Single cell electroporation using proton beam fabricated biochips 1

SINGLE CELL ELECTROPORATION USING PROTON BEAM FABRICATED BIOCHIPS SUREERAT HOMHUAN NATIONAL UNIVERSITY OF SINGAPORE 2010 SINGLE CELL ELECTROPORATION USING PROTON BEAM FABRICATED BIOCHIPS SUREERAT HOMHUAN (B.Sc (Hons.), Prince of Songkla University) A THESIS SUBMITTED FOR THE DEGREE OF PhD DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2010 Abstract Electroporation introduces polar molecules into a host cell through its membrane by giving quick electrical pulses across the cell For this thesis, the design and fabrication of a novel single cell electroporation biochip by the proton beam writing technique are presented The biochip features individual mouse neuroblastoma cells positioned in between nickel micro-electrodes SYTOX® Green nucleic acid stain (S7020) was then successfully incorporated into the cell upon electrical impulses across the electrodes Green fluorescence is observed when the stain binds with the DNA inside the cell nucleus The electric field strengths, pulse durations and numbers of pulses have been considered and optimized to achieve a high transfection rate of 82.1% and survival rate of 86.7% Proton induced high resolution (~100 nm) fluorescence images of these stained electroporated cells on our biochips further proves that the stain has been successfully bound to the DNA This single cell electroporation system is a promising method for the introduction of a variety of fluorophores, including nanoparticles and quantum dots, into cells with high success rate To my beloved grandparents…for their endless love and support Acknowledgements This thesis would never have been accomplished without the involvement and support from many people I would like to take this opportunity to express my deep and sincere gratitude to the following people My utmost gratitude goes to Prof Frank Watt, one of my thesis supervisors, for his brilliant mentorship and supervision, as well as his kindness, understanding and encouragements from the beginning of my post graduate life Prof Watt taught me how to learn from my mistakes and provide his invaluable advices during the past few years I feel very fortunate to have him as my supervisor I would like to thank Asst.Prof Andrew Bettiol for providing his expertise on optics and computer software Moreover, he never hesitates to suggest his excellent ideas Without him, my work would never be possible I also thank Asst.Prof Jeroen van Kan for his profound knowledge in all aspects of micro- and nano-machining I really appreciate his sincere support and suggestions Despite his heavy work schedule, he was always willing set aside time for me The work would not be completed without the close collaborations with many scientists from other departments at NUS I would like to use this occasion to thank Assc.Prof Sheu Fwu-Shan from Faculty of Engineering, Zhang Binbin from Department of Biological Science and former collaborator Cui Huifang for providing Neuroblastoma (N2a) cells for my experiments I am also very thankful to Asst Prof Giorgia Pastorin from Department of Pharmacy, NUS, for giving me an opportunity to use her cell culture facility, and also Santosh for his support and help though out the time I was using the facility I also would like to thank Lim Shuhui for her kindness of giving some chemicals for cell culture The journey of my postgraduate study in Singapore would be tougher and boring without having lovely and helpful seniors and friends around Living in Singapore truly would have been a different experience for me had I not been fortunate enough to share all the moments with you in CIBA I would like to thank Prof Mark Breese and i Assc.Prof.Thomas Osipowicz who make the working environment friendlier I would like to thank Isaac, my best friend, for willing to help everything I have asked for; Kyle for running imaging experiments with me; Sook Fun and Siew kit for being my supportive friends My PhD life will be incomplete without my other lovely CIBA members; Min, Ee Jin, Chammika, Shao, Mr.Choo, Armin, Taw Kuei, Anna, John, Aky, Sara, Malli, Sudheer, Haidong, Zhiya, Song Jiao and Yinghui; and my former colleagues, Reshmi, Weisheng, Liping, Jenny, Samy, Cher Yi, Mangai and Danial Thank you all! You will always have a special place in my heart I am very grateful to the Royal Thai Government for the Thai MOE-NUS PhD Scholarship; my employer, the Faculty of Science at Prince of Songkla University, for granting me a leave of absence for pursuing a PhD degree; and the Thai Ambassador to Singapore, His Excellency Nopadol Gunavibool and the staffs at the Royal Thai Embassy in Singapore for their effort and assistance in looking after the well-being of all Thai students in Singapore My sincere gratitude goes to the Department of Physics at NUS for providing me an opportunity to experience post graduate study Thanks are also due to all staffs at the department for their kind assistance in administrative issues I cannot end this acknowledgement without thanking my grandparents and my other family members, on their endless love I have relied on throughout my life Without their support, I would never be here To them I dedicate this thesis Singapore, August 2010 Yours, Sureerat Homhuan ii Table of Contents Acknowledgements i Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations xii Synopsis .1 Introduction Overall Objectives of This Study Chapter Micro- and Nano- Fabrication Techniques 1.1 Overview of lithography 1.2 Microlithography .11 1.2.1 Optical lithography 12 1.2.2 Deep UV lithography 16 1.2.3 Extreme UV lithography 16 1.2.4 X-ray lithography .19 1.2.5 Ion Beam and electron beam lithography 22 1.2.5.1 Electron beam lithography .23 1.2.5.2 Focused ion beam 25 1.2.5.3 Proton Beam Writing .26 1.3 PBW for biochip application .30 1.4 Conclusion 31 Chapter Cell Electroporation 32 2.1 Introduction 33 2.2 Methods for introduction foreign materials into host cells 35 2.2.1 Methods based on biological phenomena 35 2.2.2 Methods of chemical permeabilization 38 2.2.1 Physicsl methods 39 2.3 Electroporation 44 2.3.1 Brief introduction for electroporation 46 2.3.2 Micro-electroporation in biological cells 51 2.3.3 Single-cell electroporation 53 2.4 Conclusion 54 iii Chapter Instrumentation and Technique for Biochip Fabrication 55 3.1 Instrumentation of PBW technique 56 3.1.1 Focusing system 58 3.1.2 Scanning systems .58 3.1.3 Blanking system 60 3.1.4 Target chamber 61 3.2 Resist materials for PBW 63 3.2.1 General properties of PMMA 63 3.2.2 Spin coating of PMMA resist 65 3.2.3 PMMA development 67 3.3 Fabrication of PMMA microstructures 68 3.3.1 Beam focusing 71 3.3.2 Adjustment of the focal plane 73 3.3.3 Single-loop scanning versus multi-loop scanning .75 3.3.4 Exposure strategies 75 3.4 Conclusion 78 Chapter Application of Proton Beam Fabricated Biochips for Single Cell Electroporation .79 4.1 Biochips design for single cell electroporation purpose 80 4.2 Biochip fabrication 82 4.2.1 Nickel electroplating 82 4.2.2 Substrate preparation 88 4.2.3 Structure patterning 89 4.2.3.1 UV lithography patterning .90 4.2.3.2 PBW patterning 91 4.3 Experimental instruments and methodology .97 4.3.1 Cell preparation 97 4.3.2 Fluorescent stains .101 4.3.3 Experimental setup and method .105 4.4 Results and discussion .108 4.5 Conclusion .115 Chapter Cell Fluorescence Imaging 117 Introduction 118 5.1 Introduction to fluorescence microscopy 120 5.1.1 Principles of fluorescence 120 5.1.2 Fluorescence microscopy 132 5.1.3 Purpose of the study 134 iv 5.2 5.3 5.4 5.5 5.5 Proton induced fluorescence imaging 136 Proton induced secondary electron emission 137 Equipments and method 138 Results and discussion .141 Conclusion .147 Chapter Overall Conclusion 148 Bibliography 152 Appendix 163 A Photomultiplier Tube 163 B Publications 165 v List of Tables 1.1 The steps in the lithography process 5.1 Timescale Range for Fluorescence Processes 125 vi Chapter Instrumentation and technique for biochip fabrication commercially available in both solid (i.e sheet or film) and liquid forms It was found to be very suitable for PBW because when exposed to the energetic proton beam, the PMMA resist experiences main-chain scission (figure 3.7) and the exposed region is dissolved away in mildly alkaline developing solutions Figure 3.6 Chemical structures of methyl methacrylate and PMMA Figure 3.7 Mechanism of radiation-induced chain scission in PMMA 64 Chapter Instrumentation and technique for biochip fabrication The dose required for a full PMMA exposure corresponds to 150 nC/mm2 for MeV protons However, an over exposure (above 1500 nC/mm2) can cause problems in the dissolution of the irradiated portions in the development step, since cross linking and chain scission both can occur during the exposure Keeping within the exposure dose window hence results in rapid and precise structuring with low development time 3.2.2 Spin coating of PMMA resist Figure 3.8 Deposition step of the resist on a spincoater (spin coating) To fabricate the microstructures on the substrate, it is useful to start with a liquid photoresist since it can be deposited in desired thickness The liquid photoresist has to be spun to form a thin layer on the substrate using a spincoater, as depicted in figure 3.8 The spincoating machine consists of a disk that turns at high velocities (typically between 1000-10 000 rpm) and allows for the spreading of a drop of liquid initially deposited at the centre of the disc When using highly penetrating PBW, the thin film thickness deposited on the substrate determines the height of the structures, and hence the choice of photoresist becomes important As the structures that we have fabricated in our project 65 Chapter Instrumentation and technique for biochip fabrication have 7-ȝm height, we chose 950 PMMA A resist (950 k molecular weight, 11 wt% dissolved in Anisole) because it is possible to reach such thickness with double coating The spin curve of the resist thickness corresponding to spin speeds, providing useful information needed to select correct spin speed to obtain accurate resist thickness, is shown in figure 3.9 Figure 3.9 The spin speed versus film thickness curve for 950 PMMA A resists, solids : 9%-11% in Anisole The spin-coating process needs a clean room environment because any impurities deposited on the substrate may cause the defects on the developed structure The process starts from the preparation of the substrates, where the substrates are cleaned and dried We have found that a moderate cleaning with 99% acetone is adequate for our purpose In order to achieve a uniform layer of the resist, the resist was spun at lower speed (500 rpm) for seconds before accelerating to the final spin speed (1600 rpm) and holding this speed for 45 seconds At 1600 rpm, PMMA A 11 will give a thickness of 66 Chapter Instrumentation and technique for biochip fabrication about 3.5 ȝm In order to get ȝm-thick resist, the substrate is required to be spun twice with minute-baking at 180oC in between The coated substrate are then baked for 30 minutes at 180oC and leaved cooling down at the room temperature Direct formation of micron thick PMMA layers in one step proved to be problematic due to internal stress caused by the increased thickness 3.2.3 PMMA development After the PMMA resist is exposed with the proton beam in a predetermined pattern, the resist has to be developed Although the conventional developer for PMMA is GG developer (60% diethylene glycol monobutyl ether, 20% morpholine, 5% ethanolamine and 15% water), we found it preferable to use less viscous developer, isopropanol(IPA) based developer (7:3 of IPA:DI Water in volume), for the production of high aspect ratio confined structures[30] The non aggressive IPA-water developer ensures geometric precision of structures which are defined solely by the size of the focussed proton beam The exposed area can be developed once the exposure dose reaches the required dose (i.e 150 nC/mm2 for MeV protons) Due to the possible beam current fluctuations occurring during the writing process, a slightly higher exposure dose can be applied to prevent any insufficient radiation to the exposed areas The development of latent PMMA images after the proton exposure was carried out following the steps below: The sample with exposed resist was immersed into IPA developer for minutes to dissolve exposed resist portions; 67 Chapter Instrumentation and technique for biochip fabrication The sample was then removed from the developer and rinsed with DI water for minutes to remove the dissolved PMMA, showing the desired patterns; The sample was gently blown with dry nitrogen gas 3.3 Fabrication of PMMA microstructures As mentioned in the previous section, the photoresist chosen for this work is PMMA and thin layers can be prepared in-house by spin coating With the Ionscan software, fabrication patterns can be defined digitally and read into a computer code Using this code the proton beam can be scanned magnetically or electrostatically over the resist material to form specific patterns on demand PBW has been previously used successfully for the fabrication of high aspect ratio 3D test structures with smooth and vertical sidewalls Figure 3.10 shows a typical example, that of a 3D bridge structure fabricated by PBW The characteristics of PBW are shown in this example, ie since the proton beam travels in a straight line, with very small angle scattering, it allows the production of structurally accurately high aspect ratio smooth structures In addition, by changing the proton energy, a combination of structures of different height can be produced For many applications such as an electroporation biochip, sidewall roughness and verticality is an important issue as it is a factor in determining the uniformity of the electrostatic field between the electrodes The presence of sidewall roughness in PBW can be attributed to several causes [110]: (a) the dimensions of the proton beam spot, (b) 68 Chapter Instrumentation and technique for biochip fabrication the scanning algorithm employed and the parameters used, (c) variations in the beam intensity, (d) development conditions, (e) unwanted external varying magnetic fields influencing the beam focusing, and (f) stage vibration, especially when PBW structures are fabricated in combination with stage movement Therefore, to achieve the very smooth sidewall, it is crucial to reduce the factors that can cause rough surface sidewall Figure 3.10 3-D bridge structure written with a 1.0 MeV and 2.0 MeV proton beam [111] Chiam et al [110] has achieved the sidewall Rrms of less than nm when scan parameters are optimized Here we present the detailed proton beam writing steps for our biochip fabrication We also present the detailed protocol of performing a PBW 69 Chapter Instrumentation and technique for biochip fabrication experiment for patterning the microstructures for electroporation biochip in a μm PMMA layer on the glass cover slip as a substrate We also need to utilize our previous knowledge on modifying our structured PMMA into metallic electrodes, and Figure 3.11 shows the SEM image of a test piece fabricated using nickel electroplating, and depicts a ȝm-height and 100 nm-width smooth and straight Ni wall The roughness of the sidewall of similarly plated Ni structures was measured to be about nm The electroplating procedure will be described in the next chapter Figure 3.11 SEM image of a nickel stamp fabricated using PBW and nickel electroplating, exhibiting vertical sidewalls, and smooth surface (~7 nm).[111] 70 Chapter Instrumentation and technique for biochip fabrication Beam focusing In the conventional mode of beam focusing, one first achieves an approximate focus by observing, through an optical microscope, the ion induced luminescence from a piece of quartz The beam is then allowed to scan over a resolution standard, usually a metallic mesh, and the focus is improved until the image is of an acceptable quality Then the focus is further refined by scanning the beam spot over a sharp edge of the mesh and adjusting the focus appropriately The previous focusing protocol of the beam spot was carried out by using an X-ray test mask containing u ȝm2 holes with side-wall verticality of approximately 89.1o However, this mask is not a suitable for focusing well defined proton beam due to imprecise edges, poor surface roughness, and significant edge slope [105, 112, 113] A PBW fabricated nickel free-standing resolution standard with a side-wall verticality of 89.6o has thereby replaced the X-ray mask [114], which allows faster and more accurately focusing of the beam The 2-ȝm thick standard consists of a very fine grid on a larger and coarser supporting grid 71 Chapter Instrumentation and technique for biochip fabrication Figure 3.12 Proton-induced secondary electron imaging from a free-standing nickel grid The secondary electron image has been taken by a MeV proton beam at 0.5 pA current Using the new Ni grid, the proton beam can be focused down below 100 nm spot sizes by using the Amptektron ® UHV Model MD-502 Channel Electron Multiplier (CEM) The CEM is a ceramic capillary with a special semi conductive interior surface coating, which produces a very high secondary electron yield from the surface collisions of energetic particles This forms the basis of the electron multiplier Figure 3.12 shows CEM imaging maps taken during the process of focusing a corner of Ni grid using MeV 72 Chapter Instrumentation and technique for biochip fabrication protons scanning over a u ȝm2 (256 u 256 pixels) area, from which the beam spot size can be extracted Typical currents ranging from 50-100 pA with a beam spot of μm2 are normally used for the proton exposure step To ensure the fabrication of smooth and straight microstructures, an over-sample of up to five times the number of pixels in one direction is normally used in the direct-write process 3.3.1 Adjustment of the focal plane To pattern precise dimensions, the focal plane of the proton beam has to coincide with the top surface of the PMMA resist In the focusing system, the demagnification lens assembly requires a short working distance (from beam focus to lens), resulting in a relatively steep focusing angle For a system demagnification of 228u in x direction and 60u in y direction, a mismatch between the focal plane and the sample surface would result in increased feature sizes due to depth of focusing problems The method of reducing any depth of focusing problem relies on the use of a high magnification microscope attached to a CCD camera which projects the optical signal from the focal spot on a monitored screen There are three stages of focal plane adjustment: (1) The proton beam is roughly focused to a small spot size using quadrupole magnet controller in vertical and horizontal axes The proton induced fluorescence signals from the front surface of piece of quartz are imaged by the 73 Chapter Instrumentation and technique for biochip fabrication microscope, and in this configuration the image plane is coincident to the optical microscope image plane (2) A calibration grid, mounted next to the quartz is then moved to the proton beam spot focus position The z-position of the grid is adjusted if necessary to coincide with the optical microscope image plane The proton beam is then focused down to nanometer sizes by minimizing the edge structure of the grid using proton induced electron emission signal captured by a CEM; (3) The surface of the resist material is then moved into the beam path (the beam is turned off while moving the stage, avoiding unwanted exposure) With the proper sample mounting, the surface of the resist material is expected to be in the same plane of the proton beam as it is mounted on the same sample holder Dose normalization An accurate exposure dose per pixel can be achieved in two ways: (a) Normalization of the dose/pixel using backscattered protons for direct normalization [115] This method is used when the RBS signal count rate is relatively high The beam spot dwells on each pixel until a specified number of backscattered protons are collected, at which point the proton beam is moved to the next pixel (b) measurement of the total beam charge in a rapid and multiple repeating scanning procedure [116] In this second method, figure scanning, the beam is scanned rapidly over a figure several times until a sufficient dose has been attained This method is more suitable for a situation when the number of normalization counts for each figure 74 Chapter Instrumentation and technique for biochip fabrication is low To fabricate straight side wall and well defined structures, it is necessary to have a constant dose per scanned pixel during the exposure, ie a steady beam intensity 3.3.2 Single-loop scanning versus multi-loop scanning The current fluctuation of the proton beam during the sample exposure may sometimes vary appreciably, and can result in insufficiently irradiated resist areas Applying multiloop scanning is the way to overcome this problem In this scanning, the beam scans rapidly in repeated loops with a proportionally reduced beam dwelling time (updating time) over each pixel The final dose, which is equivalent to the required dose for PMMA, is from the accumulated dose in each scan cycle A comparison of the Rrms values for sidewalls fabricated using single-loop scans with those using multi-loop scans has been studied [110] and the result shows that the latter can improve sidewall roughness considerably In sidewalls fabricated using multi-loop scans the striations become visibly less pronounced, with the surface morphology displaying a more granular nature Therefore, in the work described in this thesis, we used the multi-loop scanning for our biochip fabrication 3.3.3 Exposure strategies While irradiating the substrate, high energy protons lose energy in a resist material mainly through collisions with atomic electrons The proton trajectory is largely unaltered by such interactions because the mass ratio between protons and electrons are very large (~1800) MeV protons maintain straight path and have a well-defined end of range in a resist material 75 Chapter Instrumentation and technique for biochip fabrication Very often it is necessary to scan pre-defined patterns on the surface of a sample This is achieved using a Scan Amplifier, which deflects the beam in a fashion similar to an electron beam being deflected in a TV monitor The scan size is set on the scan amplifier, along with the X to Y axis ratio of the area to scan This allows the irradiation of simple patterns such as squares and rectangles For irradiation of complex patterns, the scan amplifier is used with a computer running IONSCAN [107], a software package developed at CIBA This software allows any scanning modification inside the areas fixed by the scan amplifier The scan pattern is read by IONSCAN in a pixel format and each pixel is treated as a point of irradiation IONSCAN is able to control the shape of the scanned pattern and the dwell time the ion beam spends at each location (i.e controlling the dose) The dwell time is a parameter which is calculated based on the ion beam current available and dose required prior to the irradiation of the samples Varying amount of irradiation dose (number of ions/cm2) can hence be irradiated at different parts of a single sample With knowledge of the beam current, the total number of points in a pattern, and the dose required, the amount of time which the beam needs to dwell at a particular point can be calculated IONSCAN also controls a blanking system which is installed just before the switching magnet This blanking system simply deflects the beam away from the original beam axis and out of the chamber when switched on, and is used to blank the beam when no irradiation is needed More complicated patterns can therefore be irradiated within the same area fixed by the scan amplifier, the beam being blanked when moving from one figure to the other 76 Chapter Instrumentation and technique for biochip fabrication The pattern preparation prior to irradiation with IONSCAN is as follow: a) Preparation of the pattern to be irradiated in a binary (black and white) image file format b) Ionutilis software used to convert the image file into an EPL file format which may be read by IONSCAN With Ionutilis, the total number of black pixels of the pattern to be irradiated can be extracted The actual physical area that will be irradiated may be then calculated by equation (3.1) ‫ ܽ݁ݎܣ ݁ݒ݅ݐ݂݂ܿ݁ܧ‬ሺ݉ଶ ሻ ൌ ሺܵ݁‫݁ݖ݅ݏ ݊ܽܿݏ ݃݊݅ݐݐ‬ሻଶ ൈ ܰ‫ݏ݈݁ݔ݅݌ ݈ܾ݇ܿܽ ݂݋ ݎܾ݁݉ݑ‬ ሺܶ‫ݏ݈݁ݔ݅݌ ݂݋ ݎܾ݁݉ݑ݊ ݈ܽݐ݋‬ሻଶ ሺ͵Ǥͳሻ c) ‘Time of dwell per point’ defines the time which the beam will dwell at each black pixel and is calculated by equation (3.2) and (3.3) ܶ݅݉݁ ‫ ݐ݊݅݋݌ ݎ݁݌ ݈݈݁ݓ݀ ݂݋‬ൌ ܶ‫ ݁݉݅ݐ ݈ܽݐ݋‬ൌ Where ܶ‫݁݉݅ݐ ݈ܽݐ݋‬ ܰ‫ݏ݈݁ݔ݅݌ ݈ܾ݇ܿܽ ݂݋ ݎܾ݁݉ݑ‬ ‫ ܽ݁ݎܽ ݁ݒ݅ݐ݂݂ܿ݁ܧ‬ሺ݉ଶ ሻ ൈ ܴ݁‫ ݌݋݋݈ ݎ݁݌ ݁ݏ݋݀ ݀݁ݎ݅ݑݍ‬ሺ ‫ ݐ݊݁ݎݎݑܥ‬ሺ‫ܣ݌‬ሻ ൈ ͳͲିଵଶ ሺ͵Ǥʹሻ ‫ܥ‬ ሻ ݉ଶ ሺ͵Ǥ͵ሻ the Effective area can be calculated from (3.1); The Required dose per loop is the dose required for each particular photoresist divided by the number of loops; and The Current is from RBS calculation 77 Chapter Instrumentation and technique for biochip fabrication 3.4 Conclusion This chapter includes and summarizes all the description of the equipment and experimental procedures used in PBW to create structures on PMMA resist The detail about PMMA photoresist and the development after exposure were also described The information in this chapter gives background knowledge for the next chapter which will discuss the single-cell electroporation using proton beam fabricated biochips 78 ... .11 1. 2 .1 Optical lithography 12 1. 2.2 Deep UV lithography 16 1. 2.3 Extreme UV lithography 16 1. 2.4 X-ray lithography .19 1. 2.5 Ion Beam and electron beam. .. Proton Beam Fabricated Biochips for Single Cell Electroporation .79 4 .1 Biochips design for single cell electroporation purpose 80 4.2 Biochip fabrication 82 4.2 .1. .. 11 8 5 .1 Introduction to fluorescence microscopy 12 0 5 .1. 1 Principles of fluorescence 12 0 5 .1. 2 Fluorescence microscopy 13 2 5 .1. 3 Purpose of the study 13 4