APPLICATIONS OF MICROFLUIDIC CHIPS IN OPTICAL MANIPULATION & PHOTOPORATION Robert Frank Marchington A Thesis Submitted for the Degree of PhD at the University of St. Andrews 2010 Full metadata for this item is available in Research@StAndrews:FullText at: https://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/1633 This item is protected by original copyright Applications of Microfluidic Chips in Optical Manipulation & Photoporation Robert Frank Marchington A thesis presented for the degree of Doctor of Philosophy Optical Trapping & Microphotonics Groups School of Physics & Astronomy University of St Andrews June 2010 Dedicated to Mum & Mike Joe & Xanthoula Applications of Microfluidic Chips in Optical Manipulation and Photoporation Robert Frank Marchington Submitted for the degree of Doctor of Philosophy June 2010 Abstract Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies pro- mise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electri- cal, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to mani- pulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within micro- fluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. iv v The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique. Declarations Candidate’s declarations: I, Robert Frank Marchington, hereby certify that this thesis, which is approximately 41,000 words in length, has been written by me, that it is the record of work carried out by me, and that it has not been submitted in any previous application for a higher degree. I was admitted as a research student in November 2005 and as a candidate for the degree of Doctor of Philosophy in June 2006; the higher study for which this is a record was carried out in the University of St Andrews between 2005 and 2010. Date . Signature of Candidate . Supervisor’s declarations: I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of Doctor of Philosophy in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date . Signature of Supervisor . Permission for electronic publication: In submitting this thesis to the University of St Andrews I understand that I am gi- ving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested vi vii in the work not being affected thereby. I also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. I have obtained any third-party copyright permissions that may be required in order to allow such access and migration, or have requested the appropriate embargo below. Access to printed copy and electronic publication of thesis through the University of St Andrews. Date . Signature of Candidate . Signature of Supervisor . Acknowledgements I would like to begin by thanking my supervisor Professor Kishan Dholakia, for the opportunity to work in his group, for all the support and guidance he has given me, and for always taking time to help out when needed. I would also like to thank Professor Thomas Krauss for the opportunity to work in his group, and for all the useful discussions with him also. I would like to thank them both for giving me the flexibility in carving my own directions of exploration. The work here has been collaborative throughout and there is a huge array of people to thank. Firstly, the two people that everything hinged on, the legendary George Robb and Stevie Balfour, for their continued efforts in the running of the cleanroom, fixing of equipment and generally helping out in so many occasions. Also to Will and Chris for all of their time and continued help in the cleanroom and SEM, and to Chris and Mark in electronics and all of the workshop guys for making stuff work when it had to! I’d very much like to thank Peter Reece, for his inspiration, letting me come scribble all over his white board, for the brilliant discussions and ideas that would continue on to the Whey Pat. A big thanks to Klaus also, for always being helpful and for teaching me the ropes with the fibre trap. Thanks to Vene for being terrific fun and for all the days on the evanescent work. My lab partner, and office mate, Steve, thanks to you for all your time throughout on so many projects I’ve lost count, but most recently on the bacteria work. Many thanks to Dave Stevenson for all the bio chip cell zap chat throughout, and helping out so so many times and aspects. The same to Lani, thanks for looking after the cells and helping out. Thanks to Lynn and Muriel also. And thanks go to Frank for his help most recently on the photoporation work, but also generally for viii ix his interest and feedback throughout. A big thanks goes to Praveen, for his help in all aspects, from chips, to optics, for all the useful discussions, his lab work and the proof reading. Many thanks also to Doug and Andrea for their help and good times working together, which I extend to Mark who was also kind enough to proof read as well. Thanks also to Helen for her recent efforts in the microfluidics. Also, thank you Peru for being great fun and an doing excellent job on the seal tag project. Thanks to Bernie and Ailsa for giving us that opportunity. Many thanks to Wei Huang for the bacteria samples, and thanks to him and Patience for telephone reassurance after I drank them. From my time in the office, I’d like to show my appreciation of the good old days with Dave Gherardi, Phil, Graham, Dan & Dan. Especially to Graham for his particle tracking software, and his good words on Kishan and the group in the first place, that meant I applied. I express my gratitude to Michael for all his hard work on the modelling, for his insights, for taking the time for us to discuss the theory and proof reading my thesis. Thanks to Anna Chiara for being so helpful during the writing up stage, keeping the place running smoothly. Dave Walsh and Pete need thanking for their amazing homebuilt water cooler, that we installed in the finals hours, saving the day. And thanks for helping out with all the computer troubles and the shutter mounting Dave. Sorry again for breaking that fan! And thanks to Dave Stothard for his continuous source of amusement photographically and acoustically from his nearby lab, and to Tom Edwards for inspiring me to stick around in St Andrews in the first place. A Very big thanks to Yoshi for his immense input into the work in this thesis, for all of his input, his continued efforts and being a great guy to work with. On a more personal note I’d like to thank Mum and Mike for their continued support and encouragement, and particularly Mum for proof reading this entire thesis. And a very big thanks to Xanthi for all of her help in the lab, the discussions throughout, motivating me, helping at the weekends, standing in with the cell work and proof reading this thesis.