Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Microchips and their significance in isolation of circulating tumor cells and monitoring of cancers

In micro-fluid systems, fluids are injected into extremely narrow polymer channels in small amounts such as micro-, nano-, or pico-liter scales. These channels themselves are embedded on tiny chips. Various specialized structures in the chips including pumps, valves, and channels allow the chips to accept different types of fluids to be entered the channel and along with flowing through the channels, exert their effects in the framework of different reactions. The chips are generally crystal, silicon, or elastomer in texture. These highly organized structures are equipped with discharging channels through which products as well as wastes of the reactions are secreted out. A particular advantage regarding the use of fluids in micro-scales over macro-scales lies in the fact that these fluids are much better processed in the chips when they applied as micro-scales. When the laboratory is miniaturized as a microchip and solutions are injected on a micro-scale, this combination makes a specialized construction referred to as "lab-on-chip". Taken together, micro-fluids are among the novel technologies which further than declining the costs; enhancing the test repeatability, sensitivity, accuracy, and speed; are emerged as widespread technology in laboratory diagnosis. They can be utilized for monitoring a wide spectrum of biological disorders including different types of cancers. When these microchips are used for cancer monitoring, circulatory tumor cells play a fundamental role

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

Design of a microfabricated device for Ligase Detection Reaction (LDR)

The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point mutations in deoxyribonucleic acid (DNA). Developed by Francis Barany and associates at Cornell University it is used to find specific low abundant point mutations that may lead to colorectal cancer in the early stages of disease development. The research objective was to design and manufacture a microscale Ligase Detection Reaction (LDR) device in polycarbonate. The LDR module will be incorporated with other microdevices such as: Continuous Flow Polymerase Chain Reaction (CFRCR) and Capillary Electrophoresis (CE) in modular lab-on-a-chip technology. In making the microdevice, the duration of original reaction had to be scaled down from the current 2½ hours for 20 cycles for the macroscale reaction. It was found that an excess of primers in relation to PCR product was needed for efficient ligation. By changing the concentrations, volumes and time for the process the current time is down to 40 minutes for 20 cycles with indications that further time reductions are possible on the microscale. There are two mixing stages involved in the reaction. Micromixers were simulated in Fluent (v5.4, Lebanon, NH) and several test geometries selected for fabrication. Passive diffusion mixing was used based on obtaining high aspect ratios, 7 to 20. The mixers were made by SU-8 lithography, LIGA, laser ablation, and micromilling to characterize each fabrication method. It was found that LIGA was best for making the micromixers, but was the longest process. The micromixers are fabricated and tested using chemi-luminescence technique. For a successful reaction, temperatures of 0°C, 95°C and 65°C were needed. A stationary chamber was used for thermal cycling in which the sample sits while the temperature is cycled. Finite element analysis showed uniform temperatures in the rectangular 1.5μl chambers and that air slits can effectively separate the thermal cycle zone from the 0°C cooling zone and also isolate the mixing region. A test device was laid out and micromilled with the temperature zones maintained and fluid flow controlled. A commercial thin film heater and a thermoelectric module were used with PID controls to obtain the required process temperatures. Heating from 65°C to 95°C took 10 seconds, while cooling from 95°C to 65°C also took 10 seconds. The residence times at the required temperatures can be adapted to changes in the LDR

Advances in microfluidics and lab on a chip technologies

pre-printAdvances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains like biology research, infectious disease management, food safety and bio-defense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care (POC). They have also introduced new functionality, enhanced the sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods like nucleic acid amplification, next generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Advances in Microfluidics Technology for Diagnostics and Detection

Microfluidics and lab-on-a-chip have, in recent years, come to the forefront in diagnostics and detection. At point-of-care, in the emergency room, and at the hospital bed or GP clinic, lab-on-a-chip offers the potential to rapidly detect time-critical and life-threatening diseases such as sepsis and bacterial meningitis. Furthermore, portable and user-friendly diagnostic platforms can enable disease diagnostics and detection in resource-poor settings where centralised laboratory facilities may not be available. At point-of-use, microfluidics and lab-on-chip can be applied in the field to rapidly identify plant pathogens, thus reducing the need for damaging broad spectrum pesticides while also reducing food losses. Microfluidics can also be applied to the continuous monitoring of water quality and can support policy-makers and protection agencies in protecting the environment. Perhaps most excitingly, microfluidics also offers the potential to enable entirely new diagnostic tests that cannot be implemented using conventional laboratory tools. Examples of microfluidics at the frontier of new medical diagnostic tests include early detection of cancers through circulating tumour cells (CTCs) and highly sensitive genetic tests using droplet-based digital PCR.This Special Issue on “Advances in Microfluidics Technology for Diagnostics and Detection” aims to gather outstanding research and to carry out comprehensive coverage of all aspects related to microfluidics in diagnostics and detection

Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

Isolation, Expansion and Characterization of Circulating Tumor Cells using Microfluidic Technologies.

Circulating tumor cells (CTCs) are the tumor cells shed from primary tumor, which enter the bloodstream and travel to distant anatomic sites to form metastasis. Detecting CTCs, as a means of “liquid biopsy”, allows monitoring cancer progression in real time and predicting therapeutic response. The major challenge that limits the clinical utility of CTCs, especially in early stage cancer patients, is their rarity; specifically, there are only 1-10 CTCs in one milliliter of whole blood. One way to overcome this critical limitation is to ex vivo expand CTCs through culturing. In this thesis, a microfluidic CTC capture device is optimized and tested for the capture and analysis of CTCs. Using a 3D, on chip co-culture model, CTCs were successfully expanded in 70% of the 50 early stage lung and esophageal cancer patients. Cultured CTCs were characterized with immunostaining, RNA profiling, mutation analysis and invasion assays. We found concordant TP53 mutation in CTCs and matched primary tumors. Next-generation sequencing further revealed mutations in additional genes. It was found that, patients whose CTCs exhibited the greatest capacity to expand had earlier recurrence. In one of the patient samples, expanded CTCs implanted in mice resulted in a xenograft tumor, demonstrating the expanded CTCs’ ability to metastasize. Genes related to EMT and tumor microenvironment were enriched in the xenograft. In addition, building upon this co-culture model, CTCs from one of the ALK positive metastatic lung cancer patients were isolated and cultured. The cultured CTCs harbored the concordant EML4-ALK rearrangement as the tumor biopsy specimen and further served as an in vitro model for drug testing. Taken together, this study demonstrated that CTCs from early stage lung cancer are tumorigenic and mirror the phenotypic and genotypic status of primary tumors. Ex vivo culturing of CTCs will make a significant impact in the era of personalized medicine. It will bring about opportunities for individualized drug screening, such as predicting treatment response to targeted therapies and the emergence of acquired drug resistance. Cultured CTCs will also serve as tools for understanding metastatic spread of cancer cells.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120755/1/zzhuo_1.pd

Novel approaches in cancer management with circulating tumor cell clusters

© 2019 The Authors Tumor metastasis is responsible for the vast majority of cancer-associated morbidities and mortalities. Recent studies have disclosed the higher metastatic potential of circulating tumor cell (CTC) clusters than single CTCs. Despite long-term study on metastasis, the characterizations of its most potent cellular drivers, i.e., CTC clusters have only recently been investigated. The analysis of CTC clusters offers new intuitions into the mechanism of tumor metastasis and can lead to the development of cancer diagnosis and prognosis, drug screening, detection of gene mutations, and anti-metastatic therapeutics. In recent years, considerable attention has been dedicated to the development of efficient methods to separate CTC clusters from the patients’ blood, mainly through micro technologies based on biological and physical principles. In this review, we summarize recent developments in CTC clusters with a particular emphasis on passive separation methods that specifically have been developed for CTC clusters or have the potential for CTC cluster separation. Methods such as liquid biopsy are of paramount importance for commercialized healthcare settings. Furthermore, the role of CTC clusters in metastasis, their physical and biological characteristics, clinical applications and current challenges of this biomarker are thoroughly discussed. The current review can shed light on the development of more efficient CTC cluster separation method that will enhance the pivotal understanding of the metastatic process and may be practical in contriving new strategies to control and suppress cancer and metastasis