CD-based microfluidics for primary care in extreme point-of-care settings

We review the utility of centrifugal microfluidic technologies applied to point-of-care diagnosis in extremely under-resourced environments. The various challenges faced in these settings are showcased, using areas in India and Africa as examples. Measures for the ability of integrated devices to effectively address point-of-care challenges are highlighted, and centrifugal, often termed CD-based microfluidic technologies, technologies are presented as a promising platform to address these challenges. We describe the advantages of centrifugal liquid handling, as well as the ability of a standard CD player to perform a number of common laboratory tests, fulfilling the role of an integrated lab-on-a-CD. Innovative centrifugal approaches for point-of-care in extremely resource-poor settings are highlighted, including sensing and detection strategies, smart power sources and biomimetic inspiration for environmental control. The evolution of centrifugal microfluidics, along with examples of commercial and advanced prototype centrifugal microfluidic systems, is presented, illustrating the success of deployment at the point-of-care. A close fit of emerging centrifugal systems to address a critical panel of tests for under-resourced clinic settings, formulated by medical experts, is demonstrated. This emphasizes the potential of centrifugal microfluidic technologies to be applied effectively to extremely challenging point-of-care scenarios and in playing a role in improving primary care in resource-limited settings across the developing world

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

A centrifugal microfluidic platform for capturing, assaying and manipulation of beads and biological cells

Microfluidics is deemed a field with great opportunities, especially for applications in medical diagnostics. The vision is to miniaturize processes typically performed in a central clinical lab into small, simple to use devices - so called lab-on-a-chip (LOC) systems. A wide variety of concepts for liquid actuation have been developed, including pressure driven flow, electro-osmotic actuation or capillary driven methods. This work is based on the centrifugal platform (lab-on-a-disc). Fluid actuation is performed by the forces induced due to the rotation of the disc, thus eliminating the need for external pumps since only a spindle motor is necessary to rotate the disc and propel the liquids inside of the micro structures. Lab-on-a-disc systems are especially promising for point-of-care applications involving particles or cells due to the centrifugal force present in a rotating system. Capturing, assaying and identification of biological cells and microparticles are important operations for lab-on-a-disc platforms, and the focus of this work is to provide novel building blocks towards an integrated system for cell and particle based assays. As a main outcome of my work, a novel particle capturing and manipulation scheme on a centrifugal microfluidic platform has been developed. To capture particles (biological cells or micro-beads) I designed an array of V-shaped micro cups and characterized it. Particles sediment under stagnant flow conditions into the array where they are then mechanically trapped in spatially well-defined locations. Due to the absence of flow during the capturing process, i.e. particle sedimentation is driven by the artificial gravity field on the centrifugal platform, the capture efficiency of this approach is close to 100% which is notably higher than values reported for typical pressure driven systems. After capturing the particles, the surrounding medium can easily be exchanged to expose them to various conditions such as staining solutions or washing buffers, and thus perform assays on the captured particles. By scale matching the size of the capturing elements to the size of the particles, sharply peaked single occupancy can be achieved. Since all particles are arrayed in the same focal plane in spatially well defined locations, operations such as counting or fluorescent detection can be performed easily. The application of this platform to perform multiplexed bead-based immunoassays as well as the discrimination of various cell types based on intra cellular and membrane based markers using fluorescently tagged antibodies is demonstrated. Additionally, methods to manipulate captured particles either in batch mode or on an individual particle level have been developed and characterized. Batch release of captured particles is performed by a novel magnetic actuator which is solely controlled by the rotation frequency of the disc. Furthermore, the application of this actuator to rapidly mix liquids is shown. Manipulation of individual particles is performed using an optical tweezers setup which has been developed as part of this work. Additionally, this optical module also provides fluorescence detection capabilities. This is the first time that optical tweezers have been combined with a centrifugal microfluidic system. This work presents the core technology for an integrated centrifugal platform to perform cell and particle based assays for fundamental research as well as for point-of- care applications. The key outputs of my specific work are: 1. Design, fabrication and characterization of a novel particle capturing scheme on a centrifugal microfluidic platform (V-cups) with very high capture efficiency (close to 100%) and sharply peaked single occupancy (up to 99.7% single occupancy). 2. A novel rotation frequency controlled magnetic actuator for releasing captured particles as well as for rapidly mixing liquids has been developed, manufactured and characterized. 3. The V-cup platform has successfully been employed to capture cells and perform multi-step antibody staining assays for cell discrimination. 4. An optical tweezers setup has been built and integrated into a centrifugal teststand, and successful manipulation of individual particles trapped in the V-cup array is demonstrated

Microfluidic PCR with plasmonic imaging for rapid multiplexed characterization of DNA from microbial pathogens

Bloodstream infections (BSIs) are a critical concern in modern medicine due to their continued prevalence in modern hospitals, along with high costs and attributable mortality, particularly among those who are immunocompromised. The current gold standard for detection and characterization of causative pathogens involves cell culture, which can take 24-48 hours to complete, increasing time to adequate treatment and thus mortality. The rise of antimicrobial resistance in hospital acquired infections has reduced the effectiveness of broad spectrum antimicrobial treatments, resulting in a clear need for a rapid, sensitive technique for characterization of resistance markers in microbial pathogens without cell culture. Here we present the development of a microfluidic platform for polymerase chain reaction (PCR) mediated amplification of microbial gene targets in a continuous flow system for potential coupling with sample preparation systems to reduce time to diagnosis from days to within two hours. This culminated in a thermoelectric cooler mediated fluidic thermocycler with a recirculating assay region for real-time hybridization measurements to minimize assay time. We subsequently demonstrated development of a low-cost optical system for localized surface plasmon resonance imaging using a digital micromirror device and tuned nanoprism monolayers for DNA hybridization with a spectral resolution of 2nm. This LSPR imaging system was integrated in-flow into the microfluidic thermocycler, enabling detection of input E. coli DNA samples at a minimum concentration of 5fg/ [microliter]. We further demonstrated multiplex detection of target markers, indicating potential for assaying target panels for characterization of pathogens. Overall, the studies in this dissertation demonstrate a microfluidic PCR system with built-in sensitive LSPR detection of DNA hybridization. It should serve as a starting point for exploration of and expansion with fluidic sample preparation with a focus on rapid characterization of pathogens.Biomedical Engineerin

Integrated manipulation, detection and counting of cells in biofluids

The manipulation, trapping, detection and counting of cells in biological fluids is of critical importance to the areas of disease diagnosis, drug delivery and genomic applications in biomedical research. In recent times, this research has focussed on utilising the superior metering, separation, routing, mixing and incubation capabilities of centrifugal microfluidic “Lab on a Disk” (LOAD) technologies to tackle the challenge of handling numerous types of cells, proteins, genes and their reagents simultaneously. Furthermore, integrated optical detection systems are being developed in parallel to the aforementioned microfluidic technologies, to facilitate the accurate and inexpensive detection, imaging and counting of cells. This thesis describes a number of novel centrifugal microfluidic approaches towards the separation, capture and detection of white blood cells from whole blood. Firstly, a thorough review of the state-of-the art research in the areas of centrifugo-microfluidic cell handling and detection is outlined. Secondly, a series of physical size filtration and microcontact printing approaches for the capture and detection of biomimetic particles are described. Finally, the author assesses the suitability of sol-gel materials for waveguiding applications on disposable LOAD platforms and outlines areas of future work that would build upon the research undertaken in this thesis

Study of Strategies for Genetic Variant Discrimination and Detection by Optosensing

Tesis por compendio[ES] La medicina actual se dirige hacia un enfoque más personalizado basándose en el diagnóstico molecular del paciente a través del estudio de biomarcadores específicos. Aplicando este principio molecular, el diagnóstico, pronóstico y selección de la terapia se apoyan en la identificación de variaciones específicas del genoma humano, como variaciones de un único nucleótido (SNV). Para detectar estos biomarcadores se dispone de una amplia oferta de tecnologías. Sin embargo, muchos de los métodos en uso presentan limitaciones como un elevado coste, complejidad, tiempos de análisis largos o requieren de personal y equipamiento especializado, lo que imposibilita su incorporación masiva en la mayoría de los sistemas sanitarios. Por tanto, existe la necesidad de investigar y desarrollar soluciones analíticas que aporten información sobre las variantes genéticas y que se puedan implementar en diferentes escenarios del ámbito de la salud con prestaciones competitivas y económicamente viables. El objetivo principal de esta tesis ha sido desarrollar estrategias innovadoras para resolver el reto de la detección múltiple de variantes genéticas que se encuentran en forma minoritaria en muestras biológicas de pacientes, cubriendo las demandas asociadas al entorno clínico. Las tareas de investigación se centraron en la combinación de reacciones de discriminación alélica con amplificación selectiva de DNA y el desarrollo de sistemas ópticos de detección versátiles. Con el fin de atender el amplio abanico de necesidades, en el primer capítulo, se presentan resultados que mejoran las prestaciones analíticas de la reacción en cadena de la polimerasa (PCR) mediante la incorporación de una etapa al termociclado y de un agente bloqueante amplificando selectivamente las variantes minoritarias que fueron monitorizadas mediante fluorescencia a tiempo real. En el segundo capítulo, se logró la discriminación alélica combinando la ligación de oligonucleótidos con la amplificación de la recombinasa polimerasa (RPA), que al operar a temperatura constante permitió una detección tipo point-of-care (POC). La identificación de SNV se llevó a cabo mediante hibridación en formato micromatriz, utilizando la tecnología Blu-Ray como plataforma de ensayo y detección. En el tercer capítulo, se integró la RPA con la reacción de hibridación alelo especifica en cadena (AS-HCR), en formato array para genotipar SNV a partir de DNA genómico en un chip. La lectura de los resultados se realizó mediante un smartphone. En el último capítulo, se presenta la síntesis de un nuevo reactivo bioluminiscente que se aplicó a la monitorización de biomarcadores de DNA a tiempo real y final de la RPA basada en la transferencia de energía de resonancia de bioluminiscencia (BRET), eliminando la necesidad de una fuente de excitación. Todas las estrategias permitieron un reconocimiento especifico de la variante de interés, incluso en muestras que contenían tan solo 20 copias de DNA genómico diana. Se consiguieron resultados sensibles (límite de detección 0.5% variante/total), reproducibles (desviación estándar relativa < 19%), de manera sencilla (3 etapas o menos), rápida (tiempos cortos de 30-200 min) y permitiendo el análisis simultaneo de varios genes. Como prueba de concepto, estas estrategias se aplicaron a la detección e identificación en muestras clínicas de biomarcadores asociados a cáncer colorrectal y enfermedades cardiológicas. Los resultados se validaron por comparación con los métodos de referencia NGS y PCR, comprobándose que se mejoraban los requerimientos técnicos y la relación coste-eficacia. En conclusión, las investigaciones llevadas a cabo posibilitaron desarrollar herramientas de genotipado con propiedades analíticas competitivas y versátiles, aplicables a diferentes escenarios sanitarios, desde hospitales a entornos con pocos recursos. Estos resultados son prometedores al dar respuesta a la demanda de tecnologías alternativas para el diagnóstico molecular personalizado.[CA] La medicina actual es dirigeix cap a un enfocament més personalitzat basant-se en el diagnòstic molecular del pacient a través de l'estudi de biomarcadors específics. Aplicant aquest principi molecular, el diagnòstic, pronòstic i selecció de la teràpia es recolzen en la identificació de variacions específiques del genoma humà, com variacions d'un únic nucleòtid (SNV). Per a detectar aquests biomarcadors, es disposa d'una àmplia oferta de tecnologies. No obstant això, molts dels mètodes en ús presenten limitacions com un elevat cost, complexitat, temps d'anàlisis llargues o requereixen de personal i equipament especialitzat, la qual cosa impossibilita la seua incorporació massiva en la majoria dels sistemes sanitaris. Per tant, existeix la necessitat d'investigar i desenvolupar solucions analítiques que aporten informació sobre les variants genètiques i que es puguen implementar en diferents escenaris de l'àmbit de la salut amb prestacions competitives i econòmicament viables. L'objectiu principal d'aquesta tesi ha sigut desenvolupar estratègies innovadores per a resoldre el repte de la detecció múltiple de variants genètiques que es troben en forma minoritària en mostres biològiques de pacients, cobrint les demandes associades a l'entorn clínic. Les tasques d'investigació es van centrar en la combinació de reaccions de discriminació al·lèlica amb amplificació selectiva de DNA i al desenvolupament de sistemes òptics de detecció versàtils. Amb la finalitat d'atendre l'ampli ventall de necessitats, en el primer capítol, es presenten resultats que milloren les prestacions analítiques de la reacció en cadena de la polimerasa (PCR) mitjançant la incorporació d'una etapa al termociclat i d'un agent bloquejant amplificant selectivament les variants minoritàries que van ser monitoritzades mitjançant fluorescència a temps real. En el segon capítol, es va aconseguir la discriminació al·lèlica combinant el lligament d'oligonucleòtids amb l'amplificació de la recombinasa polimerasa (RPA), que en operar a temperatura constant va permetre una detecció tipus point-of-care (POC). La identificació de SNV es va dur a terme mitjançant hibridació en format micromatriu, utilitzant la tecnologia Blu-Ray com a plataforma d'assaig i detecció. En el tercer capítol, es va integrar la RPA amb la reacció d'hibridació al·lel específica en cadena (AS-HCR), en format matriu per a genotipar SNV a partir de DNA genòmic en un xip. La lectura dels resultats es va realitzar mitjançant un telèfon intel·ligent. En l'últim capítol, es presenta la síntesi d'un nou reactiu bioluminescent que es va aplicar al monitoratge de biomarcadors de DNA a temps real i final de la RPA basada en la transferència d'energia de ressonància de bioluminescència (BRET), eliminant la necessitat d'una font d'excitació. Totes les estratègies van permetre un reconeixement específic de la variant d'interès, fins i tot en mostres que només contenien 20 còpies de DNA genòmic diana. Es van aconseguir resultats sensibles (límit de detecció 0.5% variant/total), reproduïbles (desviació estàndard relativa < 19%), de manera senzilla (3 etapes o menys), ràpida (temps curts de 30-200 min) i permetent l'anàlisi simultània de diversos gens. Com a prova de concepte, aquestes estratègies es van aplicar a la detecció i identificació en mostres clíniques de biomarcadors associats a càncer colorectal i a malalties cardiològiques. Els resultats es van validar per comparació amb els mètodes de referència NGS i PCR, comprovant-se que es milloraven els requeriments tècnics i la relació cost-eficàcia. En conclusió, les investigacions dutes a terme van possibilitar desenvolupar eines de genotipat amb propietats analítiques competitives i versàtils, aplicables a diferents escenaris sanitaris, des d'hospitals a entorns amb pocs recursos. Aquests resultats són prometedors en donar resposta a la demanda de tecnologies alternatives per al diagnòstic molecular personalitzat.[EN] Current medicine is moving towards a more personalized approach based on the patients' molecular diagnosis through the study of specific biomarkers. Diagnosis, prognosis and therapy selection, applying this molecular principle, rely on identifying specific variations in the human genome, such as single nucleotide variations (SNV). A wide range of technologies is available to detect these biomarkers. However, many of the employed methods have limitations such as high cost, complexity, long analysis times, or requiring specialized personnel and equipment, making their massive incorporation in most healthcare systems impossible. Therefore, there is a need to research and develop analytical solutions that provide information on genetic variants that can be implemented in different health scenarios with competitive and economically feasible performances. The main objective of this thesis has been to develop innovative strategies to solve the challenge of multiple detection of genetic variants that are found in a minority amount in patient samples, covering the demands associated with the clinical setting. Research tasks focused on the combination of allelic discrimination reactions with selective DNA amplification and the development of versatile optical detection systems. In order to meet the wide range of needs, in the first chapter, the analytical performances of the polymerase chain reaction (PCR) were improved by incorporating a thermocycling step and a blocking agent to amplify selectively minority variants that were monitored by real-time fluorescence. In the second chapter, allelic discrimination was achieved by combining oligonucleotide ligation with recombinase polymerase amplification (RPA), which operates at a constant temperature, allowing point-of-care (POC) detection. SNV identification was carried out by hybridization in microarray format, using Blu-Ray technology as the assay platform and detector. RPA was integrated with allele-specific hybridization chain reaction (AS-HCR), in an array format to genotype SNV from genomic DNA on a chip in the third chapter. The reading of the results was performed using a smartphone. In the last chapter, a new bioluminescent reagent was synthesized. It was applied to real-time and endpoint DNA biomarker monitoring based on bioluminescence resonance energy transfer (BRET), eliminating the need for an excitation source. All the strategies allowed specific recognition of the target variant, even in samples containing as few as 20 copies of target genomic DNA. Sensitive (limit of detection 0.5% variant/total), reproducible (relative standard deviation < 19%), simple (3 steps or less), fast (short times of 30-200 min) results were achieved, allowing simultaneous analysis of several genes. As proof of concept, these strategies were applied to detect and identify biomarkers associated with colorectal cancer and cardiological diseases in clinical samples. The results were validated by comparison with reference methods such as NGS and PCR, proving that the technical requirements and cost-effectiveness were improved. In conclusion, the developed research made it possible to develop genotyping tools with competitive analytical properties and versatile, applicable to different healthcare scenarios, from hospitals to limited-resource environments. These results are promising since they respond to the demand for alternative technologies for personalized molecular diagnostics.The authors acknowledge the financial support received from the Generalitat Valenciana PROMETEO/2020/094, GRISOLIA/2014/024 PhD Grant and GVA-FPI-2017 PhD grant, the Spanish Ministry of Economy and Competitiveness MINECO projects CTQ2016-75749-R and PID2019-110713RB-I00 and European Regional Development Fund (ERDF).Lázaro Zaragozá, A. (2022). Study of Strategies for Genetic Variant Discrimination and Detection by Optosensing [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185216TESISCompendi