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Control of Molecular Motor Motility in Electrical Devices

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Control of Molecular Motor Motility in Electrical Devices Control of Molecular Motor Motility in Electrical Devices Thesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor in Philosophy By Laurence Charles Ramsey Department of Electrical Engineering & Electronics April 2014 i Abstract In the last decade there has been increased interest in the study of molecular motors. Motor proteins in particular have gained a large following due to their high efficiency of force generation and the ability to incorporate the motors into linear device designs. Much of the recent research centres on using these protein systems to transport cargo around the surface of a device. The studies carried out in this thesis aim to investigate the use of molecular motors in lab- on-a-chip devices. Two distinct motor protein systems are used to show the viability of utilising these nanoscale machines as a highly specific and controllable method of transporting molecules around the surface of a lab-on-a-chip device. Improved reaction kinetics and increased detection sensitivity are just two advantages that could be achieved if a motor protein system could be incorporated and appropriately controlled within a device such as an immunoassay or microarray technologies. The first study focuses on the motor protein system Kinesin. This highly processive motor is able to propel microtubules across a surface and has shown promise as an in vitro nanoscale transport system. A novel device design is presented where the motility of microtubules is controlled using the combination of a structured surface and a thermoresponsive polymer. Both topographic confinement of the motility and the creation of localised ‘gates’ are used to show a method for the control and guidance of microtubules. Two further studies use the actin myosin motor protein system. Both concentrate on the manipulation of actin filaments, gliding on immobilised myosin, by DC electrical fields. Motor protein is adsorbed onto several surface chemistries with varying protein adsorption properties. A range of electrical fields are applied to the motility assay and the performance is analysed in terms of the directionality and any changes in the average velocity of filaments on each surface. This enables us to attribute surface properties to particular motility characteristics and hypothesise as to the nature of protein adsorption. The same electrical motility device is used with an alternative method to allow a more detailed study of the effect of surface chemistry on the motility function and the response of the motility after exposure to an electrical field. The movement of actin filaments on myosin motors is accelerated by a DC electrical field. Upon termination of the field the motility is allowed to return to pre-field function and this section of the procedure is analysed together with the data from the previous study to draw conclusions on the protein adsorption properties of each surface. Both chapters are used to draw conclusions on the response of the motor protein system when it is adsorbed on different surface chemistries. The investigations carried out in this thesis are used to show both novel ways of controlling motor protein motility and also to highlight aspects of design that need to be taken into consideration when incorporating motor proteins into lab-on-a-chip devices. The electrical motility device in particular has proved to be a dynamic and inexpensive tool in investigating motor protein motility. i Acknowledgements I would first like to thank Prof. Dan Nicolau for giving me the opportunity to begin this project. Massive thanks goes to Dr. Harm van Zalinge for taking me on as one of his first PhD students at a particularly stressful time in the group’s history. Without the guidance and support offered my continuation in the program would not have been possible. I would like to extend a very special thank you to Dr. Jenny Aveyard (aka the hammer hanson, aka jerry vulva, aka the fun police, aka captain buzzkill, aka chunk, aka full English), your support and expertise have been a constant help along the way but it is your friendship in particular that has helped me get through some pretty tricky times over the course of my time at Liverpool. To Aleks (aka the Russian), I thank you for the constant entertainment in the office and for helping me out when I was being particularly special, if I ever need a fake passport you’ll be the first person I call. Finally I’d like to thank my family who through thick and thin have always been there to offer whatever support they could at the drop of a hat, it honestly would not of been possible to be where I am today without them. ii Table of Contents Chapter 1: Introduction...........................................................................................................1 Chapter 2: Theory. Molecular motors and their engineering potential...................................4 2.1 Motor proteins.....................................................................................................................5 2.2 Kinesin – microtubule system.............................................................................................7 2.2.1 Kinesin protein....................................................................................................9 2.2.2 Microtubules.....................................................................................................10 2.3 Myosin – actin system......................................................................................................12 2.3.1 Myosin protein..................................................................................................14 2.3.2 Actin..................................................................................................................16 2.4 The ATPase cycle.............................................................................................................18 2.4.1 Kinesin ATPase cycle, step sizes and forces....................................................18 2.4.2 Myosin ATPase cycle, step sizes and forces....................................................20 2.5 The in vitro motility assay................................................................................................22 2.6 Future prospects of molecular motors...............................................................................24 2.7 Methods for control and analysis of kinesin and myosin systems....................................28 2.7.1 Topographical confinement...........................................................................................28 2.7.2 Chemical confinement...................................................................................................30 2.7.3 Guidance by electrical fields..........................................................................................32 2.8 References.........................................................................................................................35 Chapter 3: Materials and methods.........................................................................................41 3.1 Kinesin motility with thermoresponsive PNIPAM gate...................................................42 3.1.1 Laser ablation....................................................................................................42 3.1.2 PDMS preparation............................................................................................44 3.1.3 Surface functionalisation..................................................................................45 3.1.4 Motility assay....................................................................................................45 3.2 Electrical motility device for the study of actin myosin motility on polymer surfaces....47 3.2.1 Surface functionalisation..................................................................................47 3.2.2 Electrical motility device..................................................................................51 iii 3.2.3 Motility assay....................................................................................................52 3.3 References.........................................................................................................................55 Chapter 4: Guidance and control of kinesin motility using a thermoresponsive polymer...................................................................................................................................57 4.1 Introduction.......................................................................................................................58 4.2 Application of the thermoresponsive polymer PNIPAM into a novel device for the control of microtubule motility...............................................................................................59 4.2.1 Previous kinesin motility applications..............................................................59 4.2.2 Response of motility to PNIPAM coating on globally heated surface.............61 4.3 Kinesin motility with functioning PNIPAM gate.............................................................63 4.3.1 Combinatorial gating........................................................................................63 4.3.2 Gating device....................................................................................................69 4.4 Conclusions.......................................................................................................................75
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