Towards a Theory of Droplet-Mixing Graphs in Microfluidics

In this work, we study the problem of fluid mixing in microfluidic chips. The motivation for studying this problem comes from the process of sample preparation for chemical, biological, medical and environmental experiments, which often require preparation of fluid mixtures with desired concentrations. We assume that fluids are manipulated in discrete units called droplets. The input set of droplets consist of two distinct fluids: the reactant, which is the fluid of interest, and the buffer fluid that is used to dilute it. The goal is to produce a target set of droplets with prespecified reactant concentrations. In the model we study, the mixing process in a microfluidic chip can be abstractly represented as a mixing graph. A mixing graph is a collection of micro-mixers (nodes) connected by micro-channels (edges) that converts an input set of droplets I into a set of output droplets T, by applying a sequence of 1:1 mixing operations. This graph may also produce some waste, which are superfluous droplets of fluid not used in the target set. Computational complexity of most natural questions regarding such mixing graphs remain open. For example, it is not even known whether it is decidable for a given target set to be produced without waste. Current work in the literature contains only heuristic approaches that compute mixing graphs while attempting to optimize certain objectives, including minimizing waste, reactant usage, the depth of the graphs, and more.Our first contribution is an efficient algorithm for computing mixing graphs for single-droplet targets. Our algorithm produces significantly less waste than state-of-the-art algorithms in an experimental comparison. We also provide a bound on its worst-case performance that is significantly better than those for earlier algorithms. Our second result concerns the variant of the problem where the objective is to design a mixing graph that perfectly mixes a collection of input droplets with arbitrary concentrations. We provide a complete characterization of input sets for which such graphs exist, and an efficient algorithm to construct these graphs. In addition, we provide several other results about properties of mixing graphs and the computational complexity of computing mixing graphs of fixed depth

Design and Optimization Methods for Pin-Limited and Cyberphysical Digital Microfluidic Biochips

<p>Microfluidic biochips have now come of age, with applications to biomolecular recognition for high-throughput DNA sequencing, immunoassays, and point-of-care clinical diagnostics. In particular, digital microfluidic biochips, which use electrowetting-on-dielectric to manipulate discrete droplets (or "packets of biochemical payload") of picoliter volumes under clock control, are especially promising. The potential applications of biochips include real-time analysis for biochemical reagents, clinical diagnostics, flash chemistry, and on-chip DNA sequencing. The ease of reconfigurability and software-based control in digital microfluidics has motivated research on various aspects of automated chip design and optimization.</p><p>This thesis research is focused on facilitating advances in on-chip bioassays, enhancing the automated use of digital microfluidic biochips, and developing an "intelligent" microfluidic system that has the capability of making on-line re-synthesis while a bioassay is being executed. This thesis includes the concept of a "cyberphysical microfluidic biochip" based on the digital microfluidics hardware platform and on-chip sensing technique. In such a biochip, the control software, on-chip sensing, and the microfluidic operations are tightly coupled. The status of the droplets is dynamically monitored by on-chip sensors. If an error is detected, the control software performs dynamic re-synthesis procedure and error recovery.</p><p>In order to minimize the size and cost of the system, a hardware-assisted error-recovery method, which relies on an error dictionary for rapid error recovery, is also presented. The error-recovery procedure is controlled by a finite-state-machine implemented on a field-programmable gate array (FPGA) instead of a software running on a separate computer. Each state of the FSM represents a possible error that may occur on the biochip; for each of these errors, the corresponding sequence of error-recovery signals is stored inside the memory of the FPGA before the bioassay is conducted. When an error occurs, the FSM transitions from one state to another, and the corresponding control signals are updated. Therefore, by using inexpensive FPGA, a portable cyberphysical system can be implemented.</p><p>In addition to errors in fluid-handling operations, bioassay outcomes can also be erroneous due the uncertainty in the completion time for fluidic operations. Due to the inherent randomness of biochemical reactions, the time required to complete each step of the bioassay is a random variable. To address this issue, a new "operation-interdependence-aware" synthesis algorithm is proposed in this thesis. The start and stop time of each operation are dynamically determined based on feedback from the on-chip sensors. Unlike previous synthesis algorithms that execute bioassays based on pre-determined start and end times of each operation, the proposed method facilitates "self-adaptive" bioassays on cyberphysical microfluidic biochips.</p><p>Another design problem addressed in this thesis is the development of a layout-design algorithm that can minimize the interference between devices on a biochip. A probabilistic model for the polymerase chain reaction (PCR) has been developed; based on the model, the control software can make on-line decisions regarding the number of thermal cycles that must be performed during PCR. Therefore, PCR can be controlled more precisely using cyberphysical integration.</p><p>To reduce the fabrication cost of biochips, yet maintain application flexibility, the concept of a "general-purpose pin-limited biochip" is proposed. Using a graph model for pin-assignment, we develop the theoretical basis and a heuristic algorithm to generate optimized pin-assignment configurations. The associated scheduling algorithm for on-chip biochemistry synthesis has also been developed. Based on the theoretical framework, a complete design flow for pin-limited cyberphysical microfluidic biochips is presented.</p><p>In summary, this thesis research has led to an algorithmic infrastructure and optimization tools for cyberphysical system design and technology demonstrations. The results of this thesis research are expected to enable the hardware/software co-design of a new class of digital microfluidic biochips with tight coupling between microfluidics, sensors, and control software.</p>Dissertatio

Confocal laser scanning microscopy of nanoparticles applied to immunosorbent assays

The aim of this project was to demonstrate and develop a confocal readout method for fluorescent immunosorbent assays and investigate its potential advantages in comparison to traditional immunoassays. The key point of a confocal immunosorbent assay is the ability to detect the thin layer of immunoassay in the presence of unbound fluorescent reagents without washing the overlayer. Heterogeneous and homogeneous sandwich immunoassays of human IgG model were demonstrated successfully followed by the use of an empirical decomposition method for quantitative separation of the signals of the thin fluorescent assay layer from the overlayer. The detection limits for the homogeneous and heterogeneous formats of the model were 2.2 and 5.5 ng/ml, respectively. The application of confocal microscopy in kinetic analysis of the antigen-antibody reaction of the human IgG model was studied for homogeneous and heterogeneous formats and two fluorescent labels antibodies (FITC and QDs)

Study of substrate modulation and bioreceptor anchoring for the development of high performance microarrays

Tesis por compendio[EN] The present PhD thesis is focused on the study of new approaches able to improve the performance of microarrays. Aspects such as the nature of the surfaces and the probes, functionalization of the substrates, probe printing, immobilization and target detection were considered in the fabrication process. Within all these features, modulation of the surface behavior and probe anchoring were the most challenging aspects, as the interface is key for the immobilization of the receptors and the later detection, which will determine the performance of the final device. In this work, two microarray types have been developed, one for oligonucleotides and another one for antibodies. Then, a characterization of the reached achievements is done. All the routes have in common the use of light to catalyze the attachment of bioreceptors on the surface substrates, employing click-chemistry reactions. In the first chapter, the state of the art of microarray technology is overviewed, with special focus in the main aspects of microarray design. In the second chapter, the goals for this PhD thesis are settled. These general objectives are addressed in the following experimental chapters. In the third chapter, the effect of hydrophobicity and probe multi-point attachment on the microarray performance are studied. Thus, modulation of glass slide surfaces with alkenyl and alkynyl motifs for the anchoring of mono and multithiolated oligonucleotide probes by thiol-ene and thiol-yne photocoupling reactions, respectively, was accomplished. Surfaces modified with the most hydrophobic silane (alkynyl), or anchoring polythiolated probes, revealed better performances. These microarray systems were applied to the discrimination of SNPs and to detect bacterial genome PCR products. In the fourth chapter, a rational design for the preparation of microarrays of antibodies, is done. The immobilization approach displays the oriented anchoring of thiol-bearing antibody fragments to alkenylated glass slides by thiol-ene photocoupling reaction. Multiplexed detection of cardiac biomarkers is demonstrated. The designed microarray shows higher recognition capacity in comparison to whole antibody microarrays. In the fifth chapter, improvement of a novel methodology for the anchoring of thiolated oligonucleotides has been developed. Due to the interest on modifying highly hydrophobic surfaces, a new photoinduced reaction is set up. Thanks to the features of the named "fluor-thiol photocoupling reaction", immobilization of thiolated probes to surfaces containing C-F bonds in a fast, easy and biocompatible with aqueous media way, was achieved. Hydrophobicity of the surfaces was controlled to get successful hybridizations. Because of the high hydrophobicity of the surfaces, a huge confinement of the probes is accomplished, which allows the approximation of the analytes only where the probe is linked, keeping a high repulsion in the remaining surface. The perfluorinated glass slides improved the immobilization densities and detection capacity, regarding to the alkenylated and alkynylated surfaces, and allowed the discrimination of SNPs and detection of bacterial PCR products, as well. In the sixth chapter, other surfaces different than glass are explored. Thus, polyvinylidene fluoride membranes were employed as substrates for the development of oligonucleotide microarrays. Therefore, a fast, easy and mild functionalization process by UV irradiation and organosilane chemistry, was developed. Then, alkenyl functionalized and non-functionalized membranes were applied to microarray technology by covalent anchoring through thiol-ene and fluor-thiol photocoupling reactions, respectively. Promising results were obtained with both surfaces.[ES] La presente tesis tesis doctoral se centra en el estudio de nuevas aproximaciones capaces de mejorar el rendimiento de los microarrays. Aspectos como la naturaleza de las superficies y las sondas, la funcionalización de los sustratos, la impresión, la inmovilización y la detección de las sondas se consideraron en el proceso de fabricación. Dentro de todas estas características, la modulación de la superficie y el anclaje de la sonda fueron los aspectos más desafiantes, ya que la interfaz es clave para la inmovilización de los receptores y la posterior detección, lo que determinará el rendimiento del dispositivo final. En este trabajo, se han desarrollado dos tipos de microarrays, uno para oligonucleótidos y otro para anticuerpos. Luego, se ha realizado una caracterización de los logros alcanzados. Todas las rutas tienen en común el uso de la luz para catalizar la unión de los biorreceptores en los sustratos de la superficie, empleando reacciones de la química clic. En el primer capítulo, se facilita una visión general del estado del arte de la tecnología de microarrays con un enfoque especial en los aspectos principales del diseño de microarrays. En el segundo capítulo, se establecen los objetivos de esta tesis doctoral. Estos objetivos generales se abordan en los siguientes capítulos experimentales. En el tercer capítulo, se estudia el efecto de la hidrofobia y el uso de sondas con múltiples puntos de unión, en el rendimiento del microarray. De este modo, se llevó a cabo la modulación de superficies vidrio con grupos alquenilo y alquinilo para el anclaje de sondas de oligonucleótidos mono y multitioladas mediante las reacciones de foto anclaje del tiol-eno y tiol-ino, respectivamente. Las superficies modificadas con el silano más hidrofóbico (alquinilo) y las sondas politioladas ancladas, revelaron mejores rendimientos. Estos sistemas de microarrays se aplicaron a la discriminación de SNPs y a la detección de productos de PCR de bacterias. En el cuarto capítulo, se realiza un diseño racional para la preparación de microarrays de anticuerpos. El enfoque de inmovilización muestra el anclaje orientado de los fragmentos de anticuerpos que contienen tiol sobre superficies de vidrio alqueniladas mediante reacción de foto anclaje del tiol-eno. De esta forma, se demuestra la detección multiplexada de biomarcadores cardíacos. El microarray diseñado muestra una mayor capacidad de reconocimiento en comparación con los microarrays de anticuerpos completos. En el quinto capítulo, se ha desarrollado una nueva metodología para mejorar el anclaje de oligonucleótidos tiolados. Dado el interés en modificar superficies altamente hidrófobas, se establece una nueva reacción fotoinducida. Gracias a las características de la llamada "reacción de fotoacoplamiento de fluor-tiol", se logró la inmovilización de sondas tioladas a superficies que contienen enlaces C-F de una manera rápida, fácil y biocompatible con medios acuosos. La hidrofobicidad de las superficies se controló para obtener hibridaciones exitosas. Debido a la alta hidrofobicidad de las superficies, se logra un gran confinamiento de las sondas, lo que permite la aproximación de los analitos solo donde está unida la sonda, manteniendo una alta repulsión en la superficie restante. Las superficies de vidrio perfluoradas mejoraron las densidades de inmovilización y la capacidad de detección, con respecto a las superficies alqueniladas y alquiniladas, y también, permitieron la discriminación de SNPs y la detección de productos de PCR bacterianos. En el sexto capítulo, se exploran otras superficies diferentes al vidrio. Por lo tanto, membranas de fluoruro de polivinilideno se emplearon como sustratos para el desarrollo de microarrays de oligonucleótidos. Para ello, se desarrolló un proceso de funcionalización rápido, fácil y suave, mediante el empleo de irradiación UV y la química de los organosilanos.[CA] La present tesi doctoral es centra en l'estudi de noves aproximacions capaces de millorar el rendiment dels microarrays. Aspectes com ara la naturalesa de les superfícies i les sondes, la funcionalització dels substrats, la impressió, la immobilització i la detecció de les sondes es van considerar en el procés de fabricació. Dins de totes aquestes característiques, la modulació de la superfície i l'ancoratge de la sonda van ser els aspectes més desafiadors, ja que la interfície és clau per a la immobilització dels receptors i la posterior detecció, la qual cosa determinarà el rendiment del dispositiu final. En aquest treball, s'han desenvolupat dos tipus de microarrays, un per a oligonucleòtids i un altre per a anticossos. Després, s'ha realitzat una caracterització dels resultats aconseguits. Totes les rutes tenen en comú l'ús de la llum per a catalitzar la unió dels biorreceptores en els substrats de la superfície, emprant reaccions de la química clic. En el primer capítol, es facilita una visió general de l'estat de l'art de la tecnologia de microarrays amb un enfocament especial en els aspectes principals del disseny de microarrays. En el segon capítol, s'estableixen els objectius d'aquesta tesi doctoral. Aquests objectius generals s'aborden en els següents capítols experimentals. En el tercer capítol, s'estudia l'efecte de la hidrofòbia i l'ús de sondes amb múltiples punts d'unió, en el rendiment del microarray. D'aquesta manera, es va dur a terme la modulació de superfícies de vidre amb grups alquenil i alquinil per a l'ancoratge de sondes de oligonucleòtids mono i multitiolades mitjançant les reaccions de foto ancoratge del tiol-doble enllaç i tiol-triple enllaç, respectivament. Les superfícies modificades amb el silà més hidrofòbic (alquinil) i les sondes politiolades ancorades, van revelar els millors rendiments. Aquests sistemes de microarrays es van aplicar a la discriminació de SNPs i a la detecció de productes de PCR de bacteris. En el quart capítol, es realitza un disseny racional per a la preparació de microarrays d'anticossos. L'enfocament d'immobilització mostra l'ancoratge orientat dels fragments d'anticossos que contenen el grup tiol sobre superfícies de vidre alquenilades mitjançant reacció de foto ancoratge del tiol-doble enllaç. D'aquesta forma, es demostra la detecció multiplexada de biomarcadors cardíacs. El microarray dissenyat mostra una major capacitat de reconeixement en comparació amb els microarrays d'anticossos complets. En el cinqué capítol, s'ha desenvolupat una nova metodologia per a millorar l'ancoratge de oligonucleòtids tiolats. Donat l'interés de modificar superfícies altament hidròfobes, s'estableix una nova reacció fotoinduïda. Gràcies a les característiques de l'anomenada "reacció de fotoacoplament de fluor-tiol", es va aconseguir la immobilització de sondes tioladas a superfícies que contenen enllaços C-F d'una manera ràpida, fàcil i biocompatible amb medis aquosos. La hidrofobicitat de les superfícies es va controlar per a obtindre bones hibridacions reeixides. A causa de l'alta hidrofobicidad de les superfícies, s'aconsegueix un gran confinament de les sondes, la qual cosa permet l'aproximació dels anàlits únicament on està unida la sonda i manté una alta repulsió en la superfície restant. Les superfícies de vidre perfluorades van millorar les densitats d'immobilització i la capacitat de detecció, respecte a les superfícies alquenilades i alquinilades, i també van permetre la discriminació de SNPs i la detecció de productes de PCR bacterians. En el sisé capítol, s'exploren altres superfícies diferents al vidre. Per tant, membranes de fluorur de polivinilidé es van emprar com a substrats per al desenvolupament de microarrays d'oligonucleòtids. Per a això, es va desenvolupar un procés de funcionalització ràpid, fàcil i suau, mitjançant l'ús d'irradiació UV i la química dels organosilanAgradecer al Ministerio de Economía y Competitividad de España, por su programa de becas doctorales FPIJiménez Meneses, P. (2020). Study of substrate modulation and bioreceptor anchoring for the development of high performance microarrays [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137993TESISCompendi