Single cell electroporation using proton beam fabricated biochips 1

For this thesis, the design and fabrication of a novel single cell electroporation biochip by the proton beam writing technique are presented.. 1 Thesis synopsis Chapter 1 describes an

USING PROTON BEAM FABRICATED BIOCHIPS

SUREERAT HOMHUAN

NATIONAL UNIVERSITY OF SINGAPORE

2010

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

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

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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

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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

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Table of Contents

Acknowledgements i

Table of Contents iii

List of Tables vi

List of Figures vii

List of Abbreviations xii

Synopsis 1

Introduction 3

Overall Objectives of This Study 6

Chapter 1 Micro- and Nano- Fabrication Techniques 7

1.1 Overview of lithography .8

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 2 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

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Chapter 3 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 4 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 5 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

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5.2 Proton induced fluorescence imaging 136

5.3 Proton induced secondary electron emission 137

5.4 Equipments and method .138

5.5 Results and discussion .141

5.5 Conclusion .147

Chapter 6 Overall Conclusion 148

Bibliography 152

Appendix 163

A Photomultiplier Tube 163

B Publications 165

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List of Tables

1.1 The steps in the lithography process 8

5.1 Timescale Range for Fluorescence Processes 125

electrons an electromagnetic radiation (EUV and X-rays) with matter 23 1.6 Simulation of a 1000 20 keV electrons and 2 MeV protons impinging into

10 μm thick PMMA 29 2.1 Living cell membrane The membrane consists of hydrophilic heads internal

and external which is polar, therefore, the polar molecules from outside the cells are unable to freely pass through the membrane 34 2.2 Three primary mechanisms of endocytosis that are exhibited by a typical

cell ; (from the left) receptor mediated endocytosis, pinocytosis and phagocytosis 37 2.3 A strategy for treating glioblastoma multiforme in situ using a delivery

method based on magnetic resonance imaging-guided stereotactic implantation of retrovirus vector-producing cells (VPCs) The retroviral vectors produced by the cells were used to transfer a gene encoding a prodrug, herpes simplex thymidine kinase (HSV-tk), into tumor cells 38 2.4 Functioning for the gene gun or particle gun for putting DNA into living

cells 41 2.5 Schematic of a cell between conventional electroporation electrodes A

spherical cell was induced by an external electric field to induce transmembrane voltage across the cell membran 46 2.6 The Illustrations of the hypothetical structures of a lipid bilayer membrane

that may be involved in electroporation (a) the membrane is shocked by a quick pulse (b) the reconstruction of the membrane after pulse shock (c) the membrane allow the outside molecules to pass through it before it reseals itself and continue to grow 48 3.1 Top view of the accelerator facility at the CIBA The PBW facility dedicated

for micro-and nanofabrications is on the 10 o beam line The new clear

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microscope beam line designed for microscopy purposes is on the 30 o beam

line High resolution RBS spectrometer is on 45 o beam line 57

3.2 Schematic of an ion focusing system 58

3.3 Schematic diagram of the scanning and control hardware setup for PBW procedure at the CIBA 59

3.4 Proton Beam Writing end station setup 61

3.5 Side view of the interior outlook of PBW target chamber at 10 O beamline 62

3.6 Chemical structures of methyl methacrylate and PMMA 64

3.7 Mechanism of radiation-induced chain scission in PMMA 64

3.8 Deposition step of the resist on a spincoater (spin coating) 65

3.9 The spin speed versus film thickness curve for 950 PMMA A resists, solids : 9%-11% in Anisole 66

3.10 3-D cantilever structure written with a 1.0 MeV and 2.0 MeV proton beam 69

3.11 SEM image of a nickel stamp fabricated using PBW and nickel electroplating, exhibiting vertical sidewalls, and smooth surface (~7 nm) 70

3.12 Proton-induced secondary electron imaging from a free-standing nickel grid The secondary electron image has been taken by a 2 MeV proton beam at 0.5 pA current 72

4.1 Design of the biochips for single-cell electroporation The structure consists of 8 conducting circular shape pads linked to 4 electrode pairs in the centre of the chip with conducting lines The electroporation experiments are conducted to the adherent cells in between electrodes 80

4.2 Illustration of typical setup for nickel plating The electrical voltage is given across two electrodes, the nickel pellets are at anode giving Ni 2+ to the solution while the conductive substrate is at cathode receiving the ion deposited on the surface 83

4.3 UV mask contains structures of circular pads and conducting lines The structures are not covered with chrome which will let the UV through The pattern is not included the electrodes in the centre as they will be written by PBW 90 4.4 The image file created for Ionscan program to write electrode structures on

the substrate The same file was written on one substrate for 8 times,

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creating 8 electrodes connected to the conducting lines which were patterned

by UV lithography 92 4.5 The final electroporation biochips ready for cell culture before

electropermeabilization experiments 92 4.6 Process flow of the biochips fabrication (a) Cr was sputtered on the glass

slip followed by Au for approximately 30 and 10 nm respectively 7um thick PMMA (495 PMMA A11 resist; solids: 11% in Anisole) is then spin coated on the substrate (b) The circular shapes and the conducting lines are patterned by UV lithography (c) The electrodes are especially patterned by PBW to achieve straight side walls and high aspect ratio, which cannot be achieve by standard UV lithography (d) The exposed resist is developed (e) Nickel is electroplated to form the patterns (f) The remaining resist and conductive layer are removed 93 4.7 SEM pictures of structures on chips fabricated by Proton Beam Writing The

chip was sputtered by Au before the SEM imaging to make the chip conductive (a) one of four pairs of the electrodes in the centre of the chip Fig (b) was taken in between the gap; top and bottom were ~7 μm-thick Nickel 94 4.8 Microscope images of structures on chips fabricated by Proton Beam

Writing Fig (a), (b) and (c) were taken with different magnifications (5x, 10x and 20x) showing the structures were electroplated with Nickel 95 4.9 Cell recovery and cell passage protocols The cell recovery is the method for

recover the cell from being kept alive at very low temperature (-150 o C) The freeze cells were immediately added into the fresh cell culture medium when they melted The successfully recovered cells from this step will attach to the bottom of the culture flask The second protocol is cell passage This protocol is for keeping cells alive The protocol is normally repeated every 3

to 4 days The cells may be passed to the biochips at the last step of this protocol 98 4.10 Neuroblastoma cells are grown in between the electrodes ready for

electroporation The cells are incubated at 37 o C for 72 hours 100 4.11 Stained dead N2a cells with SYTOX ® Green and Ethidium homodimer II

(a) Bright field microscope image of live and dead cells (b) Dark field image with halogen lamp incorporated with the blue filter The dead cells stained with SYTOX® Green are excited and fluoresce in green color (c) Dark field image with halogen lamp incorporated with the green filter The dead cell stained with Ethidium homodimer II are excited and fluoresce in red color Live cells do not fluoresce in both colors 104 4.12 Experimental Setup (a) the overall system consists of the pulse generator,

inverted microscope, 3D manipulators and 3D stage controller (b) The chip

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is monitored and performed the experiments on the microscope stage With the connected camera, the videos and images can be captured while doing the experiments (c) the schematic representation of the chip while performing the experiment The small probe tips are connected to a pair of the electrode before the pulses are given and passed through the centre of the chip where there were cells in between the chosen electrode gap 106 4.13 Illustration the utilization of SYTOX® Green and Ethidium homodimer II to

determine electroporated and dead cells The SYTOX ® Green was added in the solution before the electroporation was performed, both electroporated and dead cells were stained with this dye The Ethidium homodimer II, added 30 minutes after electroporation, was used for testing viability of cells; only dead cells were stained with this dye This results in 2 colour fluorescence in dead cells 107 4.14 Example result from one electroporation experiment Optical images have

been taken using an inverted microscope with 20X magnification (a) Cells are successfully grown in between a pair of electrode gap before the experiment (b) Fluorescent image of cells shows green-fluorescent cells which uptook SYTOX ® Green and (c) fluorescent image of cells shows red- fluorescent cells which uptook DEAD Red TM (b) and (c) were taken after cells were electroporated with 10 4.25-Volt electric pulses 111 4.15 Pulse parameter optimization (a) Pulse amplitude optimization (3.75, 4.00,

4.25, 4.50, 4.75 and 5.00 V/50m with 4 ms 10 pulses) The optimized pulse amplitude is at 4.25 V/50m (equal to 0.85 kV/cm -1 ), it gives highest cell viability and very good transfection rate (b) number of pulses optimization (1, 2, 4, 6, 8 and 10 pulses with 4 ms- 4.25 V/50m pulse ) 4 pulses give very high in both transfection and viability rates (c) Pulse duration optimization (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 ms with 4 of 4.25 V/50m pulses) 2 ms pulse gives highest transfection rate at 82.1 %, and viability rate at 86.7% 114 5.1 Simulation of the radial deposition of energy for 2 MeV protons (left) and

100 keV electrons (right) for a 5- m-thick layer of PMMA 119 5.2 Illustration of one form of a Jablonski diagram The singlet ground (S(0))

state, as well as the first (S(1)) and second (S(2)) excited singlet states as a stack of horizontal lines The thicker lines represent electronic energy levels, while the thinner lines denote the various vibrational energy states (rotational energy states are ignored) Transitions between the states are illustrated as straight or wavy arrows, depending upon whether the transition

is associated with absorption or emission of a photon (straight arrow) or results from a molecular internal conversion or non-radiative relaxation process (wavy arrows) Vertical upward arrows are utilized to indicate the instantaneous nature of excitation processes, while the wavy arrows are reserved for those events that occur on a much longer timescale 122

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5.3 Illustration of absorbtion and excitation spectrums of common fluorophores

FITC 127 5.4 Fluorescence lifetime decay profiles 129 5.5 Example of conventional fluorescence microscope 133 5.6 Comparison of the spatial and temporal resolutions of biological imaging

techniques Average sizes of biological features are given in logarithmic size scale; specific sizes vary widely among different species and cell lines The temporal resolution is not applicable (NA) for Electron Microscopy (EM) or Near-Field Scanning Optical Microscopy (NSOM) because they image static samples Ground-State Depletion (GSD) and Saturated Structured- Illumination Microscopy (SSIM) have not been shown on biological samples, and their temporal resolution are not determined (ND) Endoplasmic Reticulum (ER); Magnetic Resonance Imaging (MRI); Optical Coherence Tomography (OCT); Photoactivated Localization Microscopy (PARM), Stochastic Optical Reconstruction Microscopy (STORM); Positron-Emission Microscopy (PET); Stimulated Emission Depletion (STED); Total Internal Reflection Fluorescence (TIRF); Ultrasound (US); Wide Field Microscopy(WF); Proton Induced Fluorescence (PIF) 135 5.7 The electroporation biochip made on the undoped silicon wafer 138 5.8 Illustration of the PIF and PISE system at 10 degree chamber The

photomultiplier tube (Hamamatsu R7402) is mounted on the sample holder

in front of the sample, while the CEM is mounted in the chamber 140 5.9 Images of one electrode pair with 300 μm scan size, and ~100 nm beam spot

size (a) microscope image (b) fluorescent image taken from upright microscope (c) PIF image (d) PISE (e) SEM 142 5.10 Images of a cell inside the electrode gap with 30 μm scan size, and ~100 nm

beam spot size (a) microscope image (b) fluorescent image taken from upright microscope (c) PIF image (d) PISE (e) SEM 144 5.11 Images of a cell inside the electrode gap with 20 μm scan size, and ~100 nm

beam spot size An electrode is clearly seen in the image as the cell is close

to this electrode (a) microscope image (b) fluorescent image taken from upright microscope (c) PIF image (d) PISE (e) SEM 145 5.12 Images of a cell outside the electrode gaps with 20 μm scan size, and ~100

nm beam spot size (a) microscope image (b) fluorescent image taken from upright microscope (c) PIF image (d) PISE (e) SEM 146

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LIST OF ABBREVIATIONS

CEM Channel electron multiplier detector

CIBA Centre for Ion Beam Applications

DMEM Dulbecco’s Modified Eagle’s medium

DMSO Dimethylsulfoxide

IPA Isopropanol

MMA Methylmethacrylate

NSOM Near-Field Scanning Optical Microscopy

PISE Proton Induced Secondary Electron

PM Photomultiplier

SEDPs spatial energy-deposition profiles

STED Stimulated Emission Depletion

STORM Stochastic Optical Reconstruction Microscopy

TIRF Total Internal Reflection Fluorescence

US Ultrasound

UV Ultraviolet

1

Thesis synopsis

Chapter 1 describes an overview of current micro-and nano-lithographic techniques

available for biochip fabrication: The overview includes descriptions of conventional

lithographic procedures, UV lithography, electron beam lithography and proton beam

writing (PBW), where the principles, applications, advantages and drawbacks of each

technique are given In particular, fabrication strategies based on PBW technique are

described in more detail, indicating that PBW has the potential to be the most suitable

technique for 3D biochip structures, particularly those which require smooth and vertical

sidewall features at a small scale

In Chapter 2, a brief introduction is presented indicating the importance of

introducing foreign material into living cells The current methods used to induce these

materials are then described, followed by detail description of the electroporation

technique Previous studies in single cell electroporation are reviewed in this chapter,

together with the potential advantages of using PBW for the single cell electroporation

device fabrication

Chapter 3 includes and summarizes details of the PBW equipment and experimental

procedures used in PBW to create structures in PMMA resist A brief description of

PMMA photoresist and resist processing are also mentioned, as well as the exposure

strategies for fabricating precise structures

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Chapter 4 presents the results from single cell electroporation using our biochip

The design and fabrication of biochip are reported, together with descriptions about the

setup for electroporation and cell preparation The last part of the chapter gives the

optimization results for pulse amplitude, number of pulses and pulse duration The

discussion and conclusions for these experiments are also given here, as well as possible

further applications in several fields, for example biomedical science, biotechnology and

genetic engineering

In Chapter 5, bioimaging using protons, is reported Proton technology is not only

able to fabricate microstructures through PBW, but is also able to image cells at high

resolution Due to the proton’s penetrating properties, Proton Induced Fluorescence

imaging technique has potentially better resolutions than other current imaging

techniques such as confocal or optical fluorescence The fluorescence images of

electroporated and normal cells are presented here, and a comparison between

conventional fluorescence microscope images and proton induced fluorescent images are

given Protons can also induce secondary electrons which provide surface images, and

these images are compared with corresponding images using SEM

In the final chapter, Chapter 6, an overall conclusion of the research projects is

given The electroporation results and the fabrication of the single cell electroporation

biochips are evaluated, and the fluorescence imaging results from all the presented

techniques are discussed In addition, prospective developments of the fabrication, the

design, and the experimental parameters are presented together with their contributions to

advancing cell studies and bioimaging technologies

3

Introduction

Research into Micro-Electro-Mechanical Systems (MEMS) started in the early 1970s and has since received a lot of attention MEMS is the technology of very small devices generally ranging in size from 20 μm to around a mm Although, this technology can be applied to several applications, biomedical application is currently one of the most important research topics [1, 2] MEMS for biomedical applications is sometimes called Biomedical or Biological Micro-Electro-Mechanical System (BioMEMS) [3] BioMEMS uses chemical, mechanical and electrical functions of microstructures to mimic bodily functions and to interact with human body BioMEMS is also involved in many different life science applications such as biology, biophysics and medicine Some advantages of BioMEMS include biocompatibility, reproducibility, short response time and small size Moreover, BioMEMS has an ability to interact with fluids and response to electrical stimulus for drug delivery applications

Nowadays, BioMEMS is considered as the technology to interface the micro to the nano world Besides, this technology enables us to probe, measure, and explore the biological world such as single cells Many existing technologies are now being optimized, and several new micro- and nano- fabrication approaches are simultaneously being explored

The devices and integrated systems using BioMEMS are also known as chip and micro-total analysis systems (micro-TAS or μTAS) These systems are used in

lab-on-a-4

many research areas such as tissue engineering, surface modification, and diagnostic applications Among all applications for BioMEMS, diagnostics represents the largest and most researched An increasing number of BioMEMS devices for diagnostic applications have been developed by many groups of researchers in the last few years These devices differ significantly in their design and fabrication techniques and also in the areas of their applications BioMEMS for diagnostic applications are also sometimes referred to as ‘BioChips’ This type of device is used to detect cells, microorganisms, viruses, proteins, DNA and related nucleic acids

In general, the use of micro- and nano- scale detection technologies is justified by

(i) A reduction in the sensor element to the scale of the target species, thereby

providing a higher sensitivity

(ii) A reduction in reagent volumes and associated costs

(iii) A reduction in the interaction time due to small volumes

(iv) Enhanced amenability due to portability and miniaturization of the system

[4]

Advances in biochip design and fabrication are giving scientists new methods for unraveling the complex biochemical processes occurring inside cells, with the larger goal

of understanding and treating human diseases

It is believed that biochip research is evolving at a relatively slow rate The primary reason is due to the fact that the development of this technology involves interdisciplinary integration of a very diverse range of background knowledge such as physics, chemistry, biology, medical science, material science, and engineering Due to

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the complexity and the interdisciplinary nature of this area, it is crucial to apply a diverse range of expertise in both the fabrication and application areas of biochip devices Many great discoveries are anticipated in this exciting interdisciplinary research area

6

Overall Objectives of this Study

The purpose of this study is listed below

1) Design and fabricate biochips for single cell electroporation using the new technique of proton beam writing (PBW), a technique capable of direct-writing three dimensional straight side wall structures Although PBW is a new and potentially powerful technique for 3D fabrication, the technique is not yet commercially available and its development is still in its infancy Part of this study therefore is to investigate the capabilities of PBW for biochip fabrication, as well as material selection, chip design, technical constraints, and the integration of detection systems into the biochip These factors raise challenges to the execution

of the fabrication strategy

2) Design, fabricate, and test a biochip that allows single cell electroporation in Mouse Neuroblastoma (N2a) cells using the fluorescent probe SYTOX ® Green as the electroporated material

3) Study and optimize the important electrical pulse parameters for successful electroporation into the N2a cells, in order to achieve the highest transfection and viability rates

4) Study the new technique of cell fluorescent imaging using protons, by imaging the fluorescence stained electroporated N2a cells on the biochip Investigations were also carried out on simultaneous imaging by the detection of proton induced secondary electrons The results were compared with standard fluorescence microscopy and scanning electron microscopy

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Chapter 1

Micro- and Nano-fabrication techniques

Technologies for the fabrication of microcomponents, microsensors, micromachines and microelectromechanical systems (MEMS) are being developed rapidly There are many techniques currently used in microstructure production, most of which are based around surface techniques (i.e optical lithography, electron beam lithography, low energy ion beam lithography and laser ablation) and applied mostly in silicon based technology These techniques are often restricted to produce structures with depths of only a few micrometers Ion Beam Lithography (IBL) is a technique that uses an ion beam (i.e either low energy heavy ions or fast protons) to write designed structures directly into the resist materials IBL, especially using a fast proton beam, is a novel technique able to fabricate high aspect ratio micro-and nano-structure with straight and smooth sidewalls

A review on current microfabrication techniques is presented in this chapter, including a detailed discussion on fast proton beam direct writing (proton beam writing - PBW)

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1.1 Overview of lithography

Lithography is a technique used to create structures in a resist material which is normally coated on substrates such as silicon wafers The resist material is exposed to ionising radiation which chemically damages (through scissioning or cross linking) the resist in a selective pattern The resist is then treated with a specific chemical developer in order to remove the damaged resist (positive process) or alternatively to remove the undamaged resist if the damaged resist is more stable through cross-linking (negative process) In this way, structures can be formed The lithography steps of this critical process are listed in Table 1.1

Table 1.1 The steps in the lithography process (The steps in italic are optional)

Adhesion promotion

 Resist coat

 Softbake

 Alignment

 Exposure



Post-exposure bake

 Development



Resist coat: Resists are typically comprised of organic polymers applied from a solution To coat the wafers with resist, a small volume of the liquid resist is first dropped onto a substrate The substrate is then spun about its axis at a high rate, flinging off the excess resist and leaving behind, as the solvent evaporates, a thin (0.1-2 μm, typically) film of solid resist

Softbake: After the resist coating has been applied, the coating may contain unwanted solvent which may interfere with subsequent processing Baking the resist coating and substrate is then undertaken to drive off residual solvent

Alignment: Complex structures are fabricated by a series of patterning steps Typically, these start with a lithography operation followed by an etch or ion implantation Between patterning steps, there may be film depositions, planarizations, and other processes Each new pattern must be placed on top of preceding layers, and

Post-exposure bake: This is an optional baking step used to drive additional chemical reactions or the diffusion of components within the resist film

Development: This is the step by which resist is removed using chemical developers

Measurement and inspection: This is an optional operation where it is determined

if the resist features on the wafer are sized correctly, properly overlayed and are sufficiently free from defects These measurements may be used for the purposes of process quality control

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Hardbake: Because wafers with photoresist patterns are frequently subjected to etch

or ion implantation operations immediately following the lithography step, a final bake is often used to drive out volatile organic materials and water in order to preserve the vacuum integrity of the etch and ion implantation equipment The temperature required for this step is usually so high that it can degrade the photochemical properties of the resist, rendering it difficult to expose the resist further Consequently, the hardbake is one

of the last steps in the lithography process, although it may precede measurement and inspection

1.2 Microlithography

The miniaturisation of machines, actuators and sensors etc and the integration of MEMS devices are considered a high-technology growth area of enormous potential The majority of sub-micron fabrication procedures involve two types of interaction; electron magnetic radiation (e.g optical, UV or X-ray photons) or charged particles (electrons, low energy heavy ions, high energy light ions) [5] The patterning methods can be classified into 2 categories; direct write lithography (usually using focused charged particles) and patterned lithography (usually using broad beams of photons, UV, X-rays, electrons) The difference between these two methods is that direct write lithography does not require a mask, although the process is much slower Microlithography refers specifically to lithographic patterning methods capable of structuring material on a fine scale Microlithographic technologies are briefly reviewed below

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1.2.1 Optical lithography

The name optical lithography comes from an early application where the exposing energy was visible light While those wavelengths can still be used, since visible wavelengths stop at about 400 nm, the push to reduce the size of feature sizes has lead to the use of shorter wavelengths to increase resolution Ultraviolet (UV) and deep ultraviolet (DUV) sources are now used Such sources include excimer lasers which operate at wavelengths

of 248 nm, 193 nm and less However, at these shorter UV wavelengths, particularly 193

nm, optical materials and even air absorb the energy very well and there are still many problems to be overcome when using this wavelength

There are two types of photoresists: positive and negative For positive resists, the resist is exposed through a mask with UV light, which changes the chemical structure of the exposed resist so that it becomes more soluble in the developer The exposed resist is then dissolved by the developer solution, leaving windows of the bare underlying material The mask used contains an exact copy of the required pattern to be fabricated on the wafer

Negative resists behave in just the opposite manner Exposure to the UV light causes the negative resist to become polymerized, and more difficult to dissolve Therefore, the negative resist remains on the surface wherever it is exposed, and the developer solution removes only the unexposed portions Masks used for negative photoresists, therefore, contain the inverse (or photographic "negative") of the pattern to be transferred The figure below shows the pattern differences generated from the use of positive and negative resist

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A schematic illustration of optical lithography is shown in figure 1.1 The substrate wafer is firstly spin-coated with a thin layer the resist The wafer is then soft-baked to remove the solvents from the photoresist coating Soft-baking plays a critical role in photo-imaging: The photoresist coatings become photosensitive only after soft-baking

Figure 1.1 the patterns generated from different photoresists

One of the most important steps in the photolithography process is mask alignment

A mask or "photomask" is a square glass plate with a patterned emulsion of metal film on one side The mask is aligned with the wafer, so that the pattern can be transferred onto the wafer surface For multi-mask processes, each subsequent mask must be aligned to the previous pattern

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Once the mask has been accurately aligned with the pattern on the wafer's surface, the photoresist is exposed through the pattern on the mask with a high intensity ultraviolet light The exposed regions of the resist are subsequently removed using chemicals If the developed resist layer is sufficiently thin so that the silicon substrate is exposed during the development process, then subsequent chemical etching can result in micro structure formation in the silicon substrate Although photolithography is essentially a surface micromachining technique and therefore two-dimensional, various wet and dry etching techniques have been successful in producing 3-D microstructures [6]

Depending on the type of separation between mask and substrate wafer, there are mainly three exposure systems, known as contact, proximity and projection (figure 1.2 (a), (b) and (c)) The first two systems are also called shallow printing, and are the simplest and cheapest methods of imaging In contact printing, the mask is placed in close contact with the resist and covers the entire substrate with the Cr side facing the resist This however can lead to large number of defects on both the Cr and resist surfaces Also by repeating hard contact, it severely reduces the mask lifetime In proximity printing, a small gap is introduced to eliminate the above problem, although this increases the diffraction effects between two surfaces which can lead to distorted features, particularly when the features are small In the projection exposure system, a complex lens system is used to focus an image of a mask onto the surface of the substrate (see figure 1.2c) The advantage of this system is that the images on the wafer are smaller than the actual dimensions of the images on the mask

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ܦܱܨ ൌܰܣ݇ଶߣଶሺͳǤʹሻ

Where k 2 is a constant for a specific lithographic process As resolution is increased through the use of higher-NA tool, the depth of focus may be comparable to the height of the device topography Multiple-layer resist and top-surface imaging, which present a planarized top surface for exposure, can provide substantial relief in this respect at the expense of process complexity [8]

1.2.2 Deep UV lithography

Preserving the typical configuration of a conventional contact UV printer, deep UV lithography (DUV) adopts light ranging from 150 ~ 365 nm for the generation of submicrometer patterns in photoresist with a high aspect ratio of up to 15 [9] DUV is a compromise between the relatively well understood and ubiquitous photolithography, which is used to manufacture shallow microcomponents of dimensions > 250 nm, and the expensive X-ray lithography (LIGA) process, which has the potential to manufacture sub-

250 nm 3-D microcomponents [6] The shorter wavelengths of UV are used to minimise diffraction effects Special types of resist are used which are transparent to UV, thereby allowing the UV to penetrate deep into the resist for 3-D exposures A detailed report of DUV photoresist is given by Moreau: Aspect ratios of 10 can be achieved using deep UV lithography [10], which are comparable with LIGA [5]

1.2.3 Extreme UV lithography

If wavelengths of light in the range of 11-14 nm are used (which are absorbed in glass), it

is possible to construct reflecting optics of moderate efficiency (> 60%) using multilayer

Reflection occurs at interfaces between materials of different indices of refraction, and the larger the difference in refractive index then the greater the reflectivity At wavelengths < 50 nm, all materials have indices of refraction  1 Thus, it is difficult to create a highly reflective interface At EUV wavelengths, it has proven possible to make mirrors with moderate reflectivity, in the range of 60-70%, by the use of multilayers Multilayer reflectors are made by depositing alternating layers of high-Z and low-Z materials, giving a small but effective difference between refractive indices at each

interface By making the periodicity d of the multilayer stack satisfy the Bragg condition,

݀ ൌʹܿ݋ݏߠ݉ߣ ǡ ݉ ൌ ͳǡʹǡ ǥሺͳǤͶሻ

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Where  is defined in figure 1.3, the net effect of small reflectivity at each interface is moderately high reflectivity overall when the stack has a sufficient number of layers

Figure 1.3 Multilayer reflector

The most developed EUV Multilayer thin-films (MLs) consist of alternating layers

of molybdenum (Mo) and Si, and they perform best for wavelengths of approximately 13

nm EUV absorption in standard optical photoresist is very high; the absorption depth in organic resists is less than 10 nm, so the printing occurs in a very thin imaging layer at the surface of the resist Hence new resists and processing techniques are required EUV masks are made of a patterned EUV radiation absorber placed on top of a ML reflector deposited on a solid substrate, such as Si wafer [11] The current challenges in mask development are the deposition of a defect-free ML coating and the techniques for repairing occurring defects In addition, the mirrors comprising lithographic systems are stringently required to exhibit exceptional performance in both surface shape and

X-ray lithography uses collimated x-rays as the exposing energy Being much shorter

in wavelength than light and DUV, x-rays provide increased lateral resolution due to reduced diffraction effects For micromanufacturing though, it is the penetrating power of the x-rays deep into the photoresist that is the prime characteristic This penetration allows microstructures with great height to be fabricated, relative to optical lithography

It should be noted that the terms "great height" and "high aspect ratio" are often used without specific numbers being applied As technology progresses, the absolute numbers associated with these terms change For example, optical lithography has been used to create structures as high as 1 millimeter in newer negative photoresists One millimeter was generally regarded as a height only attainable with x-ray lithography

Optical lithography is limited by diffraction, which is most significant when the required structures are comparable in size to the wave length of light This has driven industry to develop techniques shorter and shorter wavelengths However, at x-ray wavelengths there are no known materials for making image-forming lenses or mirrors Consequently, x-ray lithography involves the use of proximity printing, where the mask

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is brought to within a few microns of the wafer and the x rays are passed directly through the mask and onto the wafer This is in contrast to optical lithography, which has the potential for projection of the image by a lens

X-ray masks are comprised of very thin membranes (thickness< 2 μm) made from low-atomic-number material on which is created a pattern made from a heavy metal such

as gold A large percentage of the X rays pass through the low-atomic-number material membrane, but are absorbed by the heavy metal pattern, thus generating pattern contrast Silicon carbide is a typical membrane material, and silicon nitride films were used early

in the development of x-ray lithography

The use of thin membranes for X-ray masks introduces a set of challenges Such films deform because of stresses, and there has been extensive work to understand and control them [13, 14] Mask deformation is particularly problematic for X-ray lithography, because in the close proximity geometry there is no reduction of the image between the mask and the wafer This 1:1 pattern transfer necessitates very tight tolerances for the masks, as compared with 4:1 or 5:1 reduction printing On the other hand, with x-rays there are no lens distortions or feature-size-dependent pattern- placement errors, so a greater part of the overlay budget can be allocated to the mask in x-ray lithography However, thin film masks are susceptible to vibrations when stepped

or scanned, and this needs to be addressed in any x-ray exposure system [15]

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Figure 1.4 X-ray proximity lithography with a collimated light source

Of all the challenges to X-ray lithography, the greatest involves the mask Because there is no potential for image reduction, defects, linewidth variation, and misregistration are transferred from the mask to the wafer unmitigated by the reduction found in optical steppers There is also no potential for a pellicle that will keep particulate defects out of the depth-of-field On the other hand, because there are no optics, there are no lens contributions to linewidth variation and misregistration, so 1× x-ray masks need to have linewidth variations and misregistration only about one-third that of 4× reticles However, since leading edge-mask-making capability is needed to meet the requirements

of 4× reticles, this tightening of requirements by a factor of three has made 1× x-ray masks virtually impossible to make at the resolutions where X-ray lithography becomes important Consequently, most X-ray lithography programs have been scaled back considerably or terminated altogether

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1.2.5 Ion beam and electron beam lithography

The use of ion beams is becoming increasingly more important for submicron lithography They can be used not only for direct writing onto wafers [16] but also for mask fabrication [17, 18] In these operating modes ions can also be used for ion implantation, direct ion milling, or for producing bombardment damage that will enhance

a follow-up sputtering or etching step

To use ions for lithography, or any other step in microfabrication, a patterned dose of ions has to be transmitted to the surface under consideration that is, a part of the surface has to be irradiated and a part left unirradiated [19]

The resolution of ion-beam lithography is inherently good because the secondary electrons produced by an ion beam, being of low energy , have a short diffusion range and practically no backscattering occurs[20]

In principle there are three separate and distinct ion beam processes capable of fabricating structures at the sub-100 nm level : The focused ion beam (FIB) technique where a slow focused heavy ion beam (with energies typically around 30 keV is written over a surface to create a pattern through modification, deposition or sputtering; proton beam writing, where fast (typically MeV) protons are used to direct-write deep precise 3D patterns into resist, and ion projection lithography (IPL), where medium energy ions (typically 100 keV) are projected through a patterned mask for rapid fabrication The physical interactions of different ions and electromagnetic radiation with matter is shown

in figure 1.5 [21]

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Figure 1.5 The different physical interactions of fast light ions, slow heavy ions, electrons an electromagnetic radiation (EUV and X-rays) with matter [21]

1.2.5.1 Electron Beam Lithography

There are two ways in which electron beams can be used to irradiate a surface and create

a pattern [22-24] There are the parallel exposure of all pattern elements in a mask at the same time and the sequential (scanning) exposure of one pattern element (pixel) at a time

Projection systems through a mask generally have a high throughput and are less complex than scanning systems The direct write scanning systems are under computer control where a finely focused beam (or beams) of electrons is used to generate the

Although an electron beam can be used in a variety of ways, direct control by a computer offers the ability to generate patterns without the need for a mask This enables the scanning-electron-beam systems to be used for both mask making and direct wafer writing These machines combine high spatial resolution (< 0.1 m) with accurate registration (< 0.1 m) The small electron beams are either rastered or manipulated in a vector mode The vector system is more efficient, since scanning is only performed over areas that are to be exposed on the resist In the vector mode, however, the deflection system accuracy is limited, and to apply corrections a computer system is needed [20]