Going with the µFlow: Reinterpreting Energy Input in Organic Synthesis

The popularity of microflow chemistry has skyrocketed in the last 20 years, more and more chemists are switching from macro-batch reactors to miniaturized flow devices. As a result, microfluidics is paving its way into the future by consolidating its position in organic chemistry not only as a trend but as a new, effective, and sustainable way of conducting chemistry, that clearly will continue to grow and evolve. This perspective highlights the most relevant examples of innovative enhancing technologies applied to microflow reactors aimed to improve and intensify chemical processes. The extensive applicability of microflow chemistry is further illustrated by briefly discussing examples of complex integrated microsystems and scale-up technologies, demonstrating ultimately that microflow chemistry has the potential to become the ideal technology for the future

Entwicklung und Automatisierung 3D-gedruckter mikrofluidischer Systeme zur Integration und Kultivierung adhärenter Zellkulturen

Mikrofluidische Systeme werden zur Manipulation von Flüssigkeiten auf Mikroebene eingesetzt. Von ihnen profitieren insbesondere Biowissenschaften durch die Reduktion von Reagenzien und die Automatisierung ganzer Arbeitsabläufe. Die Mikrostrukturierung erlaubt zudem die Entwicklung neuartiger mikrofluidischer Zellkultursysteme wie den organ-on-a-chip Systemen. Diese Systeme zeichnen sich durch eine höhere physiologische Relevanz gegenüber klassischen in vitro Systemen aus und können zur Rekonstruktion einzelner Organfunktionen genutzt werden. Aufgrund ihrer komplizierten Fertigung wird jedoch der Zugang zu diesen Systemen für Biowissenschaftler:innen er-schwert, sodass ihr Potential noch kaum in kommerziellen Produkten realisiert werden konnte. Eine Lösung bietet die additive Fertigung (3D-Druck) mikrofluidischer Systeme, durch die die unkomplizierte Herstellung eigener Prototypen an Ort und Stelle ermöglicht wird. Um den 3D-Druck jedoch auch für die Herstellung mikrofluidischer Zellkultursysteme nutzen zu können, benötigt es deutlich mehr Biokompatibilitätsstudien zu neuen 3D-Druckmaterialien. In diesem Sinne wurde in dem ersten Teil dieser Arbeit die in vitro Biokompatibilität eines aus Polyacrylat bestehenden, hitzebeständigen 3D-Druckmaterials sowie dessen Eignung für die Heißdampfsterilisation untersucht. Dabei konnte eine Biokompatibilität gegenüber adhärenten Mausfibroblasten und Hefezellen nachgewiesen werden. Diese Ergebnis-se ermöglichen somit den Einsatz des Materials für die Zellkultur. Die Biokompatibilität blieb auch nach Heißdampfsteri-lisation unbeeinträchtigt, sodass mit diesem Material gedruckte Zellkultursysteme unkompliziert sterilisiert werden können. Im Gegensatz dazu erwies sich das Material für menschliche embryonale Nierenzellen in Suspension als schädlich, was die Bedeutung einer auf den Organismus und die Anwendung zugeschnittenen Biokompatibilitätsprü-fung verdeutlicht. Im zweiten Teil dieser Arbeit wurde das evaluierte 3D-Druckmaterial zur Herstellung eines vollautomatischen mikroflui-dischen Ventilsystems eingesetzt, dessen Nutzen anschließend durch die Automatisierung eines Zellkulturassays als Machbarkeitsstudie demonstriert wurde. Alle mikrofluidischen Komponenten inklusive Anschlüsse, Mikromischer, Mikroventile und Auslässe wurden dabei in einem Stück gefertigt. Die kostengünstige und leicht zu steuernde Aktuation der 3D-gedruckten Ventilmembranen durch Servomotoren ist ein komplett neuer Ansatz. Die Automatisierung des Sys-tems erfolgte durch einen Raspberry Pi Computer sowie selbst entwickelter Python Skripte. Durch den kompakten Com-puter wird die portable und ferngesteuerte Verwendung des Ventilsystems ermöglicht. Nachdem eine zuverlässige Mischgenauigkeit sowie die hohe Robustheit der Ventile gezeigt werden konnte, wurde das mikrofluidische Ventilsys-tem zur Automatisierung eines Zytotoxizitätsassays als Machbarkeitsstudie verwendet. Das von der Konzentration des Toxins abhängige Zellwachstum wurde dabei mittels Lebendzellmikroskopie und Bildverarbeitung automatisiert ausge-wertet. Die Ergebnisse wurden anschließend mit denen eines pipettierten Assays verglichen. Beide Assays zeigten ein fast identisches Wachstumsverhalten, das die Eignung des Systems für die Zellkultur beweist. Letztendlich konnte durch den 3D-Druck in Kombination mit der Biokompatibilitätsbestimmung eines 3D-Druckmaterials die Automatisierung von Zellkulturassays durch ein neu entwickeltes, 3D-gedrucktes mikrofluidisches Ventilsystem ermöglicht werden. Mit der Veröffentlichung der 3D-Modelle und Skripte ist es Wissenschaftler:innen nun möglich, das System an ihre eigenen Anwendungen anzupassen.Deutsche Forschungsgemeinschaft/Emmy Noether/346772917/E

An Exploration of Paul Bowles\u27 Piano-Solo Pieces

This research paper provides a general overview of the piano-solo literature by the American composer Paul Frederic Bowles (1910-1999). Thanks to recent contributions, this repertoire is now available in recordings and musical scores as it has never been before.;This paper is divided into two sections. The first covers the biography of Paul Bowles and his musical achievements as a composer, along with his research into the folk music of Morocco and his literary writings as a music critic for the journal Modern Music and for The New Herald Tribune. The second part is about Bowles\u27 piano-solo output, divided thematically into pieces with similar forms and structures.;For Bowles\u27 solo piano music, theoretical analysis and a review of existing literature help to reveal style traits; these include his preference for short character pieces, in which Bowles employs neoclassical elements, such as melodies with classical harmonies that display bitonal and pandiatonal tendencies, along with ostinato patterns and Alberti-bass accompaniments. Bowles\u27 music often displays ternary or free structures, with motivic development techniques through which themes or passages are derived from previous motives. Jazz and folk idioms are also an important aspect of Bowles\u27 piano music, particularly in his dancelike pieces, many of which display a Latin-American flavor

Microchips for Isothermal Amplification of RNA : Development of microsystems for analysis of bacteria, virii and cells

The goal of the present work was to develop a microchip for amplification and detection of mRNA by employing nucleic acid sequence-based amplification (NASBA) technology. The technology platform should in principle be adaptable for any clinical analysis using mRNA or ssDNA as a target. To demonstrate the microchip functionality, identification of human papillomavirus (HPV) type 16, the etiological agent for cervical cancer has been used. The work shows for the first time successful real-time amplification and detection employing NASBA in microsystem formats using custom-made instruments. The first silicon-glass chips contained reaction chambers of 10 nl and 50 nl, which decreased the NASBA reaction volume by a factor of 2000 and 400, respectively. Further, experiments employing cyclic olefin copolymer (COC) microchips for simultaneous amplification and detection, automatically distributed the sample into 10 parallel reaction channels with detection volumes of 80 nl. In order to detect the simultaneous amplification in the reaction channels, a second custom-made optical detection system with increased sensitivity, heat regulation and an automatic non-contact pumping mechanism, was made. Dilution series of both artificial HPV 16 oligonucleotides and SiHa cell lines showed that the detection limits for the microchips were comparable to those obtained for experiments performed in conventional routine-based laboratory-systems. For experiments related to the development of a self-contained microchip for NASBA, the detection volume was increased to 500 nl due to the advantage of an increased fluorescence signal. For the NASBA reaction, biocompatible surfaces are critical. It was not possible to amplify any target in microchips with native silicon or COC surfaces. Adsorption measurements indicated clearly that fluorescently labelled mouse IgG bound non-specifically to the hydrophobic native COC surfaces, while PEG coated COC surfaces showed adequate protein resistance. Of the coatings tested for the COC microchips, surfaces modified with PEG showed the best biocompatibility. Successful amplification was obtained with silicon microchips when the surfaces were modified with either SigmaCote™ or SiO2. In order to integrate the NASBA reagents on chip, a thorough evaluation of the reagents to be spotted and dried was performed. Because of the limited number of microchipsavailable, it was necessary to map the most critical parameters on macroscale prior to transfer to the microscale. The DMSO and sorbitol enclosed in the standard NASBA reaction mixture were difficult to dry, and therefore it was necessary to add these compounds to the oligonucleotides or the sample of extracted nucleic acids before the sample was applied on the amplification chip. The standard NASBA reagents consist of the two main solutions, mastermix and enzymes, in addition to the sample. Both the mastermix and the enzymes were stable only when spotted and dried separately. Protectants, such as PEG and trehalose were essential for recovery of enzymatic activity after drying on macroscale. The times for diffusion of modified molecular beacons in dried mastermix and of fluorescently labelled mouse IgG in the dried enzyme solution were ~60 seconds and ~10 minutes, respectively. So far, only dried enzymes with 0.05% PEG protectant have been successfully amplified on chip. Successful amplification using a rehydrated mastermix on microchip still remains. Optimal design and fabrication methods of the microchips were found to be crucial for chip performance. Rough surfaces do not only create background noise for the optical measurements, but it also contributes to generation of bubbles and problems related to manipulation of the sample within the channel network. The silicon microchips were manufactured with optically smooth surfaces. However, low surface roughness was not easily obtained for the COC microchips. Of the fabrication methods evaluated, it was the injection moulded chips which showed the smoothest surfaces, closely followed by the hot embossed chips. Milled and laser ablated chips produced the roughest surfaces. A novel non-contact pumping mechanism based on on-chip flexible COC membranes, combined with actuation pins in the surrounding instrument, was tested and evaluated. The mechanism enabled metering, isolation and movement of nanoliter sized sample plugs in parallel reaction channels. The COC chips with integrated pumps were able to simultaneously move parallel sample plugs along the reaction channels in four different positions. Each reaction channel contained a set of 4 actuation chambers in order to obtain metering, isolation and movement of the sample plug into the detection area. The pump accuracy depended on the evaporation of sample and the deformation of the COC membranes. The results presented in this work are promising with regard to the development of a complete integrated and self-contained mRNA amplification microchip for multi-parallel target testing of clinical samples

Microfluidics for studying metastatic patterns of lung cancer

The incidence of lung cancer continues to rise worldwide. Because the aggressive metastasis of lung cancer cells is the major drawback of successful therapies, the crucial challenge of modern nanomedicine is to develop diagnostic tools to map the molecular mechanisms of metastasis in lung cancer patients. In recent years, microfluidic platforms have been given much attention as tools for novel point-of-care diagnostic, an important aspect being the reconstruction of the body organs and tissues mimicking the in vivo conditions in one simple microdevice. Herein, we present the first comprehensive overview of the microfluidic systems used as innovative tools in the studies of lung cancer metastasis including single cancer cell analysis, endothelial transmigration, distant niches migration and finally neoangiogenesis. The application of the microfluidic systems to study the intercellular crosstalk between lung cancer cells and surrounding tumor microenvironment and the connection with multiple molecular signals coming from the external cellular matrix are discussed. We also focus on recent breakthrough technologies regarding lab-on-chip devices that serve as tools for detecting circulating lung cancer cells. The superiority of microfluidic systems over traditional in vitro cell-based assays with regard to modern nanosafety studies and new cancer drug design and discovery is also addressed. Finally, the current progress and future challenges regarding printable and paper-based microfluidic devices for personalized nanomedicine are summarized.publishedVersio

Disposable sensors in diagnostics, food and environmental monitoring

Disposable sensors are low‐cost and easy‐to‐use sensing devices intended for short‐term or rapid single‐point measurements. The growing demand for fast, accessible, and reliable information in a vastly connected world makes disposable sensors increasingly important. The areas of application for such devices are numerous, ranging from pharmaceutical, agricultural, environmental, forensic, and food sciences to wearables and clinical diagnostics, especially in resource‐limited settings. The capabilities of disposable sensors can extend beyond measuring traditional physical quantities (for example, temperature or pressure); they can provide critical chemical and biological information (chemo‐ and biosensors) that can be digitized and made available to users and centralized/decentralized facilities for data storage, remotely. These features could pave the way for new classes of low‐cost systems for health, food, and environmental monitoring that can democratize sensing across the globe. Here, a brief insight into the materials and basics of sensors (methods of transduction, molecular recognition, and amplification) is provided followed by a comprehensive and critical overview of the disposable sensors currently used for medical diagnostics, food, and environmental analysis. Finally, views on how the field of disposable sensing devices will continue its evolution are discussed, including the future trends, challenges, and opportunities

Innovative miniaturized electroanalytical approaches for the analysis of clinically relevant glycoproteins

El objetivo principal de esta Tesis Doctoral ha sido el desarrollo de nuevas herramientas y estrategias electroquímicas (ultra)-miniaturizadas (sensores serigrafiados y sistemas microfluídicos), combinadas con nanomateriales como transductores electroquímicos, para la determinación de biomarcadores glicoproteicos de gran relevancia en el diagnóstico de enfermedades. En el contexto del diagnóstico clínico actual, existe una tendencia hacia el diseño y desarrollo de dispositivos portátiles que permitan el análisis descentralizado de los laboratorios clínicos, y que puede quedar englobada bajo el término POCT (de sus siglas en inglés Point-of-Care Testing). Este término describe aquellas pruebas o ensayos que se realizan en lo que podría denominarse en el punto de necesidad (cerca o por el propio del paciente, incluso de forma remota). Además de los desarrollos propios de la electrónica, el desarrollo y éxito de la tecnología POCT ha dependido y depende en gran medida del diseño y desarrollo de nuevas tecnologías analíticas (ultra)-miniaturizadas. Los dispositivos basados en la transducción electroquímica han resultado ser esenciales en el desarrollo de (bio)-sensores, produciendo plataformas simples, pero precisas y sensibles, para el diagnóstico de enfermedades y ha sido uno de los enfoques más prometedores para el desarrollo de POCTs, debido en general a su instrumentación económica y a su fácil miniaturización. Esta Tesis Doctoral aborda el análisis de dos glicoproteínas: la alfa-1-ácido glicoproteína (AGP) y la transferrina (Tf). La primera se utiliza como biomarcador glicoproteico de procesos inflamatorios, y la segunda como biomarcador de una enfermedad rara denominada trastornos congénitos de la glicosilación (CDG). La detección electroquímica puede ser una buena alternativa para la determinación de glicoproteínas debido a su miniaturización inherente, sus elevadas sensibilidad y selectividad y su bajo coste. Sin embargo, la oxidación directa de las glicoproteínas presenta baja sensibilidad debido a que los carbohidratos presentes en las mismas son electroquímicamente inactivos en condiciones cercanas a las fisiológicas. Para paliar este inconveniente, se ha propuesto el uso de complejos de osmio (VI) con ligandos nitrogenados [Os (VI) L] como sonda electroquímica. El complejo de osmio (VI) reacciona con los grupos diol de los carbohidratos formándose un éster de osmato que produce dos señales electroquímicas en electrodos de carbono. El objetivo de la Tesis Doctoral ha sido el diseño y desarrollo de dos tipos de herramientas analíticas (ultra)-miniaturizadas compatibles con la tecnología POCT, sensores y sistemas microfluídicos electroquímicos, para el análisis de las glicoproteínas seleccionadas, AGP y Tf. En efecto, de forma específica se proponen, de forma evolutiva en lo que a las prestaciones analíticas requeridas por un POCT electroquímico se refiere: i) sensores electroquímicos serigrafiados basados en carbono y en carbono nanoestructurado para el análisis individual de las glicoproteínas, ii) microchip de electroforesis capilar para el análisis simultáneo de ambas y, iii) nuevos dispositivos microfluídicos desechables de flujo pasivo y con capacidad de integración de las etapas analíticas para la determinación individual de las glicoproteínas. Las herramientas electroquímicas desarrolladas han permitido no sólo la determinación rápida, fiable, in situ y con bajo coste de estos biomarcadores relevantes en el diagnóstico clínico de importantes enfermedades, sino un avance conceptual hacia la descentralización del análisis clínico y, por ende, una mejora del diagnóstico actua

Hybrid point-of-care devices for visual detection of biomarkers and drugs

Early diagnostics is a crucial part of clinical practice offering a rapid and convenient way to investigate and quantify the presence of key biomarkers related to specific pathologies and increasing the chance of successful treatments. In this regard, point-of-care testing (POCT) shows several advantages enabling simple and rapid analyses, allowing for real-time results, and permitting home testing. Metallic nanoparticles (NPs), like gold NPs (AuNPs), can be beneficially integrated into POC devices thanks to their tunable plasmonic properties which provide a naked-eye read-out. Moreover, the high sensitivity of NPs enables the detection of biomarkers in non-invasive fluids where the concentrations are typically low. These biofluids, like saliva and urine, are functionally equivalent to serum in reflecting the physiological state of the body, whilst they are easier to handle, collect, and store. In this thesis, I first reported the design and development of a colorimetric strategy based on the morphological change of multibranched plasmonic AuNPs, aimed at detecting glucose in saliva. The sensing approach relied on a target-induced reshaping process which involves the oxidation of the NP tips and the transformation into a spherical shape, characterized by a naked-eye detectable blue-to-pink color change. The platform proved to be beneficial in the early and non-invasive diagnosis of hyperglycemia. The successful technological transfer on a solid substrate paved the way for the realization of a dipstick prototype for home testing. Then, the strategy was adapted to other biomarkers, leading to the development of a multiplexing test for the simultaneous detection of three salivary analytes (cholesterol, glucose, and lactate). This multiplexing assay enabled to save reagents, costs, and time, whilst increasing the overall clinical value of the test. Exploiting the microfluidics applied on a paper sheet, I realized a monolithic and fully integrated POC device, through a low-cost and fast CO2 laser cutter. The platform showed excellent selectivity and multiplexing ability, with negligible interferences. The second part of my thesis was focused on the development of POC devices for the detection of anticancer drug contaminations in water solutions and urine samples. Antiblastic agents have revealed high toxicity for the exposed healthcare workers who prepare and administer these drugs in occupational environments. Hence, continuous monitoring is highly required, and POCT shows tremendous potential in this context. With this aim, I realized a lateral-flow (LF) device for the assessment of doxorubicin contamination, using the fluorescent properties of the drug for naked-eye detection. The pharmacological recognition of the DNA probe was exploited to overcome the lack of anti-doxorubicin antibodies. The highly sensitive strategy was successfully adapted to a real urine sample, without resorting to complex pretreatment procedures. Then, I developed a competitive LF device for the detection of methotrexate (MTX). AuNPs were employed as the label molecules and the pharmacological competition of folic acid and MTX for the capture enzyme was exploited as the recognition mechanism, instead of costly antibodies. Despite the sensitivity requires further improvements, the strategy showed fast and reliable results, demonstrating a high potential for workers’ safety control