Synthetic membranes in microfluidic interfaces

This thesis explores the development of microfluidic technology for generating and manipulating micro-sized vesicles with the incorporation of specific membrane proteins as artificial cellular systems to mimic natural existing cells. Synthetic biology (SynBio) is an emerging area of research concerned with the application of engineering methods to the creation of new biological processes and constructs. Understanding the working principle of living cellular system is one of significant issue for scientists working in this field. Cells are known as the basic unit of life: creating model synthetic analogues offers opportunities for us to deepen our insights of complex interaction and to understand features and functions of the living cells. Microfluidic technologies have provided the capabilities of compartmentalisation, monodispersity and high-throughput generation for engineering architectures resembling cell-like structures. In vitro transcription and translation (IVTT) enables the expression of specific proteins of interest within synthetic cells via encapsulation of cell-free protein expression solution has demonstrated artificial cells with the capability of containing the process of central dogma of molecular biology. The thesis investigates the building of synthetic cell-like constructs by microfluidics. The first area of investigation focusses on the fabrication of lipid/polymer vesicles transformed from ultra-thin shell double emulsions, which were prepared using microfluidics. To bring the biological function into both vesicle-based synthetic chassis, a fluorescent protein and a pore-forming membrane protein were in vitro expressed in the artificial cell chassis. The second area of study centres on the viscosity analysis of artificial cell membranes using a combination of molecular rotors and the fluorescence lifetime imaging microscopy. The membrane viscosity plays a crucial role in membrane proteins insertion that influences the cell function regulation through functional biomembranes. The alteration of lifetime of the molecular rotors trapped in the artificial membranes reports the viscosity changes in the membrane environment induced by the dewetting process. Comparisons of viscosity values over time between lipid vesicle templated by thin-shell double emulsions with GUVs produced by an oil-free method (electroformation) offers the ability of measuring the amount of oil phase (organic solvent mixture) in artificial cell membranes. In the final chapter, the research detailed the construction of lipid bilayers with asymmetric arrangement used as more complex artificial cell models compared with most of synthetic cells with symmetric composition in their bilayers. A vesicle with hybrid asymmetric bilayer is also fabricated in same microfluidic fashion where phospholipid deposits on the inner-leaflet and block copolymer coats the outer monolayer. Taken together, the work presented in this thesis shows the potential to exploit the microfluidic construction of a functioning synthetic cell from individual molecular components, which could advance new application areas in biotechnology and health. Further developments in this research will aim to develop microfluidic technologies for: (i) physically investigating cell division process using lipid vesicles as cell models; and (ii) producing complex multicompartmental systems for the use of mimicking natural cells. The asymmetric bilayers will be studied for their influences on the integration of transmembrane proteins

Droplet Microfluidics

Droplet microfluidics has dramatically developed in the past decade and has been established as a microfluidic technology that can translate into commercial products. Its rapid development and adoption have relied not only on an efficient stabilizing system (oil and surfactant), but also on a library of modules that can manipulate droplets at a high-throughput. Droplet microfluidics is a vibrant field that keeps evolving, with advances that span technology development and applications. Recent examples include innovative methods to generate droplets, to perform single-cell encapsulation, magnetic extraction, or sorting at an even higher throughput. The trend consists of improving parameters such as robustness, throughput, or ease of use. These developments rely on a firm understanding of the physics and chemistry involved in hydrodynamic flow at a small scale. Finally, droplet microfluidics has played a pivotal role in biological applications, such as single-cell genomics or high-throughput microbial screening, and chemical applications. This Special Issue will showcase all aspects of the exciting field of droplet microfluidics, including, but not limited to, technology development, applications, and open-source systems

Micromachined hot wire sensors for turbulence measurement applications

Hot wire anemometers are a useful and reliable tool for measuring turbulent flow quantities. Nevertheless, the measurement of small-scale eddies, particularly at the Kolmogorov scale, remains to be a challenge. This can be solved by microfabrication, reducing the hot wire’s dimensions to be comparable to the Kolmogorov length. Single wire and double 45° inclined hot wire sensors were microfabricated. By optimizing the etching process, freely suspended Pt wires with 150 μm length ×3 μm width ×0.3 μm thickness were successfully fabricated. They were calibrated in a small wind tunnel, operating at constant temperature mode. A very high cutoff frequency exceeding 200 kHz was measured for a wire with 3×0.3 μm² cross section. The observed flow velocity responses comply with King’s law. In addition, a type of sensor configuration with double 45° wires proved to have excellent directional responses.Hitzdrahtanemometer bieten eine effektive und zuverlässige Möglichkeit, Geschwindigkeit, Scherstress und Vortizität eines turbulenten Flussfeldes zu bestimmen. Durch die Anwendung von Mikrotechnologien, womit die Abmessungen des Hitzdrahts in den Bereich der Kolmogorov-Länge reduziert werden können, können kleinste Wirbel vermessen werden. Sensoren mit Einzeldrähten sowie mit zwei jeweils ±45° geneigten Drähten mit kleinsten Abmessungen von 150 μm Länge, 3 μm Breite und 0.3 μm Dicke wurden hergestellt. Zur Vermeidung von Unterätzungen der Si-Zinken wurden Additive zu TMAH dazugegeben, wodurch auch die durch Pt katalysierte Dekomposition von H2O2 deutlich reduziert werden konnte. Die Sensoren wurden im Konstant-Temperatur-Modus betrieben und die Dämpfung des Schaltkreises wurde mit der Root-Locus-Methode analysiert. Dabei spielen der Offset, das Verstärkungs-Bandbreite-Produkt und der Überhitzungsgrad eine wesentliche Rolle für die Stabilität. Die Schlussfolgerungen wurden mittels Spice-Simulation verifiziert. Ein Square-Wave-Test wurde benutzt, um die Grenzfrequenz abzuschätzen. Für einen Draht mit 3 × 0.3 μm² Querschnitt wurden 200 kHz gefunden. Die Sensoren wurden in einem kleinen Windkanal kalibriert. Das Geschwindigkeits-Signal folgt dem King’schen Gesetz. Für die jeweilige Anwendung muss per Design ein sinnvoller Kompromiss zwischen Richtungsempfindlichkeit und räumlicher Auflösung gefunden werden. Die Messergebnisse zeigen, dass die ±45° Doppeldraht-Sensoren eine sehr gute Richtungsempfindlichkeit aufweisen

Asymmetrical flow field-flow fractionation in the study of water-soluble macromolecules

Asymmetrical flow field-flow fractionation (AsFlFFF) was constructed, and its applicability to industrial, biochemical, and pharmaceutical applications was studied. The effect of several parameters, such as pH, ionic strength, temperature and the reactants mixing ratios on the particle sizes, molar masses, and the formation of aggregates of macromolecules was determined by AsFlFFF. In the case of industrial application AsFlFFF proved to be a valuable tool in the characterization of the hydrodynamic particle sizes, molar masses and phase transition behavior of various poly(N-isopropylacrylamide) (PNIPAM) polymers as a function of viscosity and phase transition temperatures. The effect of sodium chloride salt and the molar ratio of cationic and anionic polyelectrolytes on the hydrodynamic particle sizes of poly (methacryloxyethyl trimethylammonium chloride) and poly (ethylene oxide)-block-poly (sodium methacrylate) and their complexes were studied. The particle sizes of PNIPAM polymers, and polyelectrolyte complexes measured by AsFlFFF were in agreement with those obtained by dynamic light scattering. The molar masses of PNIPAM polymers obtained by AsFlFFF and size exclusion chromatography agreed also well. In addition, AsFlFFF proved to be a practical technique in thermo responsive behavior studies of polymers at temperatures up to about 50 oC. The suitability of AsFlFFF for biological, biomedical, and pharmaceutical applications was proved, upon studying the lipid-protein/peptide interactions, and the stability of liposomes at different temperatures. AsFlFFF was applied to the studies on the hydrophobic and electrostatic interactions between cytochrome c (a basic peripheral protein) and anionic lipid, and oleic acid, and sodium dodecyl sulphate surfactant. A miniaturized AsFlFFF constructed in this study was exploited in the elucidation of the effect of copper (II), pH, ionic strength, and vortexing on the particle sizes of low-density lipoproteins.Väitöstyössä valmistettiin asymmetrinen virtauskenttävirtausfraktiointi(AsFlFFF)laitteisto ja tutkittiin sen soveltuvuutta teollisten, biokemiallisten ja farmaseuttisten näytteiden analysointiin. Laitteistolla tutkittiin useiden muuttujien kuten pH:n, ionivahvuuden, lämpötilan ja reaktanttien sekoitussuhteen vaikutusta tutkittavan näytteen hiukkaskokoon, moolimassaan sekä aggregaatioon. Teollisten näytteiden kohdalla AsFlFFF osoittautui tehokkaaksi työkaluksi poly(N-isopropyyliakryyliamidin) (PNIPAM) hydrodynaamisen hiukkaskoon, moolimassan sekä faasinmuutoskäyttäytymisen määrityksessä, kun ajoliuoksen viskositeettia ja lämpötilaa vaihdeltiin. Tutkittiin myös natriumkloridin sekä eri moolisuhteissa lisättyjen kationisten ja anionisten polyelektrolyyttien vaikutusta poly(metakryloetyylitrimetyyliammoniumkloridin) ja poly(natriummetakrylaatti) - poly(etyleenioksidi) -blokkikopolymeerin sekä näiden välisten kompleksien hydrodynaamiseen hiukkaskokoon. AsFlFFF:llä PNIPAM- polymeerinäytteille saadut hiukkaskoot eivät poikenneet vastaavista dynaamisella valonsironnalla saaduista tuloksista. Suuria eroja ei havaittu myöskään AsFlFFF:llä ja kokoekskluusiokromatografialla PNIPAM-näytteille määritetyissä moolimassoissa. AsFlFFF osoittautui käytännölliseksi tekniikaksi lämpöherkkien polymeerien tutkimuksessa toimittaessa alle 50 oC lämpötiloissa. AsFlFFF:n käyttökelpoisuus biologisiin, biolääketieteellisiin ja farmaseuttisiin sovelluksiin osoitettiin tutkimalla lipidi-proteiini/peptidi vuorovaikutuksia ja liposomien stabiilisuutta eri lämpötiloissa. Menetelmällä tutkittiin anionisen lipidin, oleiinihapon sekä natriumdodekyylisulfaatin ja sytosomi-C:n välillä ilmeneviä hydrodynaamisia ja sähköstaattisia vuorovaikutuksia. Työn yhteydessä rakennettua miniatyrisoitua AsFlFFF-kanavaa käytettiin tutkittaessa kupari(II):n, ionivahvuuden sekä pyörresekoituksen (vortexing) alhaisen tiheyden lipoproteiinin (LDL) partikkelikokoon aiheuttamia muutoksia

Design and synthesis of microcapsules using microfluidics for autonomic self-healing in cementitious materials

A capsule-based self-healing cementitious material, capable of autonomically repairing its own cracks, can extend the service life of concrete structures and decrease the costs associate with repair and maintenance actions. However, the size, shell thickness, shell material and mechanical properties of the capsules still need to be optimised to ensure self-healing performance. Thus, the objective of this research was to explore the controlled microfluidic encapsulation to investigate the production of microcapsules for physically triggered self-healing in cementitious materials. A flow-focusing microfluidic device was used to produce double emulsions to be selectively photopolymerised to generate a core-shell structure. Subsequently, the physical triggering was assessed by embedding the produced microcapsules in cement paste, fracturing it and observing the cracked surface in the SEM. The results showed the production of microcapsules with 80-140 μm of diameter with excellent control over size and shell thickness. Using water-in-oil-in-water (w/o/w) double emulsion, microcapsules were synthesised containing water, colloidal silica solution and sodium silicate solution as core material. In addition, an oil-in-oil-in-water (o/o/w) double emulsion was used to encapsulate mineral oil and emulsified healing agents. The formation of the core-shell structure with aqueous and organic cores was characterised using optical microscopy and SEM. It was demonstrated that the water is not retained inside of the capsule, resulting in the formation of dimples and buckled capsules, particularly for shells thickness ~7 μm. On the other hand, TGA confirmed the retention of mineral oil for shells thickness of ~2 μm and the encapsulation efficiency was demonstrated to be 66%. When the capsules were added to the cement paste, four key factors were observed to prevent physical triggering: (i) thick shells, (ii) buckling of thinner shells due to the loss of water core, (iii) mechanical properties and (iv) poor interfacial bonding. As a result, a mechanical characterisation of the shell material was performed, indicating brittle fracture at room temperature, reduced Young’s modulus when compared with cementitious matrix and stress at rupture of 15-36 MPa. In addition, an innovative methodology was proposed to functionalise the surface of the microcapsules with hydrophilic groups in order to increase the interfacial bonding between the cement paste and the microcapsules. Thus, microcapsules with low tensile strength, low shell thickness, organic core and good interfacial bonding were successfully synthesised and demonstrated to rupture upon crack formation. These results experimentally demonstrate the importance of reduced shell thickness, core retention and interfacial bonding as valuable guides during the design of microcapsules for physically triggered self-healing in cementitious materials.Science without Borders/Brazil (BEX 9185/13-5

Automated Roll-To-Roll Fluidic Self-Assembly Of Microscopic Inorganic Semiconductor Chips For Applications Of Macroelectronics

University of Minnesota Ph.D. dissertation. June 2015. Major: Electrical Engineering. Advisor: Heiko Jacobs. 1 computer file (PDF); vii, 115 pages.This paper presents the implementation of an automated roll-to-roll fluidic self-assembly system based on surface tension driven self-assembly with applications in the field of macroelectronics. The reported system incorporates automated agitation, web motion, component dispensing, and recycling. The process enables the assembly and electrical connection of semiconductor dies/chips in a continuous and parallel fashion over wide area substrates. At present the method achieves an assembly rate of 15,000 chips per hour and an assembly yield exceeding 99%. The identification and modeling of the relationship between process parameters and forces on one side and assembly rates, detachment rates, error rates, and yield on the other is discussed as it lead to the discovery of the reported design. As an application we demonstrate the realization of a solid state lighting module. This particular application requires the assembly of a conductive multilayer sandwich structure which is achieved by combining the introduced assembly process with a novel lamination step. We also have demonstrated rubber-like solid-state-lighting module using developed process

Hydrodynamics

The phenomena related to the flow of fluids are generally complex, and difficult to quantify. New approaches - considering points of view still not explored - may introduce useful tools in the study of Hydrodynamics and the related transport phenomena. The details of the flows and the properties of the fluids must be considered on a very small scale perspective. Consequently, new concepts and tools are generated to better describe the fluids and their properties. This volume presents conclusions about advanced topics of calculated and observed flows. It contains eighteen chapters, organized in five sections: 1) Mathematical Models in Fluid Mechanics, 2) Biological Applications and Biohydrodynamics, 3) Detailed Experimental Analyses of Fluids and Flows, 4) Radiation-, Electro-, Magnetohydrodynamics, and Magnetorheology, 5) Special Topics on Simulations and Experimental Data. These chapters present new points of view about methods and tools used in Hydrodynamics

Engineered environments for biomedical applications: anisotropic nanotopographies and microfluidic devices

During the last two decades micro- and nano-fabrication techniques originally developed for electronic engineering have directed their attention towards life sciences. The increase of analytical power of diagnostic devices and the creation of more biomimetic scaffolds have been strongly desired by these fields, in order to have a better insight into the complexity of physiological systems, while improving the ability to model them in vitro. Technological innovations worked to fill such a gap, but the integration of these fields of science is not progressing fast enough to satisfy the expectations. In this thesis I present novel devices which exploit the unique features of the micro- and nanoscale and, at the same time, match the requirements for successful application in biomedical research. Such biochips were used for optical detection of water-dispersed nanoparticles in microchannels, for highly controlled cell-patterning in closed microreactors, and for topography-mediated regulation of cell morphology and migration. Moreover, pilot experiments on the pre-clinical translation of micropatterned scaffolds in a rat model of peripheral nerve transaction were initiated and are ongoing. Given these results, the devices presented here have the potential to achieve clinical translation in a short/medium time, contributing to the improvement of biomedical technologies

Modern Surface Engineering Treatments

Surface engineering can be defined as an enabling technology used in a wide range of industrial activities. Surface engineering was founded by detecting surface features which destroy most of pieces, e.g. abrasion, corrosion, fatigue, and disruption; then it was recognized, more than ever, that most technological advancements are constrained with surface requirements. In a wide range of industry (such as gas and oil exploitation, mining, and manufacturing), the surfaces generate an important problem in technological advancement. Passing time shows us new interesting methods in surface engineering. These methods usually apply to enhance the surface properties, e.g. wear rate, fatigue, abrasion, and corrosion resistance. This book collects some of new methods in surface engineering