene glycol acid solution and left in dark at room temperature for 16 h. The slide was then washed with copious amounts of ultrapure water to remove unreacted PEG for further modification.

2.2.2.3 Antibody-conjugation

The conjugation of antibodies to the PEG modified surface for cell capture is shown in Scheme 2.3.

Layer 4: PEG Layer 5: Antibody- attachment conjugation

O N O NH HO O O O O O

n n n O O O

NH NH NH O O O O O O 13 200 mM EDC, 13 13 400 mM HNS 1:200 (v/v) antibody in PBS, 2 h O solvent, 1 h O O P P P O O O O O O

ITO ITO ITO

Scheme 2.3 Antibody-conjugation for cell capture.

Activation of terminal acid group

A clear tape printed with a grid of squares (1 mm × 1 mm) was taped on the back side of the modified slide as a marker to aid in locating cells by microscopy. The slide was then rinsed with 70% (v/v) ethanol and placed in the Biosafety Hood in a Physical

Containment Level 2 (PC2) lab for further use. The activation of the terminated carboxyl group is the same as in Chapter 2.2.2.2.

Antibody-conjugation on activated surface

A 1:200 (v/v) antibody solution in PBS buffer was dropped onto parafilm (50

μL/cm2 ITO) and the NHS-terminated ITO surface was placed, upside down, on top of the antibody mixture. The surface was left to incubate in the dark at room temperature for

2 h before being rinsed with PBS buffer and immersed in a 25 mM tris(hydroxymethyl)aminomethane buffer for 20 min to react with the residual NHS group on the surface. The slide was then rinsed and stored in PBS buffer before cell capture.

2.2.2.4 Cell culture protocols

Cell culture media

The cell culture media consists of Dulbecco's Modified Eagle Medium (DMEM) and 10% heat-inactivated fetal bovine serum (FBS). All media was stored in fridge at 4°C and incubated in a water bath at 37°C before use.

Cell thawing

All frozen cells were stored in liquid nitrogen and transferred by dry ice box to a

37°C water bath for quick thawing. The thawed cells were then transferred to a 50 mL centrifuge tube with 10 mL cell culture media. After centrifuging at 500 × g for 5 min to remove the media, cells were then resuspended in a T25 culture flask with 5 mL cell culture media and incubated at 37°C with 5% CO2 for 3-4 days.

Cell splitting

When the cultured cells reached 80-90% of the flask (~3-4 days), the old cell culture media was removed by a 10 mL serological pipette. The cells were then washed with PBS buffer to remove the media, 1 mL Trypsin was added and incubated for about

5 min. The cells were then resuspended with 9 mL cell culture media and suspended to single cells by pipetting the media up and down for several times. After that, 1 mL cell suspension was added into a new T75 culture flask with 9 mL cell culture media for the next cell passage. To count the cells, 20 μL of the cell suspension was mixed with the same amount of Trypan blue in a microcentrifuge tube. 20 μL of the mixture was then pipetted onto a hemocytometer for counting. The average number of cells were obtained by measuring the number of cells triplicate.

2.2.2.5 Cell sample preparation and MCF-7 cells capture protocols

Cell stain

After harvesting the MCF-7 cells, a LIVE/DEAD stain dye was diluted to 1:1,000

(v/v) in an MCF-7 cell suspension containing 200,000 cells/mL. After being incubated for 30 min, the cell suspension was then centrifuged at 125 × g for 5 min and the stained cells were resuspended in the same amount of cell culture media for further use.

Preconcentration of CTCs in whole blood

After adding stained MCF-7 cells into 2 mL of whole blood cells, an enrichment cocktail for circulating tumor cells was added into the mixed sample and later incubated for 20 min to aggregate the red blood cells. The mixture was then diluted with 2% FBS in PBS buffer and carefully placed on top of the Ficoll-Paque media in a 15 mL Eppendorf tube and then centrifuged at 1,200 × g for 20 min. The enriched cells were then washed with 2% FBS in PBS buffer and centrifuged at 400 × g for 15 min (Scheme 2.4). The cell mixture was then diluted into 6 mL with cell culture media, and 3 mL of diluted cell suspension was then carefully plated into a 12-well plate with an anti-EpCAM antibody- conjugated surface for cell capture. An antifouling surface was used as a negative control.

Scheme 2.4 Pre-treatment for a mixture of blood cells. To achieve a better performance of cell capture on an antibody modified surface, red blood cells were aggregated and removed by using a pre-treatment process.

Capture of MCF-7 cells

The cell mixture was plated onto the modified surface with a 10 ml serological pipette and then incubated for 1 h at 37°C with 5% of CO2. The surface was then washed with the same amount of cell culture media 4 times and kept immersed in cell culture media for fluorescence microscopy analysis.

2.2.3 Characterisation

2.2.3.1 Contact angle goniometry

Water contact angles using ultrapure water were measured with the sessile drop method19 by a Ramé-Hart 100-00 goniometer. The imaging of a drop of ultrapure water placed onto the sample was taken by a 50 mm fixed manual focus lens. The static water

57 contact angle of the resultant images was measured by using ImageJ v1.52 (National

Institutes of Health, USA). Values reported here are the average of three measurements per substrate for three different substrates.

2.2.3.2 Electrochemical cyclic voltammetry

Electrochemical cyclic voltammetry measurements were performed using a BAS-

100 B electrochemical analyser (Bioanalytical Systems, Inc., W. Lafayette, IN). As shown in Figure 2.1, a customised polytetrafluoroethylene (PTFT) three electrode cell was used with a substrate surface as the working electrode, a platinum mesh as the counter electrode, and Ag/AgCl in 3 M sodium chloride (CH Instrument, USA) as the reference electrode. A solution of 1 mM potassium ferrocyanide and 1 mM potassium ferricyanide in PBS buffer was used as the electrolyte solution. All measured potentials (E) are reported versus the reference electrode at room temperature. Each measurement was taken in triplicate.

potentiostat

A V Counter electrode Reference Electrolyte electrode 2+ 3+ Fe , Fe

Working electrode ITO surface

Figure 2.1 A model of a typical three electrode cell for electrochemical cyclic voltammetry measurement.

2.2.3.3 X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) data was obtained using an ESCALAB

220iXL spectrometer connected a monochromatic AlKα source (1486.6 eV), hemispherical analyser and multichannel detector (6 detectors). Data were recorded in normal emission with the analysing chamber at a pressure below 10-9 mbar with a spot size around 1 mm2. The resolution of the spectrometer was ca. 0.6 eV as measured from the Ag 3d5/2 signal (full width at half maximum, FWHM). Survey scans were measured over 1100-0 eV range with a 1.0 eV step size, a 100 ms dwell time and analyser pass energy of 100 eV. High-resolution scans were run with 0.1 eV step size, dwell time of

100 ms and the analyser pass energy set to 20 eV. After background subtraction, spectra were fitted using the Avantage fitting software. All energies are reported as binding energies in eV and referenced to the C 1s signal (corrected to 285.0 eV).

2.2.3.4 Fluorescence microscopy

Fluorescence images of antibodies and cells were taken by an Olympus BX53 upright microscope equipped with TH4-200 halogen lamp and X-Cite® 120Q excitation light source. Cy3-conjugated antibodies recorded with a Cy3 filter with an excitation filter:

545/50 nm and emission filter: 610/75 nm. MCF-7 cells with LIVE/DEAD stains were recorded using a FTIC filter with an excitation filter: 470/40 nm and emission filter:

525/50 nm. All images were captured with an Olympus XM10 monochrome camera, recorded with the CellSens software (Olympus Australia, Notting Hill, Vitoria, Australia) and processed with ImageJ v.1.52 (National Institutes of Health, USA).

2.3 Results and discussion

To confirm the modification on each surface and optimise different parameters such as reaction solutions and the length of the ethylene glycol units, X-ray photoelectron spectroscopy and microscopy was performed. The characterisation methods for each layer are shown in Scheme 2.5.

Surface 5: antibody- conjugation *Microscopy Surface 4: PEG attachment *Contact angle *XPS Surface 3: 16-PHDA NH SAMs HO O *Contact angle O O *Cyclic voltammetry *XPS n n O Surface 2: Cleaned O ITO *Contact angle NH *Cyclic voltammetry NH *XPS HO O O O O O Surface 1: Bare 13 13 ITO 13 (before cleaning) O O O *Contact angle P P P OH OH O O O O O O

ITO ITO ITO ITO ITO

Scheme 2.5 Surface characterisation methods for different surfaces and parameter optimisation.

2.3.1 Contact angle goniometry

The contact angle measurement is used to describe the hydrophobic or hydrophilic behaviour of a material which provides information about the wettability of a surface.

The intrinsic value of the contact angle is usually perturbed by factors such as surface porosity roughness and heterogeneity20. If the affinity between liquid and solid is low, the value of the contact angle made by a liquid droplet deposited on a smooth surface is greater than 90° (Figure 2.2). For example, if the water contact angle is greater than 90°, the solid surface would be considered hydrophobic. If the contact angle is 0, then the liquid has spread onto the surface, otherwise known as wetting. Here, the water contact angle was observed to compare the wettability of ITO surfaces before and after cleaning, to confirm the modification of 16-PHDA SAMs and PEG attachment.

Water

Solid

Figure 2.2 Contact angle (θ) of a liquid droplet deposited on the surface of a solid.

As shown in Figure 2.3, the average contact angles of different surfaces are reported. Bare ITO surfaces as received showed a contact angle of about 86°, indicating a low affinity between water and the surface. After solvent cleaning and plasma treating, oxygen was introduced onto the surface and hydrocarbon contaminants were partial

61 removed from the surface21,22, making the surface very hydrophilic. Thus, the contact angle dropped to less than 10° on such ITO surfaces. After attaching 16-PHDA SAMs, the presence of straight-chain alkanes on the surface resulted in an increase in the contact angle to 48°, compared with the cleaned ITO surface. Thus, before modification, clean

ITO surfaces were observed to be hydrophilic while after the SAMs modification, the

ITO surfaces became more hydrophobic. After attaching polyethylene glycol, the water contact angle decreased slightly or stayed about the same (42°) compared with the 16-

PHDA SAMs layer (48°).

(b) Surface 1: Bare ITO (c) Surface 2: Cleaned (a) 100 83.8 (before cleaning) ITO

) ° 48.0 60 42.7 40

(d) Surface 3: 16-PHDA (e) Surface 3: PEG Contact Angle ( Angle Contact 20 SAMs attachment 3.4 0

(a) 1 (b) 2 (c) 3 (d) 4 er er er er ay ay ay ay L L L L

Figure 2.3 Water contact angle measurements of different layers on ITO surfaces. (a) Contact angle measurements of different surfaces; (b) Bare ITO surfaces before cleaning; (c) ITO surfaces after plasma treatment; (d) 16-phosphohexadecanoic acid modified ITO surfaces; and (e) PEG attached surface. Error bar correspond to ±1 standard deviation.

2.3.2. Cyclic voltammetry measurements

Electrochemical cyclic voltammetry is a powerful electroanalytical method to investigate the effects of surface chemistry on reduction and oxidation processes23. In

62 electrochemical cyclic voltammetry, a triangular potential waveform was applied linearly over time. As a result, a redox active chemical can be oxidised or reduced on the electrode surfaces24. For example, in a typical three electrode cell, the ITO surface is used as the working electrode which can be controlled vs a referent electrode (Ag/AgCl). As the controlling potential changed, the response current at the working electrode can be plotted as a cyclic voltammogram, which can be regarded as the electrochemical equivalent to the spectra in optical spectroscopy25–27. During the potential scanning, if the positive potential is strong enough to oxidise ferrocyanide, the reaction occurs on the surface:

-4 -3 - (1) Fe(CN)6 Fe(CN)6 + e

Thus, the anodic current appears and the oxidation current increases to a peak. As the reverse scan switches to negative, the anodic current continues for a while when the potential is still strong enough to oxidise ferrocyanide. When the electrode becomes a strong reductant, ferricyanide will be reduced by the electrode process:

-4 -3 - (2) Fe(CN)6 Fe(CN)6 + e

resulting in a cathodic current peak. Here, electrochemical cyclic voltammetry was conducted on the cleaned ITO surface and the 16-PHDA SAMs modified ITO surfaces to confirm the formation of the first modified layer.

The results of cyclic voltammetry on clean ITO and a SAM modified ITO surface are shown in Figure 2.4. The black curve is a scan of the cleaned ITO surface. The sharp redox peaks indicate easy access of the redox probe to the electrode surface, which gives

63 evidence that the ITO surface was clean and unmodified. The red curves correspond to a cyclic voltammogram of an ITO surface that was modified with 16-PHDA. As the 16-

PHDA monolayer is on the surface, the oxidisation/reduction reaction on the ITO surface was hindered by the monolayer, resulting in the redox peaks in the curve decreasing significantly. This indicated the formation of the modified layer to restrict the access of the redox probe to the electrode surface.

40 Surface 2: Cleaned ITO

20 A)

μ Surface 3: 16-PHDA 0 Current Current ( -20

-40

-200 0 200 400 600 800

Potential (mV vs Ag/AgCl)

Figure 2.4 Cyclic voltammograms of ITO surfaces before and after modification. The measurement was recorded in PBS buffer containing 1 mM potassium ferrocyanide and 1 mM potassium ferricyanide at the scan rate of 100 mV/s for cleaned ITO surface (black line) and 16-phosphohexadecanoic acid modified ITO surfaces (red line), indicating a well-formed modified layer on the ITO surfaces.

2.3.3 X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy is a surface sensitive technique used to analyse surface chemical composition and bonding. In XPS analysis, the sample is irradiated with an X-ray beam and the number of electrons that escape are obtained as a function of their

64 kinetic energy to obtain an XPS spectra28. In an XPS spectra, the peak positions and the intensities are related to the elemental composition and their quantities on the surface respectively. Here, XPS was performed to further understand the different ITO modification layers and optimise the EDC/NHS coupling solution and the length of ethylene glycol units.

ITO modification process

Bare ITO after cleaning was analysed by XPS (Figure 2.5), which resulted in peaks at electron binding energy values of 445, 455, 487, 495 and 530 eV, which corresponds to In 3d5/2, In 3d3/2, Sn 3d5/2, Sn 3d3/2 and O 1s, respectively29,30. The peak at ca. 285 eV is the carbon C 1s and is attributed to adventitious organic contamination. The XPS analysis of an ITO surface that was modified with 16- phosphohexadecanoic acid through self-assembly confirmed that this process was successful. For example, a P 2p XPS peak at ca. 133 eV is attributed to the phosphonic acid and the ratio of C 1s to In 3d5/2 provided further evidence of surface modification

(Figure 2.5 (b)). Figure 2.5 (c) illustrates the XPS spectrum of the PEGylated layer on

ITO. This spectrum is similar to Figure 2.5 (b) containing 16-PHDA SAMs layer, as both contain characteristic peaks of ITO and P 2p peak at ca. 133 eV. In addition, the presence of amines on the surface was indicated by the N 1s peak at ca. 399.5 eV.

(a) Surface 2: Cleaned ITO 6 1.48 × 10 In 3d N 1s P 2p 4000 7000

O 1s Counts/s

Sn 3d C 1s 0 3500 4600 600 400 200 404 396 136 128

(b) Surface 3: 16-PHDA SAMs 5 6 × 10

In 3d N 1s P 2p O 1s 5700 7000

C 1s Counts/s Sn 3d

0 5000 4500 600 400 200 404 396 136 128 (c) Surface 4: PEG attachment

6 1.0 × 10 O 1s

N 1s P 2p In 3d 8000

6400 C 1s Counts/s Sn 3d

4800 0 5500 600 400 200 404 396 136 128 Binding Energy (eV)

Figure 2.5 X-ray photoelectron survey spectra. Core level spectra of phosphorus 2p and nitrogen 1s of (a) cleaned ITO surfaces; (b) 16-phosphohexadecanoic acid modified ITO surfaces; and (c) PEGylated surface.

Surface optimisation

As previously discussed in Chapter 2.2.2.2, the EDC/NHS coupling was performed to conjugate the amino group in PEG onto the carboxyl terminated surface

(Scheme 2.6). NHS was used to prepare amine-reactive esters of the carboxylate group for crosslinking. The choice of a suitable solvent for EDC/NHS activation is important because NHS can hydrolyse within hours or minutes depending on water-content and the pH of the reaction solutions.

CH3 R N O R N C H CH3 CH3 H3C N + O O N N HO EDC C H CH3 H3C N + H Carboxylic acid O-acylisourea O intermediate N HO O 1 R O NHS O 1 H2N O R Amine compound N R N R O H O Stable amide Semi-stable amine- reactive NHS ester

Scheme 2.6 Reaction of EDC/NHS activation for carboxyl groups31.

In order to choose a suitable solvent, XPS was used to observe the bonding and fluorescence microscopy was used to observe the attachment of fluorescently labelled antibodies. It was found that all the solvents (shown in Table 2.2) tested allowed the attachment of antibodies, without significant differences in the XPS and fluorescence

67 results. To provide the best pH for EDC/NHS activation, the solvents used in following experiments were 50 mM MES buffer and PBS buffer.

Table 2.2 Solvents for EDC/NHS activation and amine compound attachment.

Solvent 2 Solvent 1 (for amine compound (for EDC/NHS activation) attachment) Ultrapure water Acetonitrile Ultrapure water Dimethylformamide Ultrapure water Ultrapure water 50 mM MES buffer 50 mM MES buffer 50 mM MES buffer PBS buffer

To optimise the antifouling properties, two PEG linkers of different chain length

(4 units and 8 units) were used. XPS confirmed that both PEG linkers were attached to the surfaces with approximately similar surface coverage based on relative abundances of increasing C 1s peaks. However, when comparing the cell capture results, PEG8 prevented more non-specific cell adsorption than PEG4. For all subsequent experiments, the PEG8 acid was used.

2.3.4 Fluorescence microscopy

In order to determine the success of the bioconjugation step via the presence of antibodies on the surfaces, a fluorescently tagged antibody, Cy3-conjugated donkey anti- mouse IgG antibody was used. For cell capture experiments, specific antibodies can be used to replace the fluorescently tagged antibody to selectively capture the cells.

Since the conjugation of antibodies was performed by EDC/NHS coupling, a negative control was prepared that consisted of the antifouling surface without EDC/NHS to confirm the chemical bonding of antibodies on the surfaces. As shown in Figure 2.6, the PEG attached surface (b) and the antibody attached to the surface without carboxyl group activation (c), both had a similar brightness. While with the EDC/NHS activation surface (d), the much higher fluorescence intensity was observed than the intensity of the others. The similar fluorescence between the background and the negative control suggests that the amino PEG8 acid was suitable to be used as an antifouling surface coating towards the physisorption of smaller proteins and antibody solution.

(a) (b) Surface 4: PEG attachment (c) Surface 4: PEG attachment + antibody (without activation)

2 Fluorescence intensity/mm 4

(d) Surface 5: antibody- 1 conjugation

With activation EDC/NHS activation

Figure 2.6 Fluorescence images of antibody conjugation on modified ITO surfaces. (a) Fluorescence intensities calculated of different surfaces; (b) activated PEG attached surface without antibodies; (c) PEG attached surface without activation and then immersed into a fluorescently tagged antibody solution; and (d) activated PEGylated surface immersed into a fluorescently tagged antibody solution. Images were recorded with a 10 × objective, 200 ms exposure and a gain factor of 18 dB. Scale is the same for all images (200 μm). Error bar correspond to ±1 standard deviation.

2.3.5 Cell capture results

As discussed in Chapter 1.2, several methods can be used to isolate single cells.

However, to selectively capture single cells from a complex mixture, specific antibodies were used. Here, MCF-7 cells, which is a cell line of breast cancer cells, was captured with anti-EpCAM antibodies for further analysis. To confirm the capture of target cells,

MCF-7 cells were labelled with a FTIC-tagged LIVE/DEAD stain and then observed using a fluorescence microscope.

Capture of MCF-7 cells from culture media

To confirm the performance of the antifouling coating and the selectivity for cell capture, a series of different modified surfaces were used to capture MCF-7 cells from cell culture media. As described in Scheme 2.5, a PEG-attached surface was used as the base surface to conjugate specific antibodies in order to capture MCF-7 cells. The attachment of a non-specific antibodies was used as a negative control to illustrate the specificity of the surface.

As shown in Figure 2.7, the different performance between the PEGylated surface

(c) and the non-PEGylated surface (d) shows that the PEGylated layer provided sufficient antifouling properties to prevent non-specific adsorption. However, after anti-EpCAM antibodies were conjugated on top of the surface, the cell density increased by more than

10 times (Figure 2.7 (a)) than the antifouling surface, which shows the ability of the anti-

EpCAM antibody to capture EpCAM-rich MCF-7 cells. To ensure that this increase in cell density observed in Figure 2.7 (a) was due to the specific interaction between

EpCAM on the cell membrane and the anti-EpCAM antibodies on the surface, a non- specific donkey anti-mouse IgG antibody was conjugated to the PEGylated surface as a

70 negative control. Figure 2.7 (b) shows that MCF-7 levels of these surfaces are similar to that of Figure 2.7 (c), suggesting that the increase in cell-density by use of the anti-

EpCAM modified surface does not result from non-specific interactions between the antibodies and cells. A surface without PEG attachment (Figure 2.7 (e)) was also used as a control, suggesting the cells may be captured by non-specific interactions with surfaces that do not have an antifouling layer attached.

(a) PEG attachment (b) PEG attachment (c) PEG attachment + anti-EpCAM antibody + anti-mouse IgG antibody No antibody + MCF-7 cells + MCF-7 cells + MCF-7 cells

(f) 2 (d) No PEG (e) No PEG Number of cells/mm + anti-EpCAM antibody No antibody + MCF-7 cells + MCF-7 cells 1200

800

400

0 (a) (b) (c) (d) (e)

Figure 2.7 Fluorescence images of live cells (25,000 cells/mL) plated on different modified surfaces in cell culture media for 30 min. (a) MCF-7 cells on an anti- EpCAM antibody modified surface; (b) MCF-7 cells on a surface analogous to surface (a), except replacing anti-EpCAM with a non-specific donkey anti-mouse IgG antibody; (c) MCF-7 cells on a PEG attached layer with no antibody; (d) MCF-7 cells on an anti- EpCAM conjugated 16-PHDA SAMs surface; (e) MCF-7 cells on a 16-PHDA modified with no antibody; and (f) numbers of cells calculated on different surfaces. Images were recorded with a 4 × objective, 200 ms exposure time and a gain factor of 18 dB. Scale bar is same for all images (500 μm). Error bar correspond to ±1 standard deviation.

Capture of MCF-7 cells in a mixture containing MDA-MB-231 cells

To further understand the selectivity of anti-EpCAM antibodies to capture MFC-

7 cells from a complex mixture, an EpCAM-deficient type of cells (MDA-MB-231) was used, which is also a breast-cancer cell line. As shown in Figure 2.8, a cell mixture contain different ratios of two cells were used. MCF-7 cells were labelled with a FITC- tag before mixing. By use of a mixture of 10% MCF-7 cells and 90% MDA-MB-231 cells, the capture of the EpCAM-rich MCF-7 cells was more than two times higher than the ‘contaminant’ cells which shows that this surface has the ability to selectively capture

MCF-7 cells out of a sample containing both type of cells.

(b) 100% MCF-7 cells (c) 50% MCF-7 cells (a) 2 Number of cells/ mm

900 MCF-7 MDA-MB-231

600

(d) 10% MCF-7 cells (e) 100% MDA-MB-231 cells 300

0 100% 50% 10% 0 Percentage of MCF-7 cells

Figure 2.8 Images of a mixture of MCF-7 cells and MDA-MB-231 cells (total cell: 25,000 cells/mL) plated on anti-EpCAM conjugated surfaces for 30 mins. (a) Numbers of cells calculated on different surfaces; (b) 100% of MCF-7 cells; (c) 50% of MCF-7 cells; (d) 10% of MCF-7 cells; and (e) 100% of MDA-MB-231 cells. Images were recorded with a 4 × objective and a gain factor of 18 dB. 200 ms exposure time and 2 ms exposure time were used for fluorescence images brightfield images respectively. All images were processed with ImageJ. The brightfield images shows all these two cells and the green fluorescence images shows the labelled MCF-7 cells. Scale bar is same for all images (500 μm). Error bar correspond to ±1 standard deviation.

Capture of MCF-7 cells from whole blood

As a proof of concept that such anti-EpCAM antibody modified surfaces can be used to capture circulating tumor cells from whole blood, a series of different amounts of fluorescein labelled MCF-7 cells were added to whole blood. After a pre-treatment step to concentrate the circulating tumor cells, a cell suspension was plated onto an anti-

EpCAM antibody conjugated surface and an antifouling surface as a negative control. As shown in Figure 2.9, in each set of surfaces, the antibody modified surfaces captured about 10 times more MCF-7 cells than the antifouling surfaces. For 800 MCF-7 cells added to 2 mL of whole blood, which corresponds to a ratio of 1 cancer cell in 10 million red blood cells, some MCF-7 cells could still be observed on the surface of the anti-

EpCAM antibody modified surface while fewer were detected on the control surface.

These data indicate that such surfaces perform well for selectively capturing specific circulating tumor cells from a complex mixture, such as whole blood.

5 4 3 (a) 8 × 10 MCF-7 cells (b) 8 × 10 MCF-7 cells (c) 8 × 10 MCF-7 cells in 2 mL whole blood in 2 mL whole blood in 2 mL whole blood with anti-EpCAM antibody (top) with anti-EpCAM antibody (top) with anti-EpCAM antibody (top) and on an antifouling surface and on an antifouling surface and on an antifouling surface

(d) 800 MCF-7 cells (e) 2 in 2 mL whole blood Number of cells/mm with anti-EpCAM antibody (top) and on an antifouling surface 1000 With anti-EpCAM antibodies No antibody (antifouling surfaces)

100

1 in 10,000 1 in 100,000 1 in 1,000,000 1 in 10,000,000 Numbers of MCF-7 cells in blood cells

Figure 2.9 Images of a mixture of MCF-7 cells whole blood plated onto anti- EpCAM conjugated surfaces and antifouling surfaces for 30 mins. (a) About 8 × 105 MCF-7 cells added into 2 mL of whole blood; (b) 8 × 104 MCF-7 cells added into 2 mL of whole blood; (c) 8 × 103 MCF-7 cells added into 2 mL of whole blood; (d) 800 MCF-7 cells added into 2 mL of whole blood; and (e) number of cells calculated on different surfaces plotted on a logarithmic scale. Images were recorded with a 4 × objective, 200 ms exposure time and a gain factor of 18 dB. Scar bar is same for all images (500 μm). Error bar correspond to ±1 standard deviation.

2.4 Conclusions

Here, an ITO coated glass slide for was modified with 16-PHDA SAMs, an antifouling layer and anti-EpCAM antibodies. The formation of such as characterised by using contact angle goniometry, electrochemical cyclic voltammetry, XPS measurement and fluorescence microscopy analysis. All characterisation results showed the successful formation of each layer. The performance of the surface for antifouling properties were investigated by capturing MCF-7 cells using an antifouling surface, an anti-EpCAM conjugated surface and a non-specific antibody conjugated surface. The cell capture results suggested that the antifouling layer was able to block at least 90% of non-specific cell attachment under these conditions. To confirm the selectivity of the antibody conjugated surface, a breast cancer cell line which express less EpCAM antigen on the surface was used to mix with MCF-7 cells. By using anti-EpCAM antibody, a modified surface was able to capture more than 2 times more MCF-7 cells in a mixture containing

10% of MCF-7 cells an antifouling surface, which indicated that SAMs can be formed that are highly selective for MCF-7 cells. To further investigate the selectivity and evaluate the performance of such modified surfaces for capturing CTCs in blood, different amounts of fluorescently tagged MCF-7 cells were added into whole blood. The anti-EpCAM modified surface can be used to capture 800 MCF-7 cells in 2 mL of whole, which corresponds to 1 CTC in 10,000,000 red blood cells. In addition, the anti-EpCAM antibody conjugated surface was able to capture about 10 times (2.6-10.8 times) more

MCF-7 cells in each condition compared to an antifouling surface that does not have antibodies immobilised on the surface.

In conclusion, these results indicated that all proposed layers were well-formed on the ITO coated surface and the modified surface showed good performance for cell capture selectivity in a complex whole blood mixture. Such a surface that can selectively capture CTCs should be useful for performing chemical analysis by MALDI-MS on single CTC cells directly captured from blood.

2.5 References

(1) Ming, Y.; Li, Y.; Xing, H.; Luo, M.; Li, Z.; Chen, J.; Mo, J.; Shi, S. Circulating Tumor Cells: From Theory to Nanotechnology-Based Detection. Front. Pharmacol. 2017, 8, 35. (2) Bankó, P.; Lee, S. Y.; Nagygyörgy, V.; Zrínyi, M.; Chae, C. H.; Cho, D. H.; Telekes, A. Technologies for Circulating Tumor Cell Separation from Whole Blood. J. Hematol. Oncol. 2019, 12 (1), 48. (3) Alix-Panabières, C.; Pantel, K. Circulating Tumor Cells: Liquid Biopsy of Cancer. Clin. Chem. 2013, 59 (1), 110-118. (4) Micalizzi, D. S.; Maheswaran, S.; Haber, D. A. A Conduit to Metastasis: Circulating Tumor Cell Biology. Genes Dev. 2017, 31 (18), 1827-1840. (5) Aceto, N.; Bardia, A.; Miyamoto, D. T.; Donaldson, M. C.; Wittner, B. S.; Spencer, J. A.; Yu, M.; Pely, A.; Engstrom, A.; Zhu, H.; et al. Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis. Cell 2014, 158 (5), 1110-1122. (6) Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W. M. M.; et al. Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. N. Engl. J. Med. 2004, 351 (8), 781-791. (7) Cohen, S. J.; Punt, C. J. A.; Iannotti, N.; Saidman, B. H.; Sabbath, K. D.; Gabrail, N. Y.; Picus, J.; Morse, M.; Mitchell, E.; Miller, M. C.; et al. Relationship of Circulating Tumor Cells to Tumor Response, Progression-Free Survival, and Overall Survival in Patients with Metastatic Colorectal Cancer. J. Clin. Oncol. 2008, 26 (19), 3213-3221. (8) Alix-Panabières, C.; Vendrell, J.-P.; Pellé, O.; Rebillard, X.; Riethdorf, S.; Müller, V.; Fabbro, M.; Pantel, K. Detection and Characterization of Putative Metastatic Precursor Cells in Cancer Patients. Clin. Chem. 2007, 53 (3), 537-539. (9) Yu, M.; Stott, S.; Toner, M.; Maheswaran, S.; Haber, D. A. Circulating Tumor Cells: Approaches to Isolation and Characterization. J. Cell Biol. 2011, 192 (3), 373-382. (10) Nagrath, S.; Sequist, L. V.; Maheswaran, S.; Bell, D. W.; Irimia, D.; Ulkus, L.; Smith, M. R.; Kwak, E. L.; Digumarthy, S.; Muzikansky, A.; et al. Isolation of Rare Circulating Tumour Cells in Cancer Patients by Microchip Technology. Nature 2007, 450 (7173), 1235-1239. (11) Tellez-Gabriel, M.; Heymann, M.-F.; Heymann, D. Circulating Tumor Cells as a Tool for Assessing Tumor Heterogeneity. Theranostics 2019, 9 (16), 4580-4594. (12) Keller, L.; Pantel, K. Unravelling Tumour Heterogeneity by Single-Cell Profiling of Circulating Tumour Cells. Nat. Rev. Cancer 2019. (13) Adams, A. A.; Okagbare, P. I.; Feng, J.; Hupert, M. L.; Patterson, D.; Götten, J.; McCarley, R. L.; Nikitopoulos, D.; Murphy, M. C.; Soper, S. A. Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor. J. Am. Chem. Soc. 2008, 130 (27), 8633-8641. (14) Gleghorn, J. P.; Pratt, E. D.; Denning, D.; Liu, H.; Bander, N. H.; Tagawa, S. T.; Nanus, D. M.; Giannakakou, P. A.; Kirby, B. J. Capture of Circulating Tumor Cells from Whole Blood of Prostate Cancer Patients Using Geometrically Enhanced Differential Immunocapture (GEDI) and a Prostate-Specific Antibody. Lab Chip 2010, 10 (1), 27-29. (15) Coumans, F. A. W.; van Dalum, G.; Beck, M.; Terstappen, L. W. M. M. Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood. PLoS One 2013, 8 (4), e61770. (16) Lu, X.; Nicovich, P. R.; Zhao, M.; Nieves, D. J.; Mollazade, M.; Vivekchand, S. R. C.; Gaus, K.; Gooding, J. J. Monolayer Surface Chemistry Enables 2-Colour Single Molecule Localisation Microscopy of Adhesive Ligands and Adhesion Proteins. Nat. Commun. 2018, 9 (1), 3320. (17) Paniagua, S. A.; Hotchkiss, P. J.; Jones, S. C.; Marder, S. R.; Mudalige, A.; Marrikar, F. S.; Pemberton, J. E.; Armstrong, N. R. Phosphonic Acid Modification of Indium-Tin Oxide Electrodes: Combined

XPS/UPS/ Contact Angle Studies. J. Phys. Chem. C 2008, 112 (21), 7809-7817. (18) Chockalingam, M.; Darwish, N.; Le Saux, G.; Gooding, J. J. Importance of the Indium Tin Oxide Substrate on the Quality of Self-Assembled Monolayers Formed from Organophosphonic Acids. Langmuir 2011, 27 (6), 2545-2552. (19) Kingery, W. D.; Humenik, M. Surface Tension at Elevated Temperatures. I. Furnace and Method for Use of the Sessile Drop Method; Surface Tension of Silicon, Iron and Nickel. J. Phys. Chem. 1953, 57 (3), 359-363. (20) Drioli, E.; Criscuoli, A.; Curcio, E. Chapter 2. Membrane Materials. In Membrane Science and Technology; 2005, 11. (21) Yu, S.Y.; Chang, J.H.; Wang, P.S.; Wu, C.I.; Tao, Y.T. Effect of ITO Surface Modification on the OLED Device Lifetime. Langmuir 2014, 30 (25), 7369-7376. (22) You, Z. Z.; Dong, J. Y. Oxygen Plasma Treatment Effects of Indium-Tin Oxide in Organic Light- Emitting Devices. Vacuum 2007, 81 (7), 819-825. (23) Daubinger, P.; Kieninger, J.; Unmüssig, T.; Urban, G. A. Electrochemical Characteristics of Nanostructured Platinum Electrodes - a Cyclic Voltammetry Study. Phys. Chem. Chem. Phys. 2014, 16 (18), 8392-8399. (24) Sandford, C.; Edwards, M. A.; Klunder, K. J.; Hickey, D. P.; Li, M.; Barman, K.; Sigman, M. S.; White, H. S.; Minteer, S. D. A Synthetic Chemist’s Guide to Electroanalytical Tools for Studying Reaction Mechanisms. Chem. Sci. 2019, 10 (26), 6404-6422. (25) Mabbott, G. A. An Introduction to Cyclic Voltammetry. J. Chem. Educ. 1983, 60 (9), 697. (26) Nicholson, R. S. Theory and Application of Cyclic Voltammetry for Measurement of Electrode Reaction Kinetics. Anal. Chem. 1965, 37 (11), 1351-1355. (27) Wipf, D. O.; Kristensen, E. W.; Deakin, M. R.; Wightman, R. M. Fast-Scan Cyclic Voltammetry as a Method to Measure Rapid, Heterogeneous Electron-Transfer Kinetics. Anal. Chem. 1988, 60 (4), 306-310. (28) Yang, D.; Velamakanni, A.; Bozoklu, G.; Park, S.; Stoller, M.; Piner, R. D.; Stankovich, S.; Jung, I.; Field, D. A.; Ventrice, C. A. Chemical Analysis of Graphene Oxide Films after Heat and Chemical Treatments by X-Ray Photoelectron and Micro-Raman Spectroscopy. Carbon N. Y. 2009, 47 (1), 145-152. (29) Lin, A. W. C.; Armstrong, N. R.; Kuwana, T. X-Ray Photoelectron/Auger Electron Spectroscopic Studies of Tin and Indium Metal Foils and Oxides. Anal. Chem. 1977, 49 (8), 1228-1235. (30) Fan, J. C. C.; Goodenough, J. B. X-Ray Photoemission Spectroscopy Studies of Sn-Doped Indium- Oxide Films. J. Appl. Phys. 1977, 48, 3524. (31) Hermanson, G. T. The Reactions of Bioconjugation. In Bioconjugate Techniques; Elsevier, 2013, 229- 258.

Chapter 3

MALDI Mass Spectrometry Analysis

After capturing specific MCF-7 cells from a mixture of blood cells (see Chapter

2), the isolated single cells were then directly characterised by using matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In this chapter, MALDI-MS imaging was performed to analyse molecular metabolite ions formed from single cells, particularly for lipids of the phosphatidylcholine class. To optimise the performance of

MALDI-MS, different matrix deposition methods were compared. After obtaining

MALDI imaging results, optical images were captured to aid in locating cells.

3.1 Introduction

As discussed in Chapter 1.3, by using mass spectrometry analysis, more chemical information can typically be obtained from a complex mixture than with other chemical analysis methods. Mass spectrometry can be used in single cell analysis to study heterogeneity in cell populations without external labels and with excellent molecular sensitivity and specificity. Compared with secondary ion mass spectrometry1,2, ‘soft’ ionisation mass spectrometry has been used to detect lipids and metabolites in single cells to avoid fragments. For example, in liquid extraction surface analysis mass spectrometry, a small droplet of solvent can be used to soluble analytes at solid-liquid interface3. Such

79 system can generate a stable, prolonged electrospray of up to 15 min from a single-cell extract, which significantly improved sensitivity and reproducibility4. The direct analysis can also shorten preparation and analysis time while minimising the processing of artefact peaks5,6. While in a traditional liquid extraction surface analysis, the minimum pixel size is limited by the diameter of the solvent droplet, ~ 1 mm7, which is larger than the average size of single cancer cells (with the diameter of ~ 20 μm). Besides, the sensitivity of such analysis might dependent on the analyte and be affected by ion suppression of co- extracted matrix molecules8. Thus, liquid extraction surface analysis can be used as a verification step to complement the MALDI imaging data9,10. With the development of laser optics and improved matrix deposition methods in recent years, a spatial resolution of 10 μm can be obtained by use of a commercial MALDI-MS instrument which can be used for single cell analysis11. MALDI mass spectrometry has become a powerful technique in a large number of single cell analysis12–15. By using MALDI imaging, lipid expressions in healthy and diseased cells could be compared, and most importantly, the responding upon chemical stimulation (e.g. drug treatment) could be observed, which may be missed using a population-level technique16,17.

In this project, the mass spectra of single circulating tumor cells should be possible to obtained immediately after cell capture from blood. In addition, owing to transparency of ITO coated glass slides, the results of MALDI imaging can be directly connected with optical imaging to obtain additional information from a single cell, such as the relative concentration of an overexpressed cancer biomarker on the surface of a cell (e.g. carbonic anhydrase IX), the relative cellular uptake of a fluorescent drug molecule or nanoparticle drug delivery system.

In this chapter, captured MCF-7 cells were performed using mass spectrometry analysis by MALDI-TOF imaging and were integrated with optical imaging. Different matrix deposition methods were applied to improve the imaging results. For single cell mass spectra, MALDI-TOF/TOF was performed to aid in ion identification.

3.2 Materials and experimental methods

3.2.1 Materials

3.2.1.1 Chemicals and MALDI targets

Chemicals

A list of the chemicals used in the experimental procedures of this chapter is presented in Table 3.1. All chemicals were used as received, unless otherwise specified.

TM Ultrapure water (18 MΩ ·cm) was collected through a Millipore water purification system and was used to prepare solutions and buffers.

Table 3.1 General chemicals

Item Formula Supplier Specification or Grade

Acetonitrile CH3CN Chem-supply, AU HPLC

9-Aminoacridine C13H10N2 Sigma-Aldrich, AU HPLC α-Cyano-4- hydroxycinnamic acid C10H7NO3 Sigma-Aldrich, AU HPLC (HCCA) 2,5-Dihydroxybenzoic C H O Sigma-Aldrich, AU HPLC acid (DHB) 7 6 4 Dulbecco's Phosphate BioPerformance Gibco, US Buffer Saline (DPBS) Certified, pH 7.4

30%, semiconductor Hydrogen peroxide H O Sigma-Aldrich, AU 2 2 grade

Isopropyl alcohol (CH3)2CHOH Chem-supply, AU 99.50%

Sulfuric acid H2SO4 Sigma-Aldrich, AU 98%

Trifluoroacetic acid CF3CO2H Sigma-Aldrich, AU 99%

Solvents and matrix solutions

The MALDI matrix solutions were prepared by dissolving HCCA (7 mg/mL) or

DHB (30 mg/mL) in TA 30 solvent (30:70 (v/v) acetonitrile:water with 0.1% trifluoroacetic acid). All matrix solutions were sonicated for 30 min and filtrated using

0.22 μm syringe filters. These solutions were then used to prepare surfaces for MALDI analysis as described below. All matrix solutions were stored at 4°C for no longer than one month.

MALDI targets

A MALDI target plate holder (MTP 384; ground steel BC; part No. # 8280784),

ITO coated glass slides (75 × 25 mm, 0.9 mm thickness, Part number: #237001) and an

MTP slide adapter II (part No. 235380) were all obtained from Bruker (Sydney, AU).

Cells, proteins and lipids

MCF-7 cell lines were purchased from CellBank Australia (Westmead, NSW,

Australia). The calibrant peptides (Part No. 8222570) were obtained from Bruker. The peptides and their mass to charge ratio is shown in Table 3.2 and a MALDI-MS spectrum of the calibrants is shown in Figure 3.1. The standard lipids, 1,2-dimyristoyl-sn-glycero-

3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were obtained from Avanti Polar Lipids (Alabaster, AL).

Table 3.2 Mass calibrants for MALDI-TOF mass spectrometry18

Peptide [M+H]+ Monoisotopic [M+H]+ Average Bradykinin 1-7 757.3992 757.86 Angiotensin II 1046.5418 1047.19 Angiotensin I 1296.6848 1297.49 Substance P 1347.7354 1348.64 Bombesin 1619.8223 1620.86 ACTH clip 1-17 2093.0862 2094.43 ACTH clip 18-39 2465.1983 2466.68 Somatostatin 28 3147.471 3149.57

Inten. [a.u.] 1619.892

5 2094.188 2.5 × 10

5 2 × 10 1347.797 2466.308

5 1.5 × 10 1298.784

5 1 × 10 1046.591 4 5 × 10 3149.605 757.455 0 500 1000 1500 2000 2500 3000 3500

m/z

Figure 3.1 MALDI mass spectrum of calibrant mixture of standard peptides listed in Table 3.2. The peptide ions were formed from an ITO coated glass slide using HCCA as a matrix.

3.2.1.2 Cleaning protocols

All glassware used during the experiment such as beakers, petri dishes, and sample tubes were cleaned by piranha cleaning for 30 min then rinsed by copious amounts of ultrapure water and dried and stored in oven at 120°C before use. The MTP 384 target

83 plate was cleaned with 2-propanol to remove any matrix/sample spots and then submerged in 2-propanol and TA 30 and sonicated for 10 min. The MALDI target plate was then dried using a steam of high-purity nitrogen. The cleaning protocol of ITO coated glass slide for MALDI imaging is the same as that described in Chapter 2.2.1.3.

3.2.2 Equipment and methods

In MALDI-TOF imaging mass spectrometry, a small matrix molecule, which can absorb laser energy to aid in the formation of analyte ions, is typically deposited on the target surface at relatively high concentrations. Here, different matrix deposition methods were compared to determine the optimal conditions for obtaining high quality spectra.

3.2.2.1 Sample preparation

For MALDI MS of standards for instrument calibration, an MTP 384 target plate and sample was deposited on the plate by use of the dried droplet method as described in full in Chapter 3.2.2.2. For MALDI imaging performed on an ITO coated glass slide, calibration samples, standard lipids or cell suspensions were spotted on top of the ITO surface. Single cell samples were obtained as described in Chapter 2.2.2.2. All slides were dehydrated in a vacuum desiccator for at least 15 min and optical images were obtained by using an optical microscope (Nikon Super Coolscan 4000 ED, Nikon, AU) before the matrix was applied and used for MALDI MS.

3.2.2.2 Matrix deposition methods

As described in Chapter 1.3.4.4, a number of different methods have been developed for the preparation of samples and matrices for MALDI imaging MS. Here, three different approaches were used that are described below.

Dried droplet approach

For standards, the sample solution and matrix solution were mixed in 1:1 (v/v) ratio for 5 min. The matrix/analyte mixture (0.5 µL) was then deposited onto the target spot and allowed to dry in a vacuum desiccator. For single cell samples from Chapter

2.2.2, 0.5 µL matrix was applied on top of the cells directly and allowed to dry.

Commercial matrix sprayer

ImagePrep from Bruker Daltonics was used to spray matrix solutions onto ITO coated glass slides for MALDI-MS. A diagram of the ImagePrep system is shown in

Figure 3.2. An inverted solution reservoir that contains the matrix solution is in contact with a porous metal membrane sprayer head. The membrane can vibrate by application of a low voltage to a piezoelectric device to facilitate the generation of an aerosol of the matrix solution which is used to coat a surface with the matrix solution. That is, a fine mist can be deposited onto ITO coated glass slides.

Spray head

ITO slide

Matrix solution

Optical sensor

Figure 3.2 ImagePrep for matrix deposition on an ITO coated glass slide for MALDI imaging. A matrix solution was filled into the solution reservoir and the matrix solution to the ITO slide through a mesh on the metal spray head. A light sensor below the ITO slide was used to monitor the formation of matrix crystals on the surface.

As shown in Figure 3.2, an optical sensor was connected to monitor the amount of light scattered from the crystalline matrix layer, allowing the monitoring of the relative thickness of the matrix layer in real-time. For example, if a new layer crystallises, the density of the matrix crystals increases, thus more light will scatter. As a result, sensor voltage plateaus at a higher level than normal. Figure 3.3 shows a complete light scatter curve for all five phases of a typical HCCA surface preparation.

Light sensor voltage (V) 3.2

0.8 00:00:00 00:27:02 Time

Figure 3.3 Zoom review of an entire ImagePrep deposition encompassing five phases. The individual drops in light sensor voltage were caused by matrix solution being sprayed onto a dry matrix layer, which was partially re-dissolved by the new matrix. Subsequently light scatter decreased form the initial ‘dry’ plateau sensor voltage. The matrix then dried completely, and a final plateau was reached where sensor voltage and therefore matrix crystal was higher.

Matrix sublimation

Sublimation is the process of a solid passing directly into a gas without the intermediate formation of a liquid phase. Most organic molecules with a significant vapor pressure either under vacuum or atmospheric pressure can undergo this endothermic process at specific temperatures and pressures, if thermal decomposition does not interfere19. In the matrix sublimation deposition method for MALDI-MS, a sublimation apparatus was used that consists of two nested, inverted glass dome shaped flasks (Figure

3.4). On the bottom of the upper container, the MALDI target plate was fixed using copper tape such that it faced downwards towards the bottom container. The upper container was filled with ice to cool the substrate and aid in the deposition of the matrix.

About 200 mg of matrix powder was evenly added on the bottom flask and both flasks

87 were sealed under vacuum using an O-ring system and a vacuum pump line. During the experiment, the sublimation flask containing the matrix was heated using a sand bath.

Sublimation was initiated by opening the sublimation chamber to vacuum to create a low pressure in the apparatus. After 10-20 min, the sublimation was stopped, which resulted in ~0.5 mg/mm2 matrix being applied to the analyte and surface of the substrate. The sample was then removed for MALDI MS analysis.

Sublimation Connected to apparatus vacuum Ice ITO slide MALDI Matrix

Hot plate with sand bath

Figure 3.4 A model of sublimation chamber used to deposit matrix on ITO coated glass slides for MALDI imaging mass spectrometry experiments.

3.2.2.3 Surface characterisation

As described in Chapter 1.3.4.4, the size and uniformity of the matrix crystals formed on the MALDI surfaces is known to be significant for the quality and resolution of the imaging mass spectra. In order to characterise the matrix/analyte co-crystals on the surface, the matrix deposited surfaces were analysed using a scanning electron microscope (Tabletop microscopes TM 4000, Hitachi, US).

3.2.2.4 MALDI-TOF standard operating conditions

All MALDI-TOF data were acquired with the Bruker UltrafleXtreme MALDI-

TOF/TOF that is equipped with a nitrogen laser (337 nm). All mass spectra were acquired in positive ion mode. For analysis of small molecules such as peptides and lipids, the reflector mirror was set to 25.00 kV and the total acceleration voltage to 13.60 kV. The first acceleration plate and lens voltages in the source were adjusted to optimise signal intensity and mass resolution for a selected calibration ion. Unless otherwise stated, the voltages were 22.40 kV and 8.00 kV respectively. Laser fluence was carefully adjusted using a gradient neutral density filter to obtain the maximum signal-to-noise ratio. For spectra acquired using the delayed extraction mode, the pulse voltage was optimised for resolution depending on the mass range of the individual sample distribution.

MALDI imaging mass spectrometry

MALDI imaging mass spectrometry analysis was performed on the ITO coated glass slides which were fitted into a slide adapter II MALDI target. An ultrafleXtreme

MALDI TOF/TOF mass spectrometer operating in reflective mode was used for imaging acquisition. Scanned slides were loaded into the Flex Imaging software (v4.0, Bruker

Daltonics), which was used to generate an auto execute sequence and set ‘teaching’ points for each individual imaging experiment. Auto execute parameters were set by Flex

Control software (v3.4, Bruker Daltonics) and a fixed laser power was selected for individual samples. Results of MALDI imaging mass spectrometry were analysed in Flex

Imaging and Flex Analysis software (v3.4, Bruker Daltonics).

3.3 Results and discussion

3.3.1 Optimisation of the matrix deposition methods

In order to obtain a homogeneous layer of small matrix crystals, the dried droplet, commercial sprayer and sublimation approaches for preparing MALDI target surfaces were investigated. The crystal sizes and the sizes of gaps between crystals were measured by using scanning electron microscopy. As shown in Figure 3.5, for both MALDI matrices, HCCA and DHB, the sublimation method formed the finest crystals and the gaps between each crystal were smaller than 15 μm, which is less than the average diameter of MCF-7 cells (~20 μm). For the other two matrix deposition methods, the gaps between matrix crystals were significantly larger and did not uniformly cover the ITO surface. That is, the sublimation deposition method resulted in the most complete coverage of the surface such that any single cells should not be readily ‘missed’ by

MALDI imaging mass spectrometry.

(a) HCCA (b) HCCA (c) HCCA Dried droplet commercial sprayer Sublimation

(d) DHB (e) DHB (f) DHB Dried droplet commercial sprayer Sublimation

Figure 3.5 Brightfield images of matrix crystals deposited onto an ITO surface. (a), (b) and (c) were formed by HCCA; and (d), (e) and (f) were formed by DHB. (a) and (d) were formed by dried droplet; (b) and (e) were formed by commercial sprayer, ImagePrep; and (c) and (f) were formed by sublimation. Scale bars are 200 μm for all images.

To further understand the performance of different matrix deposition methods, a mixture solution of 100 μM standard phosphatidylcholine compounds, DMPC and DOPC, was applied to an ITO coated glass slide by a pipette spotter. The MALDI matrix DHB was then applied on top of the analyte droplets by the dried droplet method and sublimation for MALDI imaging mass spectrometry. Images of these standard spots were acquired with 30 × 30 μm plate movement/pixel in positive-ion mode and a comparison of these data is shown in Figure 3.6. The image of the phosphatidylcholine standard with sublimed matrix was more uniform in appearance relative to the image of dried droplet

91 method. The sublimed matrix standard also gave a significantly more intense signal

(more ions upon laser irradiation) when compared to the sample with dried droplet and commercial matrix deposition.

(a) DHB dried droplet 100% Inten. [a.u.]

+ [DMPC + H] 0 60 1 mm

+ + [DMPC + Na] [DOPC + H] + 20 [DOPC + Na]

0 600 650 700 750 800 850 900 m/z

(b) DHB sublimation 100% + Inten. [a.u.] [DMPC + Na] 500 0 400 1 mm

300 + [DMPC + Na] 200 + [DOPC + Na] + 100 [DOPC + H]

0 600 650 700 750 800 850 900 m/z

Figure 3.6 Mass spectrometric image of 100 μM standard phosphatidylcholine. DHB was applied by dried droplet and sublimation methods for matrix deposition. The image was acquired with 30 μm pixel size, smoothed, and is displayed relative to the intensity scale shown to right of the image. All species were detected primarily as the protonated ion, [M + H]+ or [M + Na]+.

3.3.2 MALID-MS for MCF-7 cell analysis

Single MCF-7 cell analysis was performed by MALDI-TOF MS imaging. As shown in Figure 3.7, single MCF-7 cells were captured from whole blood by an anti-

EpCAM antibody conjugated ITO surface (see Chapter 2). As discussed in Chapter

3.3.1, the matrix sublimation method provides a more homogenous surface covering with smaller matrix crystals than the other approaches that were investigated. After being washed thoroughly with PBS buffer to remove any remaining cell culture media, captured

MCF-7 cells were dried in a vacuum desiccator and then DHB matrix was applied to the surface for MALDI mass spectrometry analysis by sublimation. Thus, the cells were analysed without homogenisation and/or relocation, which eliminates any time- consuming sample processing steps and associated sample losses.

Figure 3.7 Fluorescence image of MCF-7 cells captured on a modified ITO surface from whole blood (1 MCF-7 cell per 10,000 blood cells). Image was recorded with a 4 × objective, 200 ms exposure time and a gain factor of 18 dB. Scale bar is 500 μm.

After analysing captured MCF-7 cells by MALDI imaging MS, a representative single pixel mass spectrum of a single cell is shown in Figure 3.8. The mass range was obtained from 550 to 900 m/z, which covered most of detected analytes. Due partly to the existence of quaternary amine groups on the headgroup of phosphatidylcholine and the relatively high abundance of such molecules, PC class lipids were readily detected in

93 positive mode20. The protonated molecular ions of lipids were then confirmed by tandem

MS (Figure 3.9).

Intens. +

[PC(34:1) + Na] + [PC(34:1) + +

[PC(34:0) + K] + [PC(34:0) +

+ + ] [PC(32:0) + Na] + [PC(32:0)

K 100%

[PC(36:2) + Na] + [PC(36:2) [PC(32:1) + Na] + [PC(32:1) [PC(36:1) + K] + [PC(36:1) [PC(30:0) + Na] + [PC(30:0)

[PC(28:1) + + [PC(28:1) 0 [PC(38:4) + Na] + [PC(38:4)

600 700 800 900 m/z

Figure 3.8 Mass spectrum of single MCF-7 cell. Spectrum result was acquired in positive mode with DHB as the MALDI matrix and a spatial resolution of 25 μm by MALDI-MS imaging. Heatmaps showed the relative intensity at 782.71 m/z on the surface. Scale bar is 50 μm. All labelled peaks were assigned based on MALDI- TOF/TOF and comparison to bulk cell data from the literature.

Due to the low concentration of analytes in single cell, lipids were identified by

MS/MS fragmentation patterns and characteristic fragment ions performed on a bulk

MCF-7 cell suspension. For example, Figure 3.9 showed the MS/MS identification of the sodium adduct ion of PC (34:1). In positive mode, m/z 782.71 was assigned as

[PC(34:1) + Na]+ based on MS/MS fragmentation patterns and characteristic fragment ions. The ions at m/z 577.84 were corresponded to the diglyceridelike cations ([M + Na -

205]+), resulting from the loss of sodiated cholinephosphate. Ions at m/z 184 were

94 produced from the cholinephosphate. Generally, potassium adducts were observed with a m/z 163 fragment and a sodium adducts with a m/z 147 fragment.

184.077 Intens.

761.475 577.847 86.141 700.828

200 400 600 800 m/z

Figure 3.9 MALDI-TOF MS/MS identification of m/z 782 as PC (34:1) in positive ion mode. MS/MS spectrum of m/z 782.71 with structurally specific CID ions (m/z) 86, 184, 577, 701 and 761.

The MS/MS method is typically conducted on a LIFT TOF/TOF mass spectrometer for high sensitivity and high signal-to-noise ion detection21. Here, analytes are ionised and accelerated into the first TOF analyser, where selected mass ions and derived fragments then pass to the ion source of the second TOF analyser. However, one of the major disadvantages to LIFT OTF/TOF analysis is the presence of unexpected artificial fragment ions. These ions are likely produced from being in the ‘ion family’ with the same velocity when entering the ‘LIFT’ device, rather than being filtered by the

‘timed ion selector’ device22. In addition, the detection of lipid species in a complex bio- sample may be limited by the precursor ion selection window of the instrument. Thus, the assignment of lipid peak using TOF/TOF may not be accurate.

As shown in Table 3.3, ion assignment is based on comparison to the ion assignments reported in literature23,24 for bulk cell populations and should be considered

95 tentative. In this project, protonated, sodiated and potassiated lipids were all observed in single cell mass spectra. To identify the lipids in the single cells, different adduct species have been found with a certain lipid.

To further confirm the lipid peak assignments, related studies have been compared with this project. For example, in a study of MCF-7 cells as well as other three different cell lines of breast cancer cells by MALDI TOF MS, more than 100 endogenous compounds has been detected including PC(32:0), PC(32:1), PC(36:2), PC(38:4), etc23.

In a lipid analysis of six breast cancer cell lines including MCF-7 cells and MDA-MB-

231 cells, PC(28:0), PC(30:0), PC (32:1), PC (34:1), PC(36:2) and PC(38:4) has been detected by MALDI Fourier transform ion cyclotron resonance mass spectrometry in positive ion mode25. In an imaging study of Hela cells by a high resolution atmospheric- pressure MALDI MS, PC(30:0), PC(32:1), PC(32:0), PC(34:1), PC(34:2) and PC(36:2) were observed26.

Table 3.3 Tentative assignments of lipids detected from bulk MCF-7 cells by MALDI-TOF/TOF. An MCF-7 cell suspension of 200,000 cells/mL in PBS buffer was used as the sample. DHB was used as the MALDI-MS matrix and was sublimated on the cell surface.

Assignment Structurally Measured Calculated specific Molecular Ref m/z m/z Ion form Compound CID ion formula (m/z) + 700.47352 700.48878 [M+Na] 25,27 PC(28:0) C36H72NO8P 184, 378 716.4897 716.47271 [M+K]+ + 706.44227 706.53813 [M+H] + 184, 394, 24,25,26 PC(30:0) C38H76NO8P 728.4272 728.52008 [M+Na] 706 744.44337 744.49401 [M+K]+ + 754.4908 754.53573 [M+Na] 104, 476, 23,25,26, PC(32:1) C40H78NO8P 770.50703 770.50966 [M+K]+ 732 734.49543 734.56943 [M+H]+ + 104, 184, 23,24,26,27, [M+Na] PC(32:0) C41H80NO8P 756.57991 756.55138 478, 735 772.49659 772.52531 [M+K]+

+ 780.55439 780.55138 [M+Na] 26,27 PC(34:2) C42H80NO8P 184, 758 796.57063 796.52531 [M+K]+ 760.55909 760.58508 [M+H]+ + 86, 184, 24,25,26, 782.54402 782.56711 [M+Na] PC(34:1) C42H82NO8P 577, 701 + 798.56019 798.54096 [M+K] + 784.63307 784.58268 [M+Na] 24,27 PC(34:0) C42H84NO8P 184, 762 800.56799 800.55661 [M+K]+ + 808.60762 808.58268 [M+Na] 23,25,26,27 PC(36:2) C44H84NO8P 184, 787 824.62379 824.55661 [M+K]+ + 810.69667 810.59833 [M+Na] 27 PC(36:1) C44H86NO8P 184, 789 826.61342 826.57226 [M+K]+

+ 184, 627, 23,25,27 832.58217 832.58268 [M+Na] PC(38:4) C46H84NO8P 752, 811

This demonstrated the ability for MALDI-MS to detect lipids in a single cell on the UNSW instrument, which has also been confirmed previously using other MALDI-

MS instruments. Lipids are known to have important biological functions in the brain relating to communication, metabolism, and transport28,29. Recently, increasing evidence showed the connection between the level of lipid metabolism and cancer and metabolic diseases30,31. Due to the higher proliferative rates and the larger demand of nutrient, more metabolites are produced in cancer cells and which require large quantities of proteins, lipids, and nucleotides, as well as energy in the form of ATP32. To meet such abnormal demand, tumor cells reprogram their metabolic pathways, and which lead to the significantly different of metabolism in the tumor cells from that of the tissues from which they are derived33. Thus, the alteration of lipid metabolism has been increasingly recognised as a hallmark of cancer cells34, suggesting that lipid profiles could potentially be used as an identifier of diseased cells. However, the specific function of many individual lipids is unknown35. Reasons for this include the difficulty in measuring large numbers of lipids in complex samples and the identification of specific lipid isomers. To

97 understand the lipid metabolism in cancer, the identification and measurement of cellular lipids at the molecular level is of high significance.

3.3.3 Imaging analysis for MCF-7 cells

To connect the MALDI-TOF imaging results to the microscopy images, as shown in Figure 3.10, brightfield and fluorescence images were captured by an optical microscope after MALDI MS analysis. As shown in Figure 3.10, the heatmap showed a typical PC class lipid in MCF-7 cells, PC(34:1) with one sodium adduction at m/z 782.71.

The results of all brightfield imaging, fluorescence imaging and MALDI imaging of the same areas were showed in Figure 3.10. By comparing the brightfield imaging and fluorescence imaging in the same area, the location of where MCF-7 cells were attached, and the laser ‘impact’ spots were identified. The fading of fluorescence intensity was due to the long exposure time during the measurement. By comparing the MALDI heatmap and optical imaging, the area of high mass spectra signal could be connected to the corresponding cells. That provided a proof of concept that by using such a modified ITO slide, after performing MALDI mass spectrometry analysis of captured circulating tumor cells, specific mass spectra can be matched to the corresponding cells observed in an optical microscope. This method showed the possibility to analyse the same single cell using both microscopy and mass spectrometry.

(a) Cell (1) PC(34:1)

PC(36:2) Inten. [a.u.] Inten.

600 700 800 900 m/z

(b) Brightfield (c) Fluorescence imaging after MALDI imaging after MALDI (d) MALDI imaging analysis analysis

(e) Cell (2) 100%

Inten. [a.u.] Inten. 0

PC(34:1) PC(34:0) PC(30:0)

600 700 800 900 m/z

Figure 3.10 Optical imaging and fluorescence imaging after MALDI-MS analysis, and the heatmap results of MALDI imaging of captured from whole blood (1 MCF-7 cell per 10,000 blood cells). MALDI imaging was performed on captured MCF-7 cells on a modified ITO surface. (a) and (e) were MS spectra of two different cells; and (b), (c) and (d) were brightfield imaging, fluorescence imaging and MALDI- MS imaging result respectively. For (b) brightfield imaging after MALDI-MS analysis, dark areas correspond to cells, and the light regular dots correspond to laser impact ‘craters’. For (c) fluorescence imaging after MALDI-MS analysis, the larger light spots correspond primarily to cells and smaller light dots correspond primarily to laser spots. For (d) MALDI imaging result, the DHB matrix was sublimated on the surface. Heatmaps correspond to the spatial distribution and relative intensity of PC(34:1) at m/z on the surface. The scale bar is the same for all images (70 μm). Two mass spectra show the MALDI-MS results of two labelled single pixel.

As shown in the MALDI imaging heatmap, the two labelled single pixels (Figure

3.10 (d))corresponded to the two single MCF-7 cells shown in both the brightfield and fluorescence images (Figure 3.10 (b)and (c), respectively). In both of these cells, high intensities of PC(34:1) were detected (Figure 3.10 (a) and (e), respectively). However, other lipid types were observed for these two cells. For example, besides PC(34:1), cell

(2) contained more PC lipid types such as PC(30:0) and PC(30:4), while only PC(36:2) was detected in cell (1).This observation shows that MALDI imaging has the ability to characterise small lipid variation differences within the same cell line. Thus, MALDI imaging could be suitable for the analysis of rare single cells, and has potential to detect specific biomarkers in disease cells.

As shown in Figure 3.10 (e), some extra low mass peaks (m/z less than 700) from

Cell (2) were extremely high, which may not be the peaks endogenous lipids from mammalian cell. Instead, those peaks might be related to the oxidation of surface exposed lipids4 or in-source fragments. In future experiments, cells can be snap-frozen and stored in liquid nitrogen and thawed under vacuum in a desiccator immediately prior to analysis to potentially eliminate analyte oxidation and reduce any spatial movement of analytes on the surface17.

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3.4 Conclusions

After capturing circulating tumor cells in blood, MALDI imaging analysis was then directly performed on a modified ITO slide. For improved performance of MALDI imaging, different matrix deposition methods were evaluated. The ‘dried drop’ method was the simplest to perform but resulted in the formation of large crystals on the surfaces

(~20 to ~200 μm in width), with correspondingly large ‘gaps’ between matrix crystals (>

100 μm in some cases). In comparison, the use of a commercial MALDI matrix sprayer resulted in the formation of smaller crystals on the surface. However, for the matrix DHB, surface coverage remained heterogenous with gaps on the order of 15 μm between crystals. With the matrix sublimation method, fine crystals were formed which evenly covered nearly the entire surface which is useful for single cell analysis owing to the relatively small size of circulating tumor cells (~20 μm in diameter). In addition, sublimation is a solvent-free method that can be considered environmentally more benign than the other approaches.

After performing single cell MALDI imaging, a subsequent analysis was performed by microscopy to aid in locating corresponding single cells by optical and fluorescence microscopy. These results indicate that it is feasible in the future to conduct microscopy analysis and MALDI imaging on single CTC cells captured directly from blood. In principle, the optical and fluorescence micrographs can be obtained both before and after MALDI-MS in the future to ensure the laser rastering and deposition of matrix molecules does not impact the location and morphology of captured single cells. Such an approach may potentially be used to obtain information on single cells from more dimensions, i.e. imaging and chemical information. As the spatial resolution for MALDI

101 imaging was set to 25 μm, which was slightly larger than the average size of a single circulating tumor cell, the related optical image was necessary to confirm the mass spectrum was generated from one single cell. For mass spectrometry analysis of single

MCF-7 cells, several lipids of PC class were detected and identified by MALDI-TOF

MS/MS and confirmed by comparison to literature for a bulk population of MCF-7 cells.

Owing to the ability for the combination of microscopy and mass spectrometry, morphological information and chemical information was obtained from the same single cell. This showed the potential to integrate microscopy and mass spectrometry for the analysis of the relative uptake of fluorescent drugs and fluorescently labelled nanoparticle drug delivery systems by diseased cells in order to achieve a better understanding of drug delivery and the resistance of some diseased cells to drugs.

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3.5 References

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(16) Neumann, E. K.; Comi, T. J.; Rubakhin, S. S.; Sweedler, J. V. Lipid Heterogeneity between Astrocytes and Neurons Revealed by Single-Cell MALDI-MS Combined with Immunocytochemical Classification. Angew. Chemie Int. Ed. 2019, 58 (18), 5910–5914. https://doi.org/10.1002/anie.201812892. (17) Yang, B.; Patterson, N. H.; Tsui, T.; Caprioli, R. M.; Norris, J. L. Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation. J. Am. Soc. Mass Spectrom. 2018, 29 (5), 1012–1020. https://doi.org/10.1007/s13361-018-1899-9. (18) Bruker. Instructions for Use Peptide Calibration Standard II Peptide Mixture for Calibration of Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometers (MALDI- TOF MS). (19) Murphy, R. C.; Hankin, J. A.; Barkley, R. M.; Zemski Berry, K. A. MALDI Imaging of Lipids after Matrix Sublimation/Deposition. Biochim. Biophys. Acta 2011, 1811 (11), 970–975. https://doi.org/10.1016/j.bbalip.2011.04.012. (20) Dueñas, M. E.; Essner, J. J.; Lee, Y. J. 3D MALDI Mass Spectrometry Imaging of a Single Cell: Spatial Mapping of Lipids in the Embryonic Development of Zebrafish. Sci. Rep. 2017, 7 (1), 14946. https://doi.org/10.1038/s41598-017-14949-x. (21) Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A. A Novel MALDI LIFT-TOF/TOF Mass Spectrometer for Proteomics. Anal. Bioanal. Chem. 2003, 376 (7), 952–965. https://doi.org/10.1007/s00216-003-2057-0. (22) Frankfater, C.; Jiang, X.; Hsu, F.-F. Characterization of Long-Chain Fatty Acid as N-(4- Aminomethylphenyl) Pyridinium Derivative by MALDI LIFT-TOF/TOF Mass Spectrometry HHS Public Access. J Am Soc Mass Spectrom 2018, 29 (8), 1688–1699. https://doi.org/10.1007/s13361- 018-1993-z. (23) Wang, S.; Chen, X.; Luan, H.; Gao, D.; Lin, S.; Cai, Z.; Liu, J.; Liu, H.; Jiang, Y. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Cell Cultures for the Lipidomic Analysis of Potential Lipid Markers in Human Breast Cancer Invasion. Rapid Commun. Mass Spectrom. 2016, 30 (4), 533–542. https://doi.org/10.1002/rcm.7466. (24) Wang, X.; Han, J.; Pan, J.; Borchers, C. H. Comprehensive Imaging of Porcine Adrenal Gland Lipids by MALDI-FTMS Using Quercetin as a Matrix. Anal. Chem. 2014, 86 (1), 638–646. https://doi.org/10.1021/ac404044k. (25) He, M.; Guo, S.; Li, Z. In Situ Characterizing Membrane Lipid Phenotype of Breast Cancer Cells Using Mass Spectrometry Profiling OPEN. Nat. Publ. Gr. 2015. https://doi.org/10.1038/srep11298. (26) Schober, Y.; Guenther, S.; Spengler, B.; Rö, A. Single Cell Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging. 2012. https://doi.org/10.1021/ac301337h. (27) Kaya, I.; Jennische, E.; Lange, S.; Malmberg, P. Dual Polarity MALDI Imaging Mass Spectrometry on the Same Pixel Points Reveals Spatial Lipid Localizations at High-Spatial Resolutions in Rat Small Intestine. Anal. Methods 2018, 10 (21), 2428–2435. https://doi.org/10.1039/c8ay00645h. (28) Shamim, A.; Mahmood, T.; Ahsan, F.; Kumar, A.; Bagga, P. Lipids: An Insight into the Neurodegenerative Disorders. Clin. Nutr. Exp. 2018, 20, 1–19. https://doi.org/10.1016/J.YCLNEX.2018.05.001. (29) Adibhatla, R. M.; Hatcher, J. F. Role of Lipids in Brain Injury and Diseases. Future Lipidol. 2007, 2 (4), 403–422. https://doi.org/10.2217/17460875.2.4.403. (30) Hirsch, H. A.; Iliopoulos, D.; Joshi, A.; Zhang, Y.; Jaeger, S. A.; Bulyk, M.; Tsichlis, P. N.; Shirley Liu, X.; Struhl, K. A Transcriptional Signature and Common Gene Networks Link Cancer with Lipid Metabolism and Diverse Human Diseases. Cancer Cell 2010, 17 (4), 348–361. https://doi.org/10.1016/j.ccr.2010.01.022. (31) Santos, C. R.; Schulze, A. Lipid Metabolism in Cancer. FEBS J. 2012, 279 (15), 2610–2623. https://doi.org/10.1111/j.1742-4658.2012.08644.x. (32) Tennant, D. A.; Durán, R. V.; Gottlieb, E. Targeting Metabolic Transformation for Cancer Therapy.

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Chapter 4

Summary, Conclusions and Future Work

4.1 Summary

Due to the significant role that circulating tumor cells (CTC) play in metastasis cancer research and the high level of heterogeneity among the cells in the same tissue or cell population, capture and analyse such extremely rare circulating tumor cells is necessary. However, conventional approaches to capture circulating tumor cells in blood and directly perform single cell chemical analysis are limited. The objective of this work was to develop an immuno-based chip able to capture rare MCF-7 cells in blood, and then analyse them by fluorescence microscopy and mass spectrometry. Thereby, an ITO coated glass slide commonly used for MALDI MS imaging target was modified with the antibody for MCF-7 cell capture.

In Chapter 2, the modification of the ITO surface and the cell capture performance of such a surface was investigated. Firstly, an organophosphonic acid was used to form a stable self-assembled monolayer on the ITO surface. Polyethylene glycol was then used as an antifouling layer to prevent the adsorption of non-specific proteins or cells. Subsequently, anti-EpCAM antibodies were conjugated for MCF-7 cell capture.

Characterisation with contact angle goniometry, electrochemical cyclic voltammetry,

XPS measurement and fluorescence microscopy analysis suggested the all the modification steps were successful. Such modified surface was finally used to capture

106 lab-cultured MCF-7 cells mixed in whole blood. The results indicated that the anti-

EpCAM modified surface can be used to capture 800 MCF-7 cells in 2 mL of whole blood, which corresponds to 1 CTC in 10,000,000 red blood cells. The selectivity of cell capture on an antibody-conjugated surface was 10 times more than the antifouling surface in all the concentrations tested.

In Chapter 3, the captured MCF-7 cells were then used for mass spectrometry analysis. Different MALDI matrix deposition methods were tested in order to achieve better imaging resolution and spectrum quality. Single cell chemical analysis was then performed by MALDI MS imaging and the optical images were taken afterwards to be compared with the mass spectrometry imaging results. Compounds in MCF-7 cells were identified by tandem MS using a cell suspension of MCF-7 cells. The successful detection of some phosphatidylcholine lipids showed the ability of such method for single cell chemical analysis. Using a combination of optical microscopy and mass spectrometry, morphological information and chemical information were obtained from the same single cell. In summary, this research work demonstrated an immuno-modified slide that captured extreme rare cancer cells in a mixture of whole blood and, the analysis of single cancer cells performed by optical microscopy and mass spectrometry.

4.2 Conclusions

This research project demonstrated the fabrication of an antibody-conjugated ITO slide and the application of capturing specific rare cell in a mixture of whole blood and the directly performing of fluorescence microscopy analysis and mass spectrometry

107 analysis. These results will add significant contribution in current state of knowledge for rare cell detection and for the sample preparation process in single cell MALDI MS analysis. The significance and novelty of this project include the following aspects:

Rare cell detection

By using such an immuno-based surface modification approach, rare cell capture and detection in a complex mixture was achieved using a surface that is compatible as a target for MALDI-MS. In previous studies, the first and only clinically available circulating tumor cell capture technique approved by the US Food and Drug

Administration was the CellSearch System1. However, due to the usage of ferrofluid nanoparticles, extra separating processes were required before chemical analysis and such an approach has not been integrated with any type of mass spectrometry analysis. Thus, the development of a technique that can be used to capture rare circulating tumor cells on a chip surface was important. Other chip-based circulating tumor cells isolation methods involved integrating microfluidic devices and immuno-capture2–4 to achieve a high selectivity efficiency, but detection was limited to fluorescence based approaches limiting the chemical information that can be obtained. The research reported in this thesis makes it feasible that rare cells can now be captured on a modified surface directly from blood for single cell chemical analysis by MALDI-MS.

Integration of fluorescence microscopy and mass spectrometry for single cell analysis

Fluorescence microscopy images of single cells have not been directly linked to the corresponding mass spectra of the same single cells. By modification of a conductive

108 and transparent surface that is compatible as a target for MALDI-MS and a substrate for fluorescence and optical microscopy, and the use of a grid location ruler that was affixed to the bottom of the substrate, both the fluorescence microscope image and MALDI-MS mass spectra can be obtained from the same cell. In the single-cell analysis of diseased cells, fluorescence microscopy is a widely used approach for monitoring: (i) the relative update for drug molecules, such as doxorubicin; (ii) nanoparticle drug delivery systems, and nanoparticles of different shapes and sizes; and (iii) the overexpression of cancer biomarkers that are associated with poor prognosis and metastasis, such as carbonic anhydrase IX, which is overexpressed on the surfaces of many types of solid state tumours.

Interestingly, not all diseased cells of a given cell line respond the same to such treatments.

The continued development and improvement of single cell methods that can be used to directly obtain chemical information at the level of a single cell, such as MALDI-MS, may aid in answering fundamental questions regarding why such cells uptake drug and nanoparticles to different extents, and why some cells are more ‘dangerous’ (e.g. CAIX overexpressing cancer cells) than others from the same cell line population.

4.3 Future directions

The developed method shows the potential for the multi-analysis of a captured single cell. The next step is to improve the capture efficiency to detect even lower concentrations of CTCs, and to suit clinical requirements by obtaining information important for cancer study.

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4.3.1 Improving capture efficiencies

As discussed in Chapter 1.1, the concentration of circulating tumor cells are extremely low in blood, about one circulating tumor cell in 1 mL of blood, or ~1 billion red blood cells5. However, this concentration is about 800 times lower than what achieved in this thesis, thus limiting the clinical applications of the developed method. A possible way to decrease the detection limit would be to optimise the time cells are incubated with the surface, and study how the introduction of microfluidic methods (i.e. by using continuous flowing cells) could potentially improve the capture efficiency6,7. In addition, the study of antibody-antigen interactions may also contribute to the improvement of antibody binding specificity and efficiency8.

4.3.2 Improving the cell localisation approach

One of the most important considerations for performing fluorescence microscopy and mass spectrometry analysis on the same single cell is being able to overlap the optical microscopy image with the MALDI-MS image. Thus, cell localisation is a key consideration. In previous studies, an integrated instrument involving a mass spectrometer and an optical microscope has been developed to observe and detect samples at the same time9. In this image-guided mass spectrometry approach used here, targets such as single cells were automatically located, filtered, stratified and patterned prior to

MS analysis. This alternative solution to address this problem has the advantages that advanced microscopy methods that have not yet been incorporated into the MICRO-MS platform can be readily integrated with imaging MS data.

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In this work, each cell was located by use of a transparent plastic sticker with a printed grid that was taped on the backside of the slide. To improve the localisation of cells, microcontact printing10–12 (Figure 4.1) might be used to create micro-patterns of anti-EpCAM antibodies. With this method, the captured cancer cells should be positioned in well-defined locations on the surface, making it easier to find and study them with multiple techniques (i.e. fluorescence microscopy and mass spectrometry).

PDMS stamp

Ink

Substrate

Cancer cell

Antibody

SAMs in pattern

Figure 4.1 A diagram of a potential future microcontact printing strategy to improve the localisation of single cells. With the PDMS stamp, the antibodies can be modified on the surface in a specific spatial pattern. Finally, such pattern would result in a well-organised pattern of captured cancer cells, which may assist to locate single cells in further analysis.

4.3.3 Improving the information obtained

In this work the analysis of single cells was combined with fluorescence microscopy and mass spectrometry and ~10 phosphatidylcholine lipids were detected per cell. However, the use of fluorescence microscopy was limited in evaluating the cell capture efficiency and the cell location. By using fluorescently labelled membrane lipids13–16, drugs17–19, and nanoparticle drug delivery systems further research could be

111 done to investigate the relative uptake of exogenous compounds by diseased cells of a given population. Thus, chemical information could then be obtained by both fluorescence microscopy and mass spectrometry. In addition, owing to the successful identification of more than 200 compounds in breast cancer cells by MALDI-MS imaging on bulk cell populations20, further study of single cell mass spectrometry can aim to compare the cell content in cancerous cells and in normal cells or within the same cell line by using higher resolution mass spectrometry systems. For example, with some additional modifications to the optical system to improve the laser beam quality, the lateral image resolution was demonstrated to be around 5 μm21,22. In a study of single

Hela cells, a high resolution atmospheric-pressure MALDI imaging was attached to a

Exactive Orbitrap to perform a high spatial resolution (~ 7 μm), high mass accuracy (less than 3 ppm rms), and high mass resolution (R = 100 000 at m/z = 200)23. By using a

Fourier transform ion cyclotron resonance mass spectrometer, MALDI imaging can be used to detect metabolites with the signal intensity of around 107 in one single cell24. It would also be useful to perform a statistical analysis on the MALDI-MS ion abundances and m/z values obtained from many thousands of single cells from different cell types and different fluorescent intensities to confirm that MALDI-MS can distinguish differences between such cells at the level of a single cell. Furthermore, as another major ‘soft’ ionisation technique, nanoelectrospray ionization mass spectrometry could also potentially be integrated into the workflow. For example, after capturing rare cells, a nESI emitter can be used to ‘stab’ the single cell with the assist of a micromanipulator and then nESI-MS could be performed on the cellular contents. The remaining cell can be used to conduct MALDI-TOF imaging to obtain molecular information regarding the membrane molecules.

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4.4 References

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