DNA self-assembled filaments for micro- and nanoscale propulsion

Auf der Mikroebene nutzen viele prokaryotische Mikroorganismen die Rotation helikaler Filamente, sogenannter Flagellen, um sich fortzubewegen. Die Nachahmung dieser hoch entwickelten Strukturen eröffnet einen vielversprechender Ansatz für die Konstruktion künstlich angetriebener Mikro- und Nanoroboter. Die Realisierung künstlicher Flagellen bedarf einer Methode, welche die komplexe Proteinarchitektur dieser Filamente möglichst genau imitieren kann. Neben ihrer Hauptfunktion als Träger der Erbinformation hat sich die Desoxyribonukleinsäure (DNA) in den letzten Jahren als vielseitiger Baustein im Bereich der molekularen Selbstassemblierung etabliert. In vorliegender Dissertation werden auf DNA Selbstassemblierung beruhende Filamente vorgestellt, welche als künstliche Flagellen für den Antrieb von Mikro- und Nanoschwimmern sorgen. Mikrometer lange, helikale DNA Nanotubes, welche in Form und Größe bakteriellen Flagellen gleichen, wurden unter Zuhilfenahme des „DNA tile assembly“ realisiert. Das Einfügen und/oder Entfernen von Basenpaaren ermöglichte es uns die helikale Struktur der Nanotubes auf der Nanoebene zu kontrollieren. Nanotubes mit Mikrometer großen helikalen Durchmessern und definierter Chiralität wurden anhand einer neuartigen Design Technik, dem “tile shifting”, konstruiert. Durch das Anbinden der helikalen Strukturen an magnetische Mikropartikel konzipierten wir eine neue Klasse von biokompatiblen Mikroschwimmern. Diese hybriden Strukturen lassen sich durch ein externes Magnetfeld antreiben, steuern und bewegen sich mittels eines Flagellenbündels, ähnlich wie Bakterien, fort. Mithilfe der DNA Origami Technik waren wir zudem in der Lage, DNA Nanoflagellen mit definierter Form zu konstruieren und an magnetische Nanopartikel anzubinden. Die daraus resultierenden Nanoschwimmer wurden magnetisch angeregt und zeigten eine leichte Erhöhung ihrer Diffusion gegenüber einer reinen Brownschen Molekularbewegung. Eine solch erhöhte Diffusion kann für die Fortbewegung von Nanorobotern von Vorteil sein, falls eine gerichtete Bewegung durch thermische Kräfte verhindert wird. Abschließend konstruierten wir einen Prototyp eines sich autonom fortbewegenden DNA-basierten Mikroschwimmers, indem wir den molekularen Motor F – ATPase zwischen DNA Flagellum und Mikropartikel integrierten. Nach Bereitstellung des Motortreibstoffes ATP lösten sich jedoch die künstlichen Flagellen von den Partikeln, wodurch ein Antrieb der Schwimmer verhindert wurde. Die erfolgreiche Anbindung der DNA Flagellen und die Aktivität der integrierten ATPase stellen dennoch wichtige Schritte für die Realisierung autonomer Mikroschwimmer dar. Die in dieser Dissertation gewonnen Erkenntnisse zeigen, dass DNA basierte molekulare Selbstassemblierung ein ausgezeichnetes Instrument für die Konstruktion von biokompatiblen künstlichen Filamenten ist, welche als Antriebsmechanismus künftiger Generationen von Mikro- und Nanorobotern Verwendung finden können.The rotation of helically shaped filaments, so-called flagella, is one major strategy used by many motile microorganisms to achieve propulsion. Mimicking these highly evolved structures can be a promising approach for the construction of artificially propelled micro- and nanorobots. Realizing artificial flagella, however, requires a technique, which can precisely copy the complex architecture of these protein filaments. Besides its fundamental function as carrier of the genetic information, DNA has proven to be a versatile building block in molecular self-assembly. In this dissertation, DNA-self assembled filaments are presented, which function as artificial flagella for the propulsion of micro- and nanoswimmers. Micrometer-long helically shaped DNA nanotubes, which closely resemble bacterial flagella, were constructed by applying the DNA tile assembly technique. Through the insertion and/or deletion of base pairs, we generated bending and twisting in the DNA nanotubes, which allowed us to control the tubes’ helical shape in the nanoscale regime. Nanotubes with microscale helical diameters and defined chirality were constructed by introducing a new design technique, the so-called tile shifting. By coupling the helical filaments to magnetic microparticles, we constructed a new class of fully biocompatible artificial microswimmers. The hybrid structures were actuated and steered by an external magnetic field and propelled by means of a flagellar bundle similar to motile bacteria. The DNA origami technique further allowed us to realize nanometer-sized artificial DNA flagella with defined shape and to attach them onto magnetic nanoparticles. The resulting nanoswimmers were actuated by a magnetic field and exhibited a slight enhancement of their diffusivity, in comparison to a mere Brownian motion. Such an enhanced diffusion can be of advantage for the locomotion of nanorobots if a directed motion is unfeasible due to strong thermal forces. Finally, we constructed a prototype of an autonomously propelling DNA-based microswimmer by incorporating the molecular motor F-ATPase between DNA flagella and magnetic microparticle. Unfortunately, we observed a detachment of the artificial flagella upon addition of the “motor fuel” ATP, which inhibited the propulsion of the swimmer. The successful coupling of the artificial flagella and the activity of the incorporated ATPase, nevertheless, constitute important steps towards the realization of autonomous microswimmers. The insights gained in this dissertation illustrate that DNA molecular self-assembly is an excellent tool for constructing fully biocompatible artificial flagella, which can provide future micro- and nanorobotic devices with an effective propulsion mechanism

Magnetically Driven Micro and Nanorobots

Manipulation and navigation of micro and nanoswimmers in different fluid environments can be achieved by chemicals, external fields, or even motile cells. Many researchers have selected magnetic fields as the active external actuation source based on the advantageous features of this actuation strategy such as remote and spatiotemporal control, fuel-free, high degree of reconfigurability, programmability, recyclability, and versatility. This review introduces fundamental concepts and advantages of magnetic micro/nanorobots (termed here as "MagRobots") as well as basic knowledge of magnetic fields and magnetic materials, setups for magnetic manipulation, magnetic field configurations, and symmetry-breaking strategies for effective movement. These concepts are discussed to describe the interactions between micro/nanorobots and magnetic fields. Actuation mechanisms of flagella-inspired MagRobots (i.e., corkscrew-like motion and traveling-wave locomotion/ciliary stroke motion) and surface walkers (i.e., surface-assisted motion), applications of magnetic fields in other propulsion approaches, and magnetic stimulation of micro/nanorobots beyond motion are provided followed by fabrication techniques for (quasi)spherical, helical, flexible, wire-like, and biohybrid MagRobots. Applications of MagRobots in targeted drug/gene delivery, cell manipulation, minimally invasive surgery, biopsy, biofilm disruption/eradication, imaging-guided delivery/therapy/surgery, pollution removal for environmental remediation, and (bio)sensing are also reviewed. Finally, current challenges and future perspectives for the development of magnetically powered miniaturized motors are discussed

Analysis and Modeling of Magnetized Microswimmers: Effects of Geometry and Magnetic Properties

In recent years, much effort has been placed on development of microscale devices capable of propulsion in fluidic environments. These devices have numerous possible applications in biomedicine, microfabrication and sensing. One type of these devices that has drawn much attention among researchers is magnetic microswimmers--artificial microrobots that propel in fluid environments by being actuated using rotating external magnetic fields. This dissertation highlights our contribution to this class of microrobots. We address issues regarding fabrication difficulties arising from geometric complexities as well as issues pertaining to the controllability and adaptability of microswimmers.The majority of research in this field focuses on utilization of flexible or achiral geometries as inspired by microbiological organisms such as sperm and bacteria. Here, we set forth the minimum geometric requirements for feasible designs and demonstrate that neither flexibility nor chirality is required, contrary to biomimetic expectations. The physical models proposed in this work are generally applicable to any geometry and are capable of predicting the swimming behavior of artificial microswimmers with permanent dipoles. Through these models, we explain the wobbling phenomena, reported by experimentalists. Our model predicts the existence of multiple stable solutions under certain conditions. This leads to the realization that control strategies can be improved by adjusting the angle between the applied magnetic field and its axis of rotation. Furthermore, we apply our model to helical geometries which encompass the majority of magnetic microswimmers. We demonstrate the criterion for linear velocity-frequency response and minimization of wobbling motion. One approach to improve the adaptability of swimmers to various environments is to use modular units that can dynamically assemble and disassemble on-site. We propose a model to explain the docking process which informs strategies for successful assemblies. Most studies conducted so far are to elucidate permanent magnetic swimmers, but the literature is lacking on analysis of swimmers made of soft ferromagnetic materials. In this work, we develop a model for soft-magnetic microswimmers in the saturation regime in order to predict the swimming characteristics of these types of swimmers and compare to those of hard-magnetic swimmers

Controlled Motion of Polymeric Microparticles at Interfaces and in Solution

The research scope of this thesis is the study and the characterisation of the motion of polymeric non-propulsive and propulsive microparticles on the surface and in the bulk. Characterising the motion of these particles as well as investigating different methods to control their motion and to perform useful tasks (e.g. in vivo drug delivery, cargo transport in microfluidic systems and device assembly) has provoked great interest among scientists in recent years. Since the introduction of catalytic nanorods and colloidal nanoswimmers, many attempts have been made to transform the random motion of these particles (due to Brownian phenomena) into a controlled motion, towards a desired location. The work described here is divided into three main categories: Regulating the speed and direction of the particles by modifying the substrate they move upon, steering the particles to a target using gradients of fields such as magnetic, electric, concentration, etc. and controlling the inherent propulsion direction of the particles by fabricating swimmers that are capable of producing a range of trajectories from rotation to linear translation. These methods involve controlling the speed of rolling particles by altering their affinity to the substrate, exploiting the trajectories of pH-responsive particles to produce statistical accumulation within a gradient, directing propulsive magnetic particles via an external uniform magnetic field, utilising the motion of spiralling swimmers to achieve mixing at the microscale and adjusting the area-to-volume ratio of catalytic swimmers to optimise their propulsion speed which is a function of reaction rate on their surface. In addition, the characteristics of these techniques such as their reproducibility, accuracy, autonomy and complexity are also discussed

Reactive inkjet printing and propulsion analysis of silk-based self-propelled micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures

Bio-inspired Magnetic Systems: Controlled Swimming, Fluid Pumps, and Collective Behaviour

This thesis details the original experimental investigations of magnetically actuated and controlled microscopic systems enabling a range of actions at low Reynolds number. From millimetre-robots and self-propelled swimmers to microfluidic and lab-on-a-chip technology applications. The main theme throughout the thesis is that the systems reply on the interactions between magnetic and elastic components. Scientists often take inspiration from nature for many aspects of science. Millimetre to micrometre machines are no exception to this. Nature demonstrates how soft materials can be used to deform in a manner to create actuation at the microscale in biological environments. Nature also shows the effectiveness of using beating tails known as flagella and the apparent enhancements in flow speeds of collective motion. To begin with, a swimmer comprised of two ferromagnetic particles coupled together with an elastic link (the two-ferromagnetic particle swimmer), was fabricated. The system was created to mimic the swimming mechanism seen by eukaryotic cells, in which these cells rely on morphological changes which allows them to propel resulting in approximate speeds of up to 2 body lengths per second. The aim of this system was to create a net motion and control the direction of propagation by manipulating the external magnetic field parameters. It was shown that the direction of swimming has a dependence on both the frequency and amplitude of the applied external magnetic field. A key factor discovered was that the influence of a small bias field, in this case, the Earth’s magnetic field (100 orders of magnitude smaller than the external magnetic field) resulted in robust control over the speed (resulting in typical swimming speeds of 4 body lengths per second) and direction of propulsion. Following this work, swimmers with a hard ferromagnetic head attached to an elastic tail (the torque driven ferromagnetic swimmer) were investigated. These systems were created to be analogous to the beating flagella of many natural microscopic swimmers, two examples would be sperm cells and chlamydomonas cells. These biological cells have typical speeds of 10s of body lengths per second. The main focus of this investigation was to understand how the tail length affects the swimming performance. An important observation was that there is an obvious length tail (5.7 times the head length) at which the swimming speed is maximised (approximately 13 body lengths per second). The experimental results were compared to a theoretical model based on three beads, one of which having a fixed magnetic moment and the other two non-magnetic, connected via elastic filaments. The model shows sufficient complexity to break time symmetry and create a net motion, giving good agreement with experiment. Portable point-of-care systems have the potential to revolutionise medical diagnostics. Such systems require active pumps with low power (USB powered devices) external triggers. Due to the wireless and localisation of magnetic fields could possibly allow these portable point-of-care devices to come to life. The main focus of this investigation was to create fluid pump systems comprising from the previously investigated two-ferromagnetic particle swimmer and the torque driven ferromagnetic swimmer. Building on the fact that if a system can generate a net motion it would also be able to create a net flow. Utilising the geometry of the systems, it has been demonstrated that a swimmer-based system can become a fluid pump by restricting the translational motion. The flow structure generated by a pinned swimmer in different scenarios, such as unrestricted flow around it as well as flow generated in straight, cross-shaped, Y-shaped and circular channels were investigated. This investigation demonstrated the feasibility of incorporating the device into a channel and its capability of acting as a pump, valve and flow splitter. As well as a single pump system, networks of the previously mentioned pump systems were fabricated and experimentally investigated. The purpose of this investigation was to utilise the behaviour of the collective motion. Such networks could also be attached to the walls or top of the channel to create a less invasive system compared to pump based within the channel system. The final investigation involved creating collective motion systems which could mimic the beating of cilia - known as a metachronal wave. Two methods were used to create an analogous behaviour. The first was using arrays of identical magnetic rotors, which under the influence of an external magnetic field created two main rotational patterns. The rotational patterns were shown to be controllable producing useful flow fields at low Reynolds numbers. The second system relied on the magnetic components having different fixed magnetisation to create a phase lag between oscillations. The magnetic components were investigated within a channel and the separation between the components was shown to be a key parameter for controlling the induced flow. In both cases, a simple model was produced to help understand the behaviour. Finally, a selection of preliminary investigations into possible applications were conducted experimentally. These investigations included, measuring the effective surface viscosity of lipid monolayers, created cell growth microchannels, as well as systems which could be used for blood plasma separation. The properties of lipid monolayers vary with the surface density, resulting on distinct phase transitions. Slight differences in the molecular lattice are often accompanied by significant changes in the surface viscosity and elasticity. The idea was to use a swimmer as a reporter of the monolayer viscosity, resulting in a less invasive method compared to current techniques to monitor monolayer viscosity, for example torsion pendulums and channel viscometers. The reported effective surface viscosity closely matched the typical Langmuir trough measurements (with a systematic shift of approximately 17 Ų/molecule). The blood plasma separation preliminary work shows the previously investigated two-ferromagnetic particle swimmer mixing a typical volume (100 μm) blood sample with a buffer solution in 21 seconds. The system was also able to create locations with a high population of red blood cells. This resulted in a separation between the blood plasma and red blood cells. Two other preliminary results of future investigations were presented; the collective motion of free swimmers, and the fabrication of ribbon-like structures with fixed magnetic moment patterns.European CommissionEngineering and Physical Sciences Research Council (EPSRC

Enzyme Powered Nanomotors Towards Biomedical Applications

[eng] The advancements in nanotechnology enabled the development of new diagnostic tools and drug delivery systems based on nanosystems, which offer unique features such as large surface area to volume ratio, cargo loading capabilities, increased circulation times, as well as versatility and multifunctionality. Despite this, the majority of nanomedicines do not translate into clinics, in part due to the biological barriers present in the body. Synthetic nano- and micromotors could be an alternative tool in nanomedicine, as the continuous propulsion force and potential to modulate the medium may aid tissue penetration and drug diffusion across biological barriers. Enzyme-powered motors are especially interesting for biomedical applications, owing to their biocompatibility and use of bioavailable substrates as fuel for propulsion. This thesis aims at exploring the potential applications of urease-powered nanomotors in nanomedicine. In the first work, we evaluated these motors as drug delivery systems. We found that active urease- powered nanomotors showed active motion in phosphate buffer solutions, and enhanced in vitro drug release profiles in comparison to passive nanoparticles. In addition, we observed that the motors were more efficient in delivering drug to cancer cells and caused higher toxicity levels, due to the combination of boosted drug release and local increase of pH produced by urea breakdown into ammonia and carbon dioxide. One of the major goals in nanomedicine is to achieve localized drug action, thus reducing side-effects. A commonly strategy to attain this is the use moieties to target specific diseases. In our second work, we assessed the ability of urease-powered nanomotors to improve the targeting and penetration of spheroids, using an antibody with therapeutic potential. We showed that the combination of active propulsion with targeting led to a significant increase in spheroid penetration, and that this effect caused a decrease in cell proliferation due to the antibody’s therapeutic action. Considering that high concentrations of nanomedicines are required to achieve therapeutic efficiency; in the third work we investigated the collective behavior of urease-powered nanomotors. Apart from optical microscopy, we evaluated the tracked the swarming behavior of the nanomotors using positron emission tomography, which is a technique widely used in clinics, due to its noninvasiveness and ability to provide quantitative information. We showed that the nanomotors were able to overcome hurdles while swimming in confined geometries. We observed that the nanomotors swarming behavior led to enhanced fluid convection and mixing both in vitro, and in vivo within mice’s bladders. Aiming at conferring protecting abilities to the enzyme-powered nanomotors, in the fourth work, we investigated the use of liposomes as chassis for nanomotors, encapsulating urease within their inner compartment. We demonstrated that the lipidic bilayer provides the enzymatic engines with protection from harsh acidic environments, and that the motility of liposome-based motors can be activated with bile salts. Altogether, these results demonstrate the potential of enzyme-powered nanomotors as nanomedicine tools, with versatile chassis, as well as capability to enhance drug delivery and tumor penetration. Moreover, their collective dynamics in vivo, tracked using medical imaging techniques, represent a step-forward in the journey towards clinical translation.[spa] Recientes avances en nanotecnología han permitido el desarrollo de nuevas herramientas para el diagnóstico de enfermedades y el transporte dirigido de fármacos, ofreciendo propiedades únicas como encapsulación de fármacos, el control sobre la biodistribución de estos, versatilidad y multifuncionalidad. A pesar de estos avances, la mayoría de nanomedicinas no consiguen llegar a aplicaciones médicas reales, lo cual es en parte debido a la presencia de barreras biológicas en el organismo que limitan su transporte hacia los tejidos de interés. En este sentido, el desarrollo de nuevos micro- y nanomotores sintéticos, capaces de autopropulsarse y causar cambios locales en el ambiente, podrían ofrecer una alternativa para la nanomedicina, promoviendo una mayor penetración en tejidos de interés y un mejor transporte de fármacos a través de las barreras biológicas. En concreto, los nanomotores enzimáticos poseen un alto potencial para aplicaciones biomédicas gracias a su biocompatibilidad y a la posibilidad de usar sustancias presentes en el organismo como combustible. Los trabajos presentados en esta tesis exploran el potenical de nanomotores, autopropulsados mediante la enzima ureasa, para aplicaciones biomédicas, y investigan su uso como vehículos para transporte de fármacos, su capacidad para mejorar penetración de tejidos diana, su versatilidad y movimiento colectivo. En conjunto, los resultados presentados en esta tesis doctoral demuestran el potencial del uso de nanomotores autopropulsados mediante enzimas como herramientas biomédicas, ofreciendo versatilidad en su diseño y una alta capacidad para promover el transporte de fármacos y la penetración en tumores. Por último, su movimiento colectivo observado in vivo mediante técnicas de imagen médicas representan un significativo avance en el viaje hacia su aplicación en medicina

Catalytic Self-Phoretic Active Colloids

Active Janus colloids are nano to micron sized colloids, capable of propelling themselves through fluidic environments. Localised, asymmetric catalytic reactions on the colloidal bodies are used to decompose a dissolved ‘fuel’ to produce motion. Active Janus colloids have been proposed for applications in microfluidic transport. Currently active Janus colloids are restricted in their practical applications due to the randomised nature of their trajectories over time and their low yielding production methods. This thesis is focused on active Janus colloids prepared by physical vapour deposition, which utilise hemispherically coated platinum as a catalyst to decompose aqueous hydrogen peroxide. Many theories and mathematical models have been reported and are discussed in this thesis as to the precise nature of the mechanism of motion. To contribute to this discussion, active colloids were prepared with different surface functionalities on the non-catalytic section of the Janus colloids. The results indicated that the hydrophobicity of the non-catalytic face influenced the propulsive velocity of the active colloid which informs on the relationship between the fluid and the phoretic body. In an effort to produce active colloids with non-random, prescribed trajectories, the symmetry of the catalytically active layer was incrementally broken and found to introduce an additional angular velocity. The magnitude of angular velocity was controllable through production parameters. An alternative, more scalable fabrication method was developed during the course of this work. A solution based fabrication method was found to successfully produce active colloids in high concentrations which were phoretically analogous to those previously fabricated. Finally, an investigation into the effect of the active cap shape and surface coverage was conducted. Significantly, this study found that symmetrically active colloids displayed propulsive behaviour. The suggestion that asymmetry is not required for producing enhanced motion can be used to inform and simplify future fabrication methods