In this dissertation, several microfabricated devices are introduced to develop new applications in the area of chemical analysis. Electrochemical micropumps, chip-based liquid chromatography systems and multilayer capillary electrophoresis microdevices with crossover channels were fabricated using various substrates such as poly(dimethylsiloxane) (PDMS), glass, and poly(methyl methacrylate) (PMMA). I have demonstrated pressure-driven pumping of liquids in microfabricated channels using electrochemical actuation. PDMS-based micropumps were integrated easily with channel-containing PMMA substrates. Flow rates on the order of ~10 µL/min were achieved using low voltages (10 V). The potential of electrolysis-based pumping in microchannels was further evaluated for pressure driven microchip liquid chromatography (LC). Two micropumps were connected with reservoirs for sample and mobile phase, situated at the ends of microchannels for sample injection and separation, respectively. Columns micromachined in glass were coated covalently with an organic stationary phase to provide a separation medium. A pressure-balanced sample injection method was developed and allowed the injection of picoliter sample volumes into the separation channel. Fast (<40 s) separation of three fluorescently tagged amino acids was performed in a 2.5-cm-long microchip column with an efficiency of 3300 theoretical plates. Improved electrode designs that eliminate the stochastic formation of bubbles on the electrode surface will enhance pumping reproducibility. Multilayer polymeric microdevices having fluidically and electrically independent crossover channels were made using phase-changing sacrificial layers (PCSLs). High-performance electrophoretic separations of fluorescently labeled amino acids were carried out in multilayer PMMA microchips. Neither pressure nor voltage applied in a crossover channel resulted in negative effects on the separation quality in the main fluidic path. A fifty-fold reduction in crossover volumes was achieved in next-generation multilayered microchips. The ability to make minimal dead volume crossover channels facilitated the design and operation of multichannel array microdevices with a minimum number of electrical and fluidic inputs. Replicate electrophoretic separation of two peptides was performed in parallel for three independent microchannels connected to a single sample reservoir. My work demonstrates the value of PCSLs in making complex microfluidic structures that should expand the application of micro-total analysis systems.

No description available.

The ability of culturing cells in vitro has given many advantages in biological research and has become a standard methodology in drug discovery and toxicology. However traditional culturing methods give limited possibilities comparing to microfluidic systems. In order to understand the cellular mechanisms of Natural killers against virus infected cells and tumors, we developed a method for observing in parallel, high numbers of individual Natural killer-target cell conjugates in confined regions. An important advantage of this method is that it gives the possibility to keep track of large numbers of specific conjugates in a time scale of several days. Thus live cell imaging of NK-Target cell interactions in multi-well microstructures, can offer valid statistical information about NK cells processes that can lead to a better understanding of the function and regulation of the immune system. / Ανοσολογία είναι ο επιστημονικός κλάδος που διερευνά τους σύνθετους μηχανισμούς με τους οποίους το ανθρώπινο σώμα αντιδρά και καταπολεμά μολύνσεις ή δυσλειτουργίες που προέρχονται είτε από παθογόνα ή από μεταλλάξεις των κυττάρων του ίδιου του οργανισμού. Οι αντιδράσεις του ανοσοποιητικού συστήματος διαχωρίζονται σε εγγενείς και προσαρμοσμένες άνοσες αντιδράσεις ανάλογα με την ταχύτητα και την εξειδίκευση των αντιδράσεων αυτών ενάντια στα παθογόνα. Το εγγενές ανοσοποιητικό σύστημα αντιδρά άμεσα και συνήθως είναι αρκετά αποτελεσματικό ώστε να εξοντώσει το παθογόνο πριν προκαλέσει αρρώστια. Σε περιπτώσεις όπου η δραστικότητα το εγγενούς δεν είναι επαρκής, το προσαρμοσμένο ανοσοποιητικό σύστημα ενεργοποιείται με την βοήθεια του εγγενούς και χρησιμοποιώντας πολύ συγκεκριμένους μηχανισμούς με τη βοήθεια των οποίων παύει η διαδικασία της μόλυνσης. Τα φυσικά κύτταρα δολοφόνοι (Natural killer cells-NK) ανήκουν στο εγγενές ανοσοποιητικό σύστημα και παίζουν σημαντικό ρόλο στην προστασία του οργανισμού και την ρύθμιση του ανοσοποιητικού συστήματος. Βασικός στόχος της διπλωματικής εργασίας είναι η διερεύνηση των αλληλεπιδράσεων μεταξύ φυσικών κυττάρων δολοφόνων και κυττάρων στόχων. Τα κύτταρα στόχοι είναι είτε κύτταρα μολυσμένα με ιούς ή καρκινικά κύτταρα. Η αρχική υπόθεση ήταν ότι οι πληροφορίες σχετικά με τις λειτουργίες των NK κυττάρων είναι ευκολότερο να καταγραφούν και να αναλυθούν εκτενέστερα, αν μεγάλος αριθμός μεμονωμένων ζευγών ΝΚ-στόχων παρατηρηθούν ξεχωριστά σε περιορισμένο μικρόχωρο. Για την επίτευξη του σκοπού αυτού χρησιμοποιήθηκαν μικροσυσκευές πολυκυψελών εντός των οποίων καλλιεργήθηκαν ξεχωριστά για αρκετές μέρες κύτταρα στόχοι και κύτταρα δολοφόνοι, έτσι ώστε να ελεγχθεί η ζωτικότητα και η λειτουργικότητα των κυττάρων μέσα στους μικρόχωρους. Πιο συγκεκριμένα, για τον έλεγχο αυτό τα κύτταρα τοποθετήθηκαν στις κυψέλες και καλλιεργήθηκαν για 3-4 ημέρες. Κάθε μέρα μία συγκεκριμένη περιοχή της μικροσυσκευής παρατηρήθηκε σε απλό οπτικό μικροσκόπιο και τα κύτταρα μέσα στις κυψέλες μετρήθηκαν. Τα δεδομένα καταγράφηκαν σε μορφή πινάκων και επεξεργάστηκαν στο MatLab. Τα ιστογράμματα που κατασκευάστηκαν έδειξαν ότι η κατανομή των κυττάρων μέσα στις κυψέλες μεταβάλεται και ο συνολικός αριθμός τους αυξάνεται. Τα πειράματα σχετικά με τον έλεγχο του πολλαπλασιασμού των κυττάρων πραγματοποιήθηκαν για 3 διαφορετικούς τύπους, 221Cw6, Nishi και NKL. Εφόσον πρώτα έγινε ο έλεγχος βίο-συμβατότητας των κυττάρων στις μικροκυψέλες, στη συνέχεια κύτταρα δολοφόνοι και κύτταρα στόχοι επεξεργάστηκαν με ειδικές φθορίζουσες βαφές, τοποθετήθηκαν στις μικροσυσκευές και παρατηρήθηκαν με τη χρήση συνεστιακού φθορίζοντος μικροσκοπίου. Με χρήση ειδικής λειτουργίας του μικροσκοπίου, εικόνες συλλέχθηκαν κάθε1-3 λεπτά για 6-12 ώρες. Με τη χρήση της λειτουργίας αυτής ήταν δυνατή η παρακολούθηση των κινήσεων των κυττάρων μέσα στις κυψέλες και η καταγραφή της συμπεριφοράς τους και των γεγονότων κατά την διάρκεια του πειράματος. Έχοντας μεγάλο αριθμό κυψελών (60-100) σε κάθε πείραμα, υπήρξε η δυνατότητα παρατήρησης μεγάλου αριθμού γεγονότων εκ των οποίων κάποια ήταν εξαιρετικά σπάνια η ακόμα και μοναδικά. Λεπτομέρειες σχετικά με την μεθοδολογία των πειραμάτων, την καταγραφή και ανάλυση των αποτελεσμάτων, αναγράφονται αναλυτικά και επεξηγούνται στην παρούσα εργασία.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

No description available.

No description available.

There are various energy dissipation mechanisms that affect the dynamic response of microstructures used in MEMS devices. A cumulative effect of such losses is captured by an important characteristic of the structure called Quality factor or Q-factor. Estimating Q-factor at the design stage is crucial in all applications that use dynamics as their principle mode of operation. A high Q-factor indicates sharp resonance that, in turn, can indicate a broad flat response region of the structure. In addition, a high Q-factor typically indicates a high sensitivity. Microstructures used in MEMS are generally required to have much higher Q-factors than their macro counterparts. However some damping mechanisms present in microstructures can reduce the Q-factor of the structure significantly. In the present work, we investigate the dependence of Q-factor on the squeeze film damping an energy dissipation mechanism that dominates by a couple of orders of magnitude over other losses when a fluid (e.g., air) is squeezed through gaps due to vibrations of a microstructure. In particular, we show the effect of nonlinear terms in the analysis of squeeze film damping on the Q-factor of a structure. We also show the effect of rarefaction, surface roughness along with their coupled effect and with different boundary conditions such as open border effect, blocked boundary effect on the squeeze film damping. Finally, we develop similitude laws for calculating squeeze film damping force in up-scaled structures. We illustrate the effects by studying various type of microstructures including parallel plates, beams, plate and beam assemblies such as MEMS microphone, vibratory gyroscope etc. We view the contributions of this work as a significant in investigating and integrating all important effects altogether on the squeeze film damping, which is a significant factor in the design and analysis of MEMS devices.

Les cellules tumorales circulantes (CTCs) sont la principale voie de dissémination du cancer dans le corps humain au travers de la circulation sanguine. Ces cellules ont la capacité de se détacher de la tumeur primaire, de rejoindre la circulation sanguine et de survivre dans cet environnement. Une sous-population spécifique de ces cellules a la capacité de coloniser de nouveaux tissus et de former des métastases. L'importance de ces cellules rares dans la circulation sanguine a été intensément étudiée au cours des dernières décennies, et il a été constaté que les informations phénotypiques et génomiques qu'elles contiennent pourraient être corrélées avec celles obtenues à partir d'une biopsie tissulaire. De plus, le nombre et l'incidence des CTC chez les patients métastatiques pourraient être utilisés comme indicateurs pronostics. Ainsi, leur isolement à partir d'échantillons sanguins et leur analyse a été proposé en remplacement des biopsies conventionnelles, comme une alternative moins invasive et permettant un échantillonnage plus répété. In fine, la détection et l'analyse des CTC en routine clinique pourraient être utilisées pour le suivi en temps réel des thérapies et de leur efficacité pour améliorer la prise en charge des patients, un pas de plus vers une médecine de précision. Dans ce projet de thèse, nous avons développé de nouveaux micro-dispositifs pour la capture, sous flux, de cellules cancéreuses à partir de sang complet humain. Nous avons exploité les propriétés physiques des CTC, plus grandes et moins déformables que les cellules sanguines normales, pour discriminer ces cellules rares (<1 cellule par mL aux premiers stades de la maladie). Des micro-dispositifs ont été conçus tels des tamis à trois dimensions pour filtrer sélectivement les cellules cancéreuses tout en préservant l'intégrité et la viabilité des cellules. De plus, les dispositifs ont été conçus pour permettre l'accès au matériel biologique isolé et effectuer ainsi une identification des cellules in situ, e.g. par immunocytochimie, mais aussi potentiellement pour servir de plateforme pour une analyse fonctionnelle de ces cellules. Nous avons proposé deux approches totalement compatibles avec la routine clinique. La première consiste en un guide équipé de microdispositifs, conçu pour être introduit directement dans la circulation sanguine au travers d'un cathéter médical et effectuer la capture des cellules cancéreuses in vivo. La deuxième approche vise à réaliser l'isolement des CTCs en utilisant des microdispositifs intégrés à des plateformes ex vivo compatibles avec les consommables médicaux de prélèvement sanguin.[...] / Circulating tumor cells (CTCs) are believed to represent the main pathway of cancer dissemination in the human body through the circulatory system. These cells have the ability to detach from the primary tumor, enter into the bloodstream, and survive in this environment. A specific subpopulation of these cells possesses the capacity of colonizing new tissues and forming metastases. The relevance of these rare cells in the bloodstream has been intensively investigated during the last decades, finding that phenotypic and genomic information they carry could be correlated with that of solid biopsies. Moreover, the number and incidence of CTCs in metastatic patients could be used as an indicator for prognosis. Thus, their isolation from blood samples and analysis has been proposed as a surrogate to solid biopsies, having the added value of being a less invasive procedure and allow a more repeated measure. In fine, the routine analysis of CTCs in clinical practice could be used for the real-time monitoring of therapies and the adaptation of treatment in order to improve the outcome of patients, a step forward towards so-called precision medicine. In this PhD project, we have developed novel micro- devices for the capture, in flow conditions, of tumor-derived cells from human whole blood. CTCs being larger and less deformable than normal blood cells, we exploited theses physical traits to discriminate them. Sieve-like micro-devices were engineered to selectively sort out tumor-derived cells having as a priority the preservation of cell integrity and viability. In addition, devices were designed to allow direct access to the isolated biological material and thus perform in situ cell identification, such as immunocytochemistry, but also to potentially serve as a platform for functional analysis. We proposed two approaches compatible with clinical routine. The first approach consists in a customized guiding-strip equipped with integrated microfilters, designed to be introduced directly within the bloodstream through a conventional medical catheter to perform the capture of tumor-derived cells in vivo. The second approach aims to perform CTC isolation ex vivo through the integration of microfilters into a platform compatible with blood collection medical sets. [...]

Within healthcare there is a market pull for biomedical devices that can rapidly perform laboratory processes, such as diagnostic testing, in a hand-held format. For this reason, biomedical devices must become smaller, more sophisticated, and easier to use for a reasonable cost. However, despite the accelerating academic research on biomedical microdevices, and especially plastic-based microfluidic chips, there is still a gap between the inventions in academia and their benefit to society. To bridge this gap there is a need for new materials which both exhibit similar properties as industrial thermoplastics, and that enable rapid prototyping in academia. In this thesis, thiol-ene and thiol-ene-epoxy thermosets are evaluated both in terms of their suitability for rapid prototyping of biomedical microdevices and their potential for industrial manufacturing of “lab-on-chips”. The first part of the thesis focuses on material development of thiol-ene and thiol-ene-epoxy thermosets. Chemical and mechanical properties are studied, as well as in vitro biocompatibility with cells. The second part of the thesis focuses on microfabrication methods for both thermosets. This includes reaction injection molding, photostructuring, and surface modification. It is demonstrated how thiol-ene and thiol-ene-epoxy both provide advantageous thermo-mechanical properties and versatile surface modifications via “thiol-click chemistry”. In the end of the thesis, two applications for both polymer platforms are demonstrated. Firstly, thiol-ene is used for constructing nanoliter well arrays for liquid storage and on-demand electrochemical release. Secondly, thiol-ene-epoxy is used to enhance the biocompatibility of neural probes by tuning their flexibility. It is concluded that both thiol-ene and thiol-ene-epoxy thermosets exhibit several properties that are highly suitable for rapid prototyping as well as for scalable manufacturing of biomedical microdevices. / <p>QC 20171003</p>