Non-volatile liquid-film-embedded microfluidic valve for microscopic evaporation control and contactless bio-fluid delivery applications

Quick evaporation speed of microfluids can cause many unexpected problems and failures in various microfluidic devices and systems. In this dissertation, a new evaporation speed controlling method is demonstrated using a thin liquid-film based microfluidic valve. Microfluidic droplet ejectors were designed, fabricated and integrated with the liquid-film based microfluidic valve. The thin liquid film with nonvolatility and immiscibility exhibited excellent microfluidic valve functionality without any stiction problem between valve components, and provided a very effective evaporation protection barrier for the microfluids in the device. Successful evaporation control by the liquid-film-embedded (LiFE) microfluidic valve has been demonstrated. In addition, guided actuation of the microfluidic valve along predefined paths was successfully achieved using newly developed oil-repellent surfaces, which were later used for developing ‘virtual walls’ for confining low surface tension liquids within predefined areas. Moreover, bioinspired slippery surfaces for aiding the microfluidic valve along the ejector surface have also been developed. These slippery surfaces were evaluated for their effectiveness in reducing microfluidic valve driving voltages. Finally, a sliding liquid drop (SLID) shutter technique has been developed for a normally closed functionality with aid from nanostructures. The SLID shutter resolves many issues found in the previous LiFE microfluidic valve. Smooth and successful printing results of highly volatile bio-fluids have been demonstrated using the SLID shutter technique. I envision that these demonstrated techniques and developed tools have immense potential in various microfluidic applications

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target

A Quantitative MALDI-MSI Study of the Movement of Molecules in Biological Systems

The use of mass spectrometry imaging (MSI) for the analysis of 3D tissue models of human skin has been shown to provide an elegant label-free methodology for the study of both drug absorption and drug biotransformation. The main aim of the work presented in this thesis was to develop methodology for quantitative assessment of percutaneous absorption using matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). Quantitative assessment of the absorption of an antifungal agent, terbinafine hydrochloride, into the epidermal region of a commercial full thickness living skin equivalent model (Labskin) was used as a model system. Different approaches to generate robust and sensitive quantitative mass spectrometry imaging (QMSI) data were developed and compared. The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging provided a high-resolution method for the determination of terbinafine hydrochloride in Labskin. A quantitative assessment of the effect of adding a penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle was also performed, and data was compared to LC–MS/MS measurements of isolated epidermal tissue extracts. Comparison of means and standard deviations indicated no significant difference between the values obtained by the two methods. In this thesis the localisation of hydrocortisone hydrochloride in ex-vivo skin was also investigated. Hydrocortisone exhibits a low ionisation efficiency that makes its detection challenging with mass spectrometry techniques. An in-solution and on-tissue chemical derivatisation reaction using the Girard reagent T, a hydrazine based reagent, significantly increased the sensitivity and detection of the respective hydrocortisone-derivative using MALDI-MSI. In an additional study, MALDI-MSI was used to assess the metabolic activity in Labskin by employing the approach of "substrate-based mass spectrometry imaging" (SBMSI). Preliminary MALDI-MSI data detected the activity of the carboxylesterase 1 enzyme in the epidermal layer of skin. The MALDI-MSI data was supported by preliminary LC-MS/MS analysis. To investigate the reproducibility of the results future investigations are required

Development of multiwave-based bioprinting technology

Pluripotent stem cells (PSCs) are the most favourable sources of cells for tissue engineering applications due to their unique potency and self-renewal characteristics however they are quite fragile and can be directed to differentiate erroneously by the application of external forces. A novel multi-nozzle valve-based bioprinting platform was developed that was able to position droplets of bio-ink – such as cells in suspension – with high spatial accuracy and low impact. Volumes as low as 2 nL were successfully dispensed. Several different versions of the machine were created before the final machine was made integrating improvements and solutions to problems encountered during development. A complete evaluation of cell compatibility was carried out in order to quantify the response of cells to the bioprinting process. In the first ever study of this kind, the viability and pluripotency of human embryonic and induced pluripotent stem cells was investigated post-printing and were found to be almost completely unaffected by the bioprinting process. Many cells require a 3D culture environment in order to maintain their in vivo functions. A hybrid bioprinted-hanging-droplet technique was used to create uniform spheroid aggregates of programmable sizes from PSCs which could be used to direct PSC differentiation or as building blocks for tissue generation. Hydrogels can also be used to recreate the 3D in vivo cellular environment using the bioprinter. Alginate and hybrid polypeptide-DNA hydrogels were used, the latter for the first time with a bioprinting platform. Complex 3D structures could be created in a layer-by-layer approach with programmable heterogeneous properties throughout. Cells were added to the hydrogel precursor solution and used to bioprint 3D structures. The cells were found to be functional and highly viable while being encapsulated throughout the 3D structure of the bioprinted hydrogel which will allow the future creation of more accurate human tissue models. PSCs were successfully directed to differentiate into hepatocyte-like cells. It was shown that the bioprinting process did not interrupt or alter the pre-programmed differentiation of the cells which means that these cells can be patterned in 3D using the bioprinter while differentiating, greatly speeding up the creation of mini-liver tissue. Hepatic stellates and HUVECs were co-cultured with the hepatocyte-like cells in various ratios in an attempt to improve their hepatic function. However, no clear improvement in cytochrome P450 activity was observed indicating that further optimisation is required in this area

Development of multivalve-based bioprinting technology

Pluripotent stem cells (PSCs) are the most favourable sources of cells for tissue engineering applications due to their unique potency and self-renewal characteristics however they are quite fragile and can be directed to differentiate erroneously by the application of external forces. A novel multi-nozzle valve-based bioprinting platform was developed that was able to position droplets of bio-ink – such as cells in suspension – with high spatial accuracy and low impact. Volumes as low as 2 nL were successfully dispensed. Several different versions of the machine were created before the final machine was made integrating improvements and solutions to problems encountered during development. A complete evaluation of cell compatibility was carried out in order to quantify the response of cells to the bioprinting process. In the first ever study of this kind, the viability and pluripotency of human embryonic and induced pluripotent stem cells was investigated post-printing and were found to be almost completely unaffected by the bioprinting process. Many cells require a 3D culture environment in order to maintain their in vivo functions. A hybrid bioprinted-hanging-droplet technique was used to create uniform spheroid aggregates of programmable sizes from PSCs which could be used to direct PSC differentiation or as building blocks for tissue generation. Hydrogels can also be used to recreate the 3D in vivo cellular environment using the bioprinter. Alginate and hybrid polypeptide-DNA hydrogels were used, the latter for the first time with a bioprinting platform. Complex 3D structures could be created in a layer-by-layer approach with programmable heterogeneous properties throughout. Cells were added to the hydrogel precursor solution and used to bioprint 3D structures. The cells were found to be functional and highly viable while being encapsulated throughout the 3D structure of the bioprinted hydrogel which will allow the future creation of more accurate human tissue models. PSCs were successfully directed to differentiate into hepatocyte-like cells. It was shown that the bioprinting process did not interrupt or alter the pre-programmed differentiation of the cells which means that these cells can be patterned in 3D using the bioprinter while differentiating, greatly speeding up the creation of mini-liver tissue. Hepatic stellates and HUVECs were co-cultured with the hepatocyte-like cells in various ratios in an attempt to improve their hepatic function. However, no clear improvement in cytochrome P450 activity was observed indicating that further optimisation is required in this area

Manipulation of Particles and Fluid with Surface Acoustic Waves

Small biomolecules can be challenging and expensive to isolate and manipulate, but they may be the key for developing more efficient diagnostic tools for the detection of biomarkers, such as those associated with pancreatic cancer. Pancreatic cancer has a survival rate of less than 10% five years after diagnosis. For this reason, the research and establishment of cheap, effective screening tools are highly desired. One promising method of screening is separating and identifying biomarkers in a patient’s blood that are indicative of pancreatic cancer. The purpose of this project is to develop a system using surface acoustic waves (SAWs), in conjugation with microfluidics and surface functionalization, to manipulate small molecules and droplets with the goal of enabling the system to isolate particles within the blood. The separation and manipulation are achieved by passing the targeted fluidic material, in this case blood, through a microfluidic channel on top of a SAW device. The SAWs are generated by using an array of interdigital transducers (IDTs) photolithographically printed on a piezoelectric crystal to convert the electrical signal into acoustic waves. The SAW propagates, guided at the top surface of the material, and interacts with the fluid, exerting a force on the fluid and on particles in solution. Using open microfluidics to manipulate fluid droplets as an alternative to a continuous flow stream in an enclosed microfluidic channel is also an option. Functionalizing the surface using a fluorinated silane and coating with a fluorinated oil creates a liquid layer for easy droplet manipulation. The results of implementing this procedure shows dewetting of the oil layer, but despite this the liquid coating did provide a surface hydrophobic enough to allow for easy droplet sliding. This procedure combined with a heat source allows for concentration of particles at specific locations for easier particle detection. The development of the device discussed is expected to help yield a new and consistent approach to biologic particle manipulation methods that is both compact and more sensitive to facilitate the detection of diseases such as pancreatic cancer long before symptoms appear

A Combinatorial Method for Discovery of BaTiO3-based Positive Temperature Coefficient Resistors

PhDThe conventional materials discovery is a kind of empirical (“trial and error”) science that of handling one sample at a time in the processes of synthesis and characterization. However, combinatorial methodologies present the possibility of a vastly increased rate of discovery of novel materials which will require a great deal of conventional laboratory work. The work presented in this thesis, involved the practice of a conceptual framework of combinatorial research on BaTiO3-based positive temperature coefficient resistor (PTCR) materials. Those including (i) fabrication of green BaTiO3 base discs via high-throughput dip-pen printing method. Preparation and formulation of BaTiO3 inks (selection of dispersant and binder/volume fraction) were studied. The shape of drying residues and the morphogenesis control of droplet drying were discussed. (ii) investigation of a fast droplet-doping method, which induced the dopant precursor solution infiltrating into the porous BT base disc. Various characterization methods were used to examine the dopant distribution in the body of disc. (iii) devising a high-throughput electrical measurement system including an integrated unit of temperature control and automatic measurement operation, and an arrayed multichannel jig. (iv) synthesis of donor-doped BaTiO3 libraries, which involved lanthanum, erbium, yttrium as donor elements and manganese as an acceptor dopant element respectively. Their temperature dependant resistivities were also explored. The work successfully developed an integrated tool including high-throughput synthesis of a large batch of libraries and high-throughput electrical property measurement for combinatorial research on BaTiO3-based PTCR ceramics. The Abstract ii combinatorial method, thus validated, has the potential to deliver dopant-doped BTbased PTCR libraries rapidly with a very wide range of dopant mixtures and concentrations for electrical property measurement and deserves to be applied to other low level dopant ceramic systems. These approaches are novel and paving the way for other new materials selection and materials research

Cellular droplet microarray: a miniaturized technique for high-throughput screening of small molecules and proteins

Das Hochdurchsatz-Screening ist von großer Bedeutung und bildet einen der Eckpfeiler der aktuellen Life-Science-Research wie Biologie, Biotechnologie, Biochemie, Computerwissenschaft, medizinische Chemie und Pharmakologie. Das Interesse an einem Screening mit hohem Durchsatz unter akademischen, mittleren und kleinen Biotech-Unternehmen, staatlichen und gemeinnützigen Screening-Standorten, sowie Auftragsforschungsorganisationen wurde spürbar erhöht, da Hochdurchsatz-Screenings eine effiziente Analyse von einer Vielzahl von Stoffen in kürzester Zeit ermöglicht. Das steigende Interesse an dieser Methode führte zu einer schnellen Entwicklung von Screening-Technologien mit hohem Durchsatz in Kombination mit komplementären Technologien. Die Nachteile herkömmlicher Hochdurchsatz-Screenings beinhalten hohe Kosten und Materialverschwendung, lange Zykluszeiten, geringe Produktivität und die begrenzte Ausbaufähigkeit der existierenden Stoffbibliotheken. Die genannten Nachteile verdeutlichen die Wichtigkeit der Bemühungen zur Entwicklung von verbesserten miniaturisierten und automatisierten Hochdurchsatz-Screening Methoden. Droplet Microarray (DMA) ist eine dieser miniaturisierten Plattformen, welche durch die Vereinigung von Oberflächenchemie, Oberflächenfunktionalisierung und Biologie erschaffen wurde. Die hohe Tröpfchendichte auf DMA senkt die Kosten, Materialverschwendung, Zykluszeit des Screenings und erhöht die Effizienz des Verfahrens, während sie effektiv die Verwendung größerer chemischer und biologischer Bibliotheken unterstützt. Die weitere Entwicklung, Auswertung und Verbesserung der DMAs spielt nicht nur für den Fortschritt der Hochdurchsatz-Screenings eine wichtige Rolle, sondern beherbergt auch unermessliches Potential in pharmalogischen Bereichen und der Stammzellenforschung, in welcher es die Wirksamkeit von Gentransfers und das Kultivieren von undifferenzierten Stammzellen erleichtert. Pharmakologisches Priming (Drug Repurposing) wurde als adjuvante Strategie zur Verbesserung der Effizienz nicht-viraler Genübertragung angesehen. Dennoch ist die facettenreiche Anwendung des pharmakologischen Priming aufgrund der unerschwinglich hohen Kosten für Reagenzien und die Durchführung der Methode unweit verbreitet, was den aufkommenden Drang nach miniaturisierten Plattformen und Drug Repurposing erklärt. Abgesehen von der Dringlichkeit im pharmakologischen Bereich, um klinisches Potenzial zu realisieren, benötigt die Stammzellenforschung auch miniaturisierte Methoden um den Einfluss von Zelloberflächeninteraktionen und Zellkulturmedium auf das Verhalten von Stammzellen und die Effekte von Zusatzstoffen, und Oberflächenbeschichtungen einschließlich oberflächenabsorbierter Proteine auf Stammzellen zu beobachten. Das Ziel für den ersten Teil der Dissertation war es nach kleinen Molekülen (Stoffen) auf DMAs zu suchen, welche transfektionsverstärkende Effekte beinhalten, um eine mögliche Verbesserung in den Bereichen Gentransfer- und Therapie zu bieten. Eine Untersuchung der Einflüsse von 774 Food and Drug Administration (FDA)-zugelassenen Wirkstoffen auf die Transfektionseffizienz mit verschiedenen Zelltypen in miniaturisierter- und hochdurchsatzweise wurde mit Hilfe der DMAs veranlasst. Das Screening der FDA-zugelassenen Arzneimittelbibliothek identifizierte 14 einzelne Verbindungen, die eine zwei- bis fünffache Verbesserung der Transfektion aufwiesen. Diese Treffer wurden verifiziert und in großem Maßstab untersucht. Die Ergebnisse deuten darauf hin, dass der auf DMA basierende Ansatz für das Drug Repurposing beständig ist und zur Untersuchung und Entwicklung wirksamerer nicht-viraler Genübertragungssysteme verwendet werden könnte. Das Ziel des zweiten Abschnitts der Dissertation war es, den Einfluss von Oberflächeneigenschaften und sinkendem Volumen auf die Pluripotenz von human induzierten pluripotenten Stammzellen (hiPSCs) zu bestimmen. Zwei künstliche Oberflächen mit unterschiedlichen chemischen Elementen und deren dazugehörigen DMAs wurden für die Kultivierung und Pluripotenz von hiPSCs in vitro untersucht. Die Oberflächen und DMAs wurden genutzt, um human induzierten pluripotenten Stammzellen in 2 ml und 200 nL-Volumen zu kultivieren. Die Ergebnisse zeigten, dass hiPSCs eine hohe Lebensfähigkeit, sowie die erwartete Morphologie und Pluripotenz in 200 nL Tröpfchen auf Typ A und Typ B DMAs ohne Matrigelbeschichtung nach 24 h Kultivierung aufwiesen. Dies beweist, dass DMAs eine vielseitige und simple Plattform für kurzzeitige und xeno-freie Hochdurchsatz-Screenings von hiPSCs sind. Als Ziel für den dritten Teil der Dissertation wurde das Identifizieren von chemisch definierten Proteinen, welche das Kultivieren und Aufrechterhalten der Plutipotenz von hiPSCs Zellen auf DMAs, sowie Mikrotiterplatten weiter verbessern könnten, angesetzt. Es ist möglich eine Proteinbeschichtung, Zellkultur und Immunfluoreszenzfärbung auf miniaturisierter Ebene parallel mit Hilfe von DMAs durchzuführen, was zu einer Reduzierung von Versuchsfehlern und Verbrauchsmaterialien führt. Auf Grund dessen wurden DMAs als Basis zur Überprüfung von elf verschiedenen Proteinen und deren verwandten binären und ternären Kombinationen (insgesamt 231 verschiedene Gruppen) auf ihre Fähigkeit zur Erhaltung der Pluripotenz von hiPSCs genutzt. Aus diesem Raster wurden zehn Gruppen von ternären Proteinkombinationen identifiziert, welche die Proliferation und das Self-Renewal besser unterstützen könnten als mit Matrigel beschichtete Oberflächen. Die effizientesten Proteinkombinationen des primären Screenings wurden weiterhin in einer Langzeitkultur (fünf Wochen) verifiziert. Zusätzlich wurde die Formation von embryonalen Körperchen der auf den ausgewählten Proteinbeschichtungen kultivierten Zellen erzielt und es folgte die Differenzierung der hiPSCs in drei Keimblätter. Zusammengefasst, wurden DMAs als miniaturisierte Schnellscreening-Plattform verwendet, um mehrere biologische Fragen zu beantworten. Als erstes wurden 776 Wirkstoffe auf Nanoebene untersucht und somit kosten-, zeit- und arbeitssparend verwertet. Vierzehn Stoffe wiesen eine zwei- bis fünffache Verbesserung der Transfektionstechniken auf. Als zweites wurden chemische Komponente von Zellkulturoberflächen und kleinen Volumina (200nl) von Zellkulturmedium gefunden, die zur Erhaltung der Pluripotenz von hiPSCs beitragen. Als letztes wurden zehn Gruppen von ternären Proteinkombinationen identifiziert, welche das Kultivieren von undifferenzierten hiPSCs unterstützen. Zwei von ihnen wurden weiter untersucht, um eine Langzeitkultur von undifferenzierten hiPSCs zu erzielen, gefolgt von ihrer Differenzierung in drei Keimblätter. Eine Zusammenfassung und eine Aussicht auf die Zukunft befinden sich am Ende der Dissertation

Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery