The development of a novel approach to the design of microdevices Submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy by Yulia Alekseeva Faculty of Life and Social Sciences Swinburne University of Technology December 2011 Abstract The effectiveness of protein-based microdevices depends on the ability of their surfaces to provide spatial immobilization and maintain protein bioactivities. Although methodologies for the construction of microdevices for biomedical applications have been developed, the manufacturing of microdevices remains expensive due to the high cost of materials and fabrication processes. As the surfaces display structural uniformities which restrict protein-surface interactions and consequently protein immobilization, innovative approaches to the design of surfaces are required. The approaches need to allow for the minimization of fabrication costs via efficient amplification and spatial immobilization of multiplex proteins so that the bioactivity of protein-based microdevices (e.g., microarrays) can be retained. A novel approach to the design of surfaces for microdevices has been developed and evaluated in this work. This approach is based on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. The structures of a 100 nm-range are represented by „combinatorialized‟ micro/nano- channels that allow amplified protein immobilization in a highly controlled manner. The relationship between the properties of the micro/nano-channel surface topography, physico-chemistry, and protein immobilization, for five, molecularly different proteins, i.e., lysozyme, myoglobin, alpha-chymotrypsin, human serum albumin, and human immunoglobulin has been investigated. Using quantitative fluorescence measurements and atomic force microscopy, protein immobilization on microstructures has been characterized. It has been found that the combinatorial nature of the micro/nano-channels allowed a 3 to 10- fold amplification of protein adsorption, as compared to the protein adsorption on flat, chemically homogenous polymeric surfaces. An improved methodology allowing in vitro assembled micron- and nano- scale tracks of proteins (i.e., actin) which support unidirectional translocation of beads functionalized with motor proteins (i.e., myosin) was also developed. The nanotracks composed of aligned F-actin/gelsolin bundles were formed by electrostatic condensation of F-actin/gelsolin with Ba2+. ii The prospects for employment of bacterial ATP producers as replacements for the energy source, and prokaryotic actin homologues as replacements for eukaryotic actin in microdevices based on molecular motor-based systems, have been explored. A search for ATP producers among 86 environmental strains of 17 genera, including 4 species of 3 genera described in this thesis has been performed. Bacteria belonging to the genera Sulfitobacter, Marinobacter and Staleya and/or Planococcus and Kocuria have been found to be promising producers of extracellular and intracellular ATP, respectively. Substitution of eukaryotic actin with inherently stable prokaryotic actin- related proteins, i.e., MreB or FtsA, may point the way to the development of the next generation of microdevices for biomedical applications. iii Acknowledgments This thesis resulted from research supported by the Australian Research Council (ARC) and partially supported by the Defense Advanced Research Projects Agency (DARPA). I acknowledge the great support of the Research Higher Degrees Committee (RHDC) of my alma mater. I enjoyed working on my project. I had the great honor and pleasure of being supervised by a person with a very strong interdisciplinary vision, Professor Elena Ivanova. I acknowledge her incredible support and encouragement. I thank Professor Michael Gilding for his kind words of encouragement and support. I acknowledge the support provided by Professor Pam Green. I thank Professor Russell Crawford for his support. I also wish to thank Professor Dan V. Nicolau for his support; Dr Vlado Buljan and Dr Murat Kekic (the University of Sydney) for sharing their experience in molecular motor protein extraction and handling; Dr Igor Sbarski, Dr Gregory M. Demyashev, Dr Luisa Filipponi, Dr Andrea Viezzoli, Dr Dan V. Nicolau, Jr., Dr Jonathan P. Wright, Dr Duy K. Pham, Marjan Ilkov, Dr Hans Brinkies, Anya Ilkova, Dr Natasa Mitik-Dineva for assisting in laboratory experiments and data analysis. I thank all LSS and IRIS staff members who supported this study. I thank my student teammates who have shared their thoughts with me. I enjoyed a friendly scientific atmosphere of our group meetings. I acknowledge the support of Professor Tomoo Sawabe and Dr Karin Hayashi (Hokkaido University), Professor Richard Christen (the University of Nice Sophia Antipolis), Dr Nataliya I. Kalinovskaya, Dr Natalia V. Zhukova, Dr Galina M. Frolova, Professor Valery V. Mikhailov, Dr Nataliya M. Gorshkova, Dr Valeriya V. Kurilenko, Dr Olga I. Nedashkovskaya (Pacific Institute of Bioorganic Chemistry) iv and Arkady Kurilenko (Pacific Oceanological Institute). I thank Professor Victor P. Chelomin (Pacific Oceanological Institute) for fruitful scientific discussions. I would also like to thank Maryna Mews for her editorial assistance. Last but not least, I would like to express my greatest appreciation to my darling parents, Lyubov & Vladimir, for participating in my son‟s upbringing and supporting this study. Special thanks are extended to all those who helped me in gaining sole custody of my child. And lastly, I would love to thank my son, Maxim, for being a good guy. v This thesis is dedicated to my son, Maxim. vi DECLARATION I certify that the work presented in the thesis contains no material which has been submitted for another degree of any other university. To the best of my knowledge it does not contain any material previously published or written by another person except where due reference is made in the text. Contributions of the respective researchers to this study: Professor Elena Ivanova supervised the research project; Professor Dan V. Nicolau organized the project; Dr Vlado Buljan and Dr Murat Kekic (the University of Sydney) assisted in protein extraction and handling; Dr Igor Sbarski, Dr Gregory M. Demyashev, Dr Luisa Filipponi, Dr Andrea Viezzoli, Dr Dan V. Nicolau, Jr., Dr Jonathan P. Wright, Dr Duy K. Pham, Marjan Ilkov, Dr Hans Brinkies, Anya Ilkova, Dr Natasa Mitik-Dineva assisted in laboratory experiments and data analysis; Professor Tomoo Sawabe and Dr Karin Hayashi (Hokkaido University), Professor Richard Christen (the University of Nice Sophia Antipolis), Dr Nataliya I. Kalinovskaya, Dr Natalia V. Zhukova, Dr Galina M. Frolova, Professor Valery V. Mikhailov, Dr Nataliya M. Gorshkova, Dr Valeriya V. Kurilenko, Dr Olga I. Nedashkovskaya (Pacific Institute of Bioorganic Chemistry), Professor Victor P. Chelomin and Arkady Kurilenko (Pacific Oceanological Institute) assisted in microbiology-related experiments and data analysis. I declare that this thesis has been partially professionally copyedited and proofread by Maryna Mews, however, the editorial assistance did not affect its substantive content. Yulia Alekseeva vii List of Publications Book chapter 1. Critical aspects in microfluidic systems design. Alekseeva YV, Crawford RJ, Ivanova EP. In : Advances in Chemistry Research 15, Nova Publishers (NY), 2012. Journal articles 2. Protein immobilisation on micro/nanostructures fabricated by laser microablation. Nicolau DV, Ivanova EP, Fulga F, Filipponi L, Viezzoli A, Dobroiu S, Alekseeva YV, Pham DK. Biosensors and Bioelectronics 26(4):1337- 1345, 2010. 3. “Pseudoalteromonas januaria" SUT 11 as the source of rare lipodepsipeptides. Kalinovskaya NI, Dmitrenok AS, Kuznetsova TA, Frolova GM, Christen R, Laatsch H, Alexeeva YV, Ivanova EP. Current Microbiology 56(3):199-207, 2008. 4. ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces. Ivanova EP, Alexeeva YV, Pham DK, Wright JP, Nicolau DV. International Microbiology 9(1):37-46, 2006. 5. A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly(tert -butyl methacrylate). Ivanova EP, Wright JP, Pham DK, Brack N, Pigram P, Alekseeva YV, Demyashev GM, Nicolau DV. Biomedical Mater ials 1(1):24-32, 2006. 6. Characterization of unusual alkaliphilic gram-positive bacteria isolated from degraded brown alga thalluses. Ivanova EP, Wright JP, Lysenko AM, Zhukova NV, Alexeeva YV, Buljan V, Kalinovskaya NI, Nicolau DV, Christen R, Mikhailov VV. Microbiological Journal 68(4):10-20, 2006. 7. Controlling the covalent and noncovalent adsorption of proteins on polymeric surfaces in aqueous liquids. Ivanova EP, Pham DK, Alekseeva YV, Demyashev GM, Nicolau DV. Chinese Journal of Light Scattering 17(3):234-236, 2005. 8. Presence of ecophysiologically diverse populations within Cobetia marina strains isolated from marine invertebrate, algae and the environments. Ivanova EP, Christen R, Sawabe T, Alexeeva YV, Lysenko AM, Chelomin VP, Mikhailov VV. Microbes and Environments 20(4):200-207, 2005. 9. Controlled self-assembly of actin filaments for dynamic biodevices. Alexeeva YV , Ivanova EP, Pham DK, Buljan V, Sbarski I, Ilkov M, Brinkies HG, Nicolau DV. Nanobiotechnology 1(4):379-388, 2005. 10. Bacillus algicola sp. nov., a novel filamentous organism isolated from brown alga Fucus evanescens. Ivanova EP, Alex eeva YV, Zhukova NV, Gorshkova NM, Buljan