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Die vorliegende Arbeit beschäftigt sich mit der Synthese und Charakterisierung bisensitiver Hydrogelsysteme für die Realisierung hoch leistungsfähiger chemischer Transistoren in der Mikrofluidik. Dabei wurden unterschiedliche (semi)interpenetrierende Polymernetzwerke auf Basis von N Isopropylacrylamid und Acrylsäure hergestellt und ihre Quelleigenschaften und mechanischen Stabilität bei unterschiedlichen Stimuli untersucht. Hierfür wurde die TANAKA-Kinetik modifiziert, um sie auf Proben unterschiedlicher Aspektverhältnisse anpassen zu können. Es zeigte sich der wechselseitige Einfluss der Teilnetzwerke auf die Quellgeschwindigkeit und Stabilität der (semi)interpenetrierende Polymernetzwerke. Durch eine Optimierung der Synthese konnten die Volumenänderungen der sensitiven Hydrogele gesteigert werden.

Swimming is a fundamental feature in many living systems. Biological microorganisms move in the search of food, appropriate pH, temperature, mate and for many other elements crucial for life. A classic example is sperm cell, which travels thousands of its body length in the complex genital tract of females to reach the egg. Inspired by such unique character and diversified motion abilities of the biological world, researchers have always been intrigued to create small artificial microbots which could swim and perform complex tasks. In his famous talk ’There is plenty of room at the bottom’ in 1960, Richard Feynman suggested designing swallowable doctors which could travel in the blood vessels and perform the surgery. Although seemingly exquisite and far-fetched, this idea laid the foundation stone to pave the path towards building autonomously propelled artificial machines with applications ranging from targeted drug delivery to environmental remediation. However, considerable challenges are yet to be addressed before developing fully functional artificial machines, especially in the biomedical applications. For instance, directed transport in vivo, using man-made artificial machines face many obstacles starting from their fabrication, fuels for powering them and their interactions with the surroundings. Rapid changes in the environment in vivo, would make it difficult in selecting the ideal material and shape design of the microswimmer and would most probably require a flexible structure which could potentially squeeze itself and easily pass through small cavities. With most of the swimmers, in the past, being designed from inorganic materials, leave them unsuitable for biological applications. In addition, the environments inside an animal body is dominated by various complexity such as flows of bodily fluids, cavities and soft tissues. In laboratory settings, often these peculiarities are ignored as mostly the motion behavior is tested in stagnant conditions on solid substrates and it is unclear how would an artificial machine will behave in such complex environments. In this thesis, we combined the advances in microfluidics to benefit the microswimmer research manifold. In the last few years, microfluidics and micromotors have been used together in various instances because of their co-sharing regime of low Reynolds number and excellent fluid manipulation abilities at the microscale. In addition, microfluidics offer unique opportunities in designing structures with well-engineered shapes. With these points in mind, in this thesis, we used microfluidics to fabricate microswimmers and design custom made environments to mimic the complexity present in vivo, and to study the feedback of artificial swimmers in them. Specifically, in the first part of the thesis, two microfluidic strategies namely droplet microfluidics and stop-flow lithography were investigated to design hydrogel-based micromotors. Besides, in the next part, we developed complex environments and studied the motion behavior of conventional microswimmers in them. In the first subpart of the thesis, using droplet microfluidics, we designed polyacrylamide and poly (ethylene glycol) diacrylate (PEGDA) based Janus droplets using co-flowing phases with enzyme immobilized in one of the phases to confer asymmetry. The droplets were polymerized on-chip using UV polymerization. We found that the polyacrylamide and PEGDA 565 particles did not result into efficient bubble production when suspended in H2O2 solution and we explain this behaviour using the analogy of smaller pore size and possible poisoning of the enzyme by acrylamide. But, when a 10 v/v% PEGDA 700 was selected as the polymer material, it resulted in very efficient bubble evolution, although the Janus geometry was compromised which restricted swimming for these particles. The second subpart dealt with applying stop-flow lithography technique for designing hydrogel micromotors with different shapes and these shapes corresponded to different swimming modes. Exploiting laminar flow in the low Reynolds number region in the microfluidics channels, we fabricated micromotors with variable composition, shape and controlled active regions. Furthermore, we studied the different trajectories resulting from the complex interactions between swimmer body and fluid dynamics around it and connected them to the theoretical findings. We found close agreement between the experimental results and the theoretical outcomes: I-shaped structure behaved as a pump, U-shaped as a propeller and S-shaped as a rotor. Post fabrication, during real applications, the micromotors will be exposed to complex environments for instance interfaces and flows. To evaluate the feedback of microswimmers in these situations, in the next two sections, we designed custom made environments using microfluidics and we studied the response of well-studied Janus microswimmers in them. It should be noted that in the following two sections we used Janus particles rather than the bubble driven swimmers (fabricated in the first section) for simplicity. In this section, we designed an oil-water interface using a special microfluidic trap design and explored the motion behaviour of a very well-studied Pt@SiO2 Janus micromotors on them. The chip geometry facilitated on-demand merging of a droplet of particles and the ‘fuel’ (H2O2) inside the trap. Additionally, the large surface of the trap resulted in high surface energy which was compensated by partial wetting of the glass substrate. This partial wetting created patches of oil on the glass which we refer to as ‘oil dimples’. The dimples gave us the unique opportunity to directly compare the propulsion and performance of Janus motors at both interfaces (oil-water and solid-water) within the same setup and under similar experimental conditions. The swimming pattern and the speed values were found to be similar at the two interfaces and we conjecture an interplay of various factors such as microscale friction, lubrication, surface locking by the surfactant, reaction product absorption by oil and potential Marangoni influences for this similarity. In the next section, we designed a laminar flowing system using a square glass capillary and studied the response of a spherically symmetrical Janus micromotor in the conditions of flow. Previously, in the literature the response of Pt@SiO2, which is a model pusher-type micromotor, has been studied and they have been demonstrated to migrate cross-stream when the flow is imposed. In this thesis, we introduce a Cu@SiO2 colloid which we hypothesize to resemble a puller-type configuration based on theoretical flow field calculations. Additionally, in the literature, it has been predicted that pullers would exhibit upstream migration when placed under the conditions of flow. Indeed, when placed under flow, these particles migrate upstream, resembling many of the swimmers from biological world. These experimental findings are recovered theoretically using a simple squirmer model in puller configuration. The model also predicted a unique jumping behaviour for these particles, at very high flow rate. When increasing the flow rate in the experiments, we actually capture this characteristics. Finally, based on the theoretical flow field calculations and particularly their upstream response in the imposed flow, we conjecture a puller configuration for Cu@SiO2 micromotors. To sum up, this thesis made important advances by creating a number of different shapes of microswimmers and designing complex environments using microfluidics in which microswimmers can be placed and their response can be studied. Although, in this thesis we emphasized on Janus particles, in future, these custom-made environments can be used to assess the behaviour of other microswimmers including biological ones. While still many engineering and medical problems need to be solved before fully functional applications of artificial microswimmers are realized, manifestations of various shape designs and understanding their behaviours in complex surroundings are the first crucial steps.:Contents: Acknowledgements List of Abbreviations 1. Introduction 2. Fundamentals of active matter and microfluidics 2.1. Active matter 2.1.1. Physical fundamentals of motion at microscale 2.1.2. Biological microswimmers 2.2. Review Paper: Microfluidics for microswimmers 3. Aims and Motivation 4. Results and Discussion 4.1. Microfluidics for fabrication of microswimmers 4.1.1. Introduction 4.1.2. Droplet microfluidics 4.1.3. Stop-flow lithography 4.1.4. Paper - Fundamental Modes of Swimming Correspond to Fundamental Modes of Shape: Engineering I–, U–, and S– Shaped Swimmers 4.2. Microfluidics for specific environments: Interfaces 4.2.1. Introduction 4.2.2. Paper - Study of Active Janus Particles in the Presence of an Engineered Oil–Water Interface 4.3. Microfluidics for specific environments: Flow 4.3.1. Introduction 4.3.2. Paper - Upstream rheotaxis of catalytic Janus spheres 5. Summary and Final Remarks 6. Experimental Details 6.1. Fabrication of hydrogel particles using droplet microfluidics 6.2. Characterization of the hydrogel particles 6.3. Motion studies of the hydrogel particles A. Appendix A.1. Droplet microfluidics A.2. Stop-flow lithography A.3. Microfluidics for specific environments: Interfaces A.4. Microfluidics for specific environments: Flow B. List of publications Bibliography C. Erklärung

Biological cell populations, such as in tissues or microbial communities, are constantly subject to different sources of noise and variability. Despite this, multicellular systems are still able to function properly because cells coordinate with each other by communication. Using biological model systems to study this multiscalar process can be challenging because of their innate complexity. In this thesis, we address this challenge by building a synthetic multicellular system using bottom-up in vitro assembly approaches. Using this platform, we aim to study the effect of cell-to-cell communication to population variability in a minimal and simplified context. To achieve this, we require a synthetic cell population with (i) quantifiable gene expression dynamics, (ii) customizable population variability, and (iii) intercellular communication. Having these characteristics will allow us to test different initial configurations of population variability and monitor population gene expression dynamics with and without cell-to-cell communication. To generate these synthetic cell populations, reconstituted cell-free expression systems (CFES) are encapsulated into monodisperse-sized liposomes using double-emulsion microfluidics. Both transcription and translation levels are simultaneously monitored and quantified to develop models of cell-free gene expression dynamics and differentiate between bulk and encapsulated formats. Population variability was then incorporated by combining different batches of cells to create distinct subpopulations or by using a two-inlet double-emulsion microfluidic device to generate single populations with a large dispersion of encapsulated DNA template. Lastly, genetic circuits based on the quorum sensing system of Vibrio fischeri are used to implement diffusion-mediated intercellular signalling. Quorum sensing gene circuits in Escherichia coli extract-based CFES were tested in bulk and phase transfer-generated synthetic cells. Together with these experimental systems, corresponding models of synthetic cell populations that can account for population variability and secrete-and-sensing communication are developed using mixed-effects models and moment dynamics. Overall, this work leverages CFES and microfluidic technologies to reproducibly generate a simplified in vitro model of multicellular systems that can be easily monitored spatiotemporally to study multi-scalar processes.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography / Biologische Zellpopulationen, z.B. in Geweben oder mikrobiellen Gemeinschaften, sind ständig verschiedenen Quellen von Rauschen und Variabilität ausgesetzt. Trotzdem sind multizelluläre Systeme in der Lage, ordnungsgemäß zu funktionieren, weil sich die Zellen durch Kommunikation miteinander abstimmen. Die Verwendung biologischer Modellsysteme zur Untersuchung dieses multiskalaren Prozesses kann aufgrund ihrer angeborenen Komplexität eine Herausforderung darstellen. In dieser Arbeit gehen wir diese Herausforderung an, indem wir ein synthetisches multizelluläres System mit Hilfe von Bottom-up-in vitro-Assembly-Ansätzen aufbauen. Mit Hilfe dieser Plattform wollen wir die Auswirkungen der Kommunikation von Zelle zu Zelle auf die Populationsvariabilität in einem minimalen und vereinfachten Kontext untersuchen. Um dies zu erreichen, benötigen wir eine synthetische Zellpopulation mit (i) quantifizierbarer Genexpressionsdynamik, (ii) anpassbarer Populationsvariabilität und (iii) interzellulärer Kommunikation. Mit diesen Eigenschaften können wir verschiedene Ausgangskonfigurationen der Populationsvariabilität testen und die Genexpressionsdynamik der Population mit und ohne Zell-zu-Zell-Kommunikation beobachten. Um diese synthetischen Zellpopulationen zu erzeugen, werden rekonstituierte zellfreie Expressionssysteme (CFES) mit Hilfe der Doppelemulsions-Mikrofluidik in monodisperse Liposomen eingekapselt. Sowohl die Transkriptions- als auch die Translationsraten werden gleichzeitig überwacht und quantifiziert, um Modelle für die Dynamik der zellfreien Genexpression zu entwickeln und zwischen Bulk- und verkapselten Formaten zu unterscheiden. Die Variabilität der Populationen wurde dann durch die Kombination verschiedener Zellchargen zur Bildung unterschiedlicher Subpopulationen oder durch die Verwendung einer mikrofluidischen Doppelemulsionsvorrichtung mit zwei Einlässen zur Erzeugung einzelner Populationen mit einer großen Streuung der eingekapselten DNA-Vorlage einbezogen. Schließlich werden genetische Schaltkreise auf der Grundlage des Quorum-Sensing-Systems von Vibrio fischeri verwendet, um diffusionsvermittelte interzelluläre Signalübertragung zu implementieren. Quorum-Sensing-Genkreisläufe in CFES auf der Basis von Escherichia coli-Extrakten wurden in synthetischen Zellen getestet, die durch Bulk- und Phasentransfer erzeugt wurden. Zusammen mit diesen experimentellen Systemen wurden entsprechende Modelle synthetischer Zellpopulationen entwickelt, die die Populationsvariabilität und die Sekretions- und Sensing-Kommunikation mit Hilfe von Mixed-Effects-Modellen und Momentendynamik berücksichtigen können. Insgesamt nutzt diese Arbeit CFES- und Mikrofluidik-Technologien, um reproduzierbar ein vereinfachtes in vitro-Modell multizellulärer Systeme zu erzeugen, das leicht raum-zeitlich überwacht werden kann, um multiskalare Prozesse zu untersuchen.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography

Some of the major obstacles the microfluidics industry has yet to overcome in order to facilitate large scale manufacturing of devices are costly back-end processes. Among these, bonding presents some of the most obvious difficulties and is often associated with structural deformation and surface modification. Off-stoichiometric thiol-ene (OSTE) is a relatively new material and hasn’t yet achieved the same level of adoption as Polydimethylsiloxane (PDMS) which has been the go-to material in the field of microfluidics for over two decades. OSTE offers an alternative to PDMS and promises bonding without surface treatment as well as a hydrophilic surface, removing a step in the manufacturing process. In this work, the property of OSTE to bond with a variety of commonly used thermoplastic materials were tested as well as its suitability for use in pharmaceutical devices such as Lab-on-a-chip. In addition to untreated OSTE, a surface modifier was used to examine the potential for surface modification when using OSTE as a microfluidics material. From the testing performed, we demonstrated OSTE’s capacity to form robust bonds with a range of thermoplastic materials as well as comparable biocompatibility to PDMS. / Bland de största hindren som industrin ännu ej löst när det kommer till storskalig produktion av mikrofluidiska produkter är kostsamma ”back-end” processer. Av dessa presenterar bindingsprocesser några av de mest uppenbara svårigheterna och medför ofta deformationer av finstrukturer samt ändringar i ytkemi. Off-stoichiometric thiolene (OSTE) är ett relativt nytt material och har ännu inte blivit lika utbrett i sin använding som Polydimethylsiloxane (PDMS) vilket har varit standardmaterialet i mikrofluidik i över två årtionden. OSTE erbjuder ett alternativ till PDMS, med bindingsprocesser som ej kräver ytterligare ytmodifikationer och en hydrofil yta, vilket eliminerar ett steg i tillverkningsprocessen. I detta arbete undersöktes egenskapen av OSTE att binda till en rad ofta använda thermoplaster samt dess lämplighet i medicinskt bruk, i system som ”Lab-on-a-chip”. Förutom obehandlad OSTE, så användes en ytmodifierare för att undersöka möjligheten för ytmodifiering vid användingen av OSTE i mikrofluidik. Resultaten av våra tester visade OSTE’s förmåga att forma robusta bindingar till en rad thermoplaster så väl som en jämförbar biokompatibilitet till PDMS.

Microfluidics covers the science of manipulating small quantities of fluids using microscale devices with great potential in analysis, multiplexing, automation and high-throughput screening. Compared to conventional systems, microfluidics benefits from miniaturization resulting in shortened time of experiments, decreased sample and reagent consumptions as well as reduced overall costs. For microfluidic devices where further weight and cost reduction is additionally required, stimuli-responsive hydrogels are particularly interesting materials since they can convert an environmental stimulus directly to mechanical work without any extra power source. Hydrogels are used as chemostats, micropumps, and chemo-mechanical valves in microfluidics. Existing studies about hydrogels for flow control reported on hydrogels responsive to only one stimulus, including temperature, pH value, and solvent. Combining temperature and pH stimuli within one material is an interesting approach, which allows internal as well as external flow control and broadens potential applications. Among the variety of temperature- and pH-responsive monomers, N-isopropylacrylamide (NiPAAm) and acrylic acid (AA) are considered as ideal building blocks to obtain a hydrogel with pronounced stimuli response. There are different architectures for realizing a temperature- and pH-responsive hydrogel with NiPAAm and AA (e.g. copolymer gels, interpenetrating polymer networks (IPNs), semi-IPNs, or graft copolymer gels). Each approach has its inherent benefits and disadvantages. Grafted hydrogels with a temperature-responsive backbone and pH-responsive graft chains are a promising architecture overcoming drawbacks of copolymer gels (loss of thermoresponsive behavior due to the comonomer), interpenetrating polymer networks (IPNs, difficult fabrication of structured particles via soft lithography), and semi-IPNs (leakage of penetrating polymer). However, studies about multi-responsive grafted hydrogels for flow control in microfluidics are comparatively rare and further research is needed to emphasize their real potential. For this reason, the overall aim of this work was the synthesis of temperature- and pH-responsive grafted hydrogels based on NiPAAm and AA for flow control in microfluidics. This required the synthesis of a pH-responsive macromonomer by RAFT polymerization. As a suitable chain transfer agent with a carboxylic acid group for an end-group functionalization, 2-(dodecyl-thiocarbonothioylthio)-2-methylpropionic (DTP) acid was employed. The approach towards the synthesis of the pH-responsive macromonomer based on two key steps: (i) attaching a functional group, which retains during RAFT polymerization, and (ii) conducting the RAFT polymerization to synthesize the pH-responsive macromonomer. In total, four functionalizations for the macromonomer were investigated, including allyl, unconjugated vinyl, acrylamide, and styrene. End-group analysis and solubility tests revealed that macromonomers with a styrene functionalization are suitable for the synthesis of graft copolymer gels. A series of grafted net-PNiPAAm-g-PAA-styrene hydrogels with a PNiPAAm backbone and PAA-styrene graft chains (Mn = 4200 g/mol, Mw/Mn = 1.6) were prepared and characterized. The main goal was to identify suitable stimuli for an application as a chemo-mechanical valve and to show reversibility of the swelling and shrinking process. Importantly, the temperature sensitivity should be retained, while a pH response needs to be introduced. Equilibrium swelling studies quantified with the response ratio revealed that a grafting density of PAA-styrene between 0.25 and 1 mol-% provides a suitable response towards temperature, pH, salt, and solvent. Furthermore, the swelling and shrinking process is highly reproducible over four consecutive cycles for all four stimuli. In order to evaluate the swelling kinetics of grafted net-PNiPAAm-g-PAA-styrene hydrogels, the collective diffusion model extended by a volume specific surface was applied. The determined cooperative diffusion coefficients of net-PNiPAAm-g-PAA-styrene indicated faster response time with increasing PAA-styrene content. Remarkably, net-PNiPAAm-g-PAA-styrene containing 1 mol-% PAA-styrene exhibited an accelerated swelling rate by a factor of 9 compared to pure net-PNiPAAm. Rheological analysis of net-PNiPAAm-g-PAA-styrene showed that an increasing graft density leads to decreasing mechanical stability. The photopolymerization experiments showed that the gelation time linearly increases with the grafting density. Grafted net-PNiPAAm-g-PAA-styrene hydrogels were tested in two fluidic setups for flow control. A straightforward fluidic platform was developed consisting of a fluid reservoir, an inlet channel, an actuator chamber and an outlet channel. The actuator chamber was filled with crushed hydrogel particles. Accordingly, the fluid flow was directed by the active resistance of the hydrogel particles in the actuator chamber (i.e. swelling degree) and allowed flow control by the local environmental conditions. Flow rate studies showed that the fluid flow throttles when the inlet channel was provided with a solution in which the hydrogel swells (pH 9 buffer solution at room temperature). In contrast, the hydrogel-based valve opens immediately when a solution was used in which the hydrogel collapses. The advantageous properties of net-PNiPAAm-g-PAA-styrene were highlighted by using pH, salt and solvent stimulus in one experiment. Remarkably, the opening and closing function was reversible over six consecutive cycles. As part of a collaboration project with the chair of polymeric microsystems within the Cluster of Excellence Center for Advancing Electronics Dresden (A. Richter and P. Frank), membrane assures hydraulic coupling in a chemo-fluidic membrane transistor (CFMT) and grafted net-PNiPAAm-g-PAA-styrene hydrogels were combined to emphasize the potential of both systems. Flow rate studies showed that 4 different stimuli can be used to control the opening and closing state of the CFMT. Multiple opening and closing cycles revealed no considerable changes in the valve function emphasizing a high potential for an application in microfluidics.

In der vorliegenden Arbeit wird eine integrierbare Pumpentechnologie für polymerbasierte mikrofluidische Systeme entwickelt. Ausgehend von den Anforderungen für die Durchführung molekulardiagnostischer Nachweise kommen dabei Fertigungsverfahren zum Einsatz, die sich auch für Einweg-Anwendungen eignen. Das genutzte Aktorprinzip für die integrierten Mikropumpen basiert auf der Elektrolyse von Wasser. Zur besseren technologischen Integrierbarkeit wird das Wasser in Form eines Hydrogels appliziert. Der Elektrolyt wird dabei mit einer Polymermembran mit geringer Wasserdampfdurchlässigkeit verschlossen. Die Membran wird in ihrem plastischen Verformbereich genutzt. Zur Dimensionierung der Mikropumpen und des mikrofluidischen Systems werden analytische und numerische Modelle entwickelt, die eine gute Übereinstimmung mit den Messwerten zeigen. Die Funktionsfähigkeit wird anhand zweier vollständig integriert ablaufender Immunoassays demonstriert. Dabei kommt ein polymerbasierter, optischer Biosensor zum Einsatz.

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