Isothermal-based DNA biosensors for application in pharmacogenetics

Tesis por compendio[EN] The determination of genetic biomarkers is progressively becoming more extended and popular, being commercialized even in kits for personalized medicine. Establishing specific genotype variations for each patient, such as single nucleotide polymorphisms (SNPs), could be a fundamental tool in the field of diagnosis, prognosis and therapy selection. However, the use of DNA testing is not fully implemented in general healthcare, mainly due to technical and economic barriers associated to the current technologies, which are limited only to specialized centers and large hospitals. In this thesis, the main goal was to overcome these obstacles by developing simpler, faster and more affordable point-of-care (POC) genotyping systems. Allele discrimination was achieved by employing isothermal enzymatic reactions, like recombinase polymerase amplification (RPA), ligation of oligonucleotides and loop-mediated isothermal amplification (LAMP). These processes were integrated to colorimetric indicators and immunoenzymatic assays, in a microarray format. Using compact discs and polycarbonate chips as platforms, the detection was achieved through widespread electronics, like disc-reader, flatbed scanner and smartphone. To demonstrate their capacities, the resulting systems were applied for identifying SNPs in human samples, associated to therapies for tobacco smoking cessation, major depression disorder and blood clotting-related diseases. After selecting the proper conditions, all studied strategies discriminated SNPs in samples containing as low as 100 copies of genomic DNA, with an error rate below 15%. Most importantly, the developed methods have reduced assays times varying between 70 and 140 minutes, at a cost similar to a conventional PCR-based analog, but maintaining or raising amplification efficiency and eliminating the need of specialized temperature cyclers and fluorescence scanners. In conclusion, the biosensors based in isothermal reactions and consumer electronics devices greatly improve the competitivity of POC DNA analysis. It was demonstrated that the technologies developed in this thesis could support genotyping assays in low-resource areas, such as primary healthcare centers and emerging countries. Through this democratization of genetic testing and by performing adequate association studies, molecular diagnostics and personalized medicine practices could have their application extended to the clinical routine.[ES] La determinación de biomarcadores genéticos es cada vez más extensa y popular, estando incluso comercializándose kits para medicina personalizada. Establecer las variaciones específicas en el genotipo de cada paciente, como los polimorfismos de un solo nucleótido (SNP) podría ser una herramienta fundamental en el campo del diagnóstico, pronóstico y selección de la terapia. Sin embargo, el uso de pruebas de ADN no se encuentra completamente implementado en la atención médica general, principalmente debido a las barreras técnicas y económicas asociadas a las tecnologías actuales, limitadas solamente a centros especializados y grandes hospitales. En esta tesis, el objetivo principal fue superar estos obstáculos mediante el desarrollo de sistemas de genotipado point-of-care (POC), más simples, rápidos y asequibles. La discriminación alélica se logró mediante el uso de reacciones enzimáticas isotermas, como la amplificación de la recombinasa polimerasa (RPA), la ligación de oligonucleótidos y la amplificación isotérmica mediada por bucle (LAMP). Estos procesos se integraron a indicadores colorimétricos y ensayos inmunoenzimáticos en formato de micromatriz. Utilizando discos compactos y chips de policarbonato como plataforma de ensayo, se ha logrado la detección mediante dispositivos electrónicos de consumo, como un lector de discos, escáner documental y teléfono móvil. Para demostrar sus capacidades, los sistemas resultantes se aplicaron a la identificación de SNPs en muestras humanas, asociados a terapias antitabaquismo, para depresión y enfermedades relacionadas con la coagulación de la sangre. Tras seleccionar las condiciones adecuadas, todas las estrategias estudiadas discriminaron SNPs en muestras conteniendo tan solo 100 copias de ADN genómico, con una tasa de error inferior al 15%. Más importante, los métodos desarrollados han reducido los tiempos de ensayo a valores entre 70 y 140 minutos, a un coste similar a un análogo convencional basado en la reacción en cadena de la polimerasa (PCR), pero manteniendo o aumentando la eficiencia de amplificación y eliminando la necesidad de termocicladores y escáneres de fluorescencia. En conclusión, los biosensores basados en reacciones isotérmicas y dispositivos de electrónica de consumo mejoran en gran medida la competitividad del análisis POC de ADN. Se ha demostrado que las tecnologías desarrolladas en esta tesis podrían apoyar los ensayos de genotipado en áreas de recursos escasos, como centros de atención primaria y países emergentes. A través de esta democratización de las pruebas genéticas y realización estudios de asociación adecuados, el diagnóstico molecular y las prácticas en medicina personalizada podrían extender su aplicación a la rutina clínica.[CA] La determinació de biomarcadors genètics és cada vegada més extensa i popular, estant fins i tot comercialitzant-se kits per a medicina personalitzada. Establir les variacions específiques en el genotip de cada pacient, com els polimorfismes d'un sol nucleòtid (SNP) podria ser una eina fonamental en el camp del diagnòstic, pronòstic i selecció de la teràpia. No obstant això, l'ús de proves d'ADN no es troba completament implementat en l'atenció mèdica general, principalment a causa de les barreres tècniques i econòmiques associades a les tecnologies actuals, limitades solament a centres especialitzats i grans hospitals. En aquesta tesi, l'objectiu principal va ser superar aquests obstacles mitjançant el desenvolupament de sistemes de genotipat point-of-care (POC), més simples, ràpids i assequibles. La discriminació al·lèlica es va aconseguir mitjançant l'ús de reaccions enzimàtiques isotermes, com l'amplificació de la recombinasa polimerasa (RPA), la lligació de oligonucleòtids i l'amplificació isotèrmica mediada per bucle (LAMP). Aquests processos es van integrar a indicadors colorimètrics i assajos inmunoenzimàtics en format de micromatriu. Utilitzant discos compactes i xips de policarbonat com a plataforma d'assaig, s'ha conseguit la detecció mitjançant dispositius electrònics de consum, com un lector de discos, escàner documental i telèfon mòbil. Per a demostrar les seues capacitats, els sistemes resultants es van aplicar a la identificació de polimorfismes en mostres humanes, associats a teràpies antitabaquisme, per a depressió i malalties relacionades amb la coagulació de la sang. Després de seleccionar les condicions adequades, totes les estratègies estudiades van ser capaces de discriminar SNPs en mostres contenint tan sols 100 còpies d'ADN genòmic, amb una taxa d'error inferior al 15%. Més important, els mètodes desenvolupats han reduït els temps d'assaig a valors entre 70 i 140 minuts, a un cost similar a un anàleg convencional basat en la reacció en cadena de la polimerasa (PCR), però mantenint o augmentant l'eficiència d'amplificació i eliminant la necessitat de termocicladors i escàners de fluorescència. En conclusió, els biosensors basats en reaccions isotèrmiques i dispositius d'electrònica de consum milloren en gran manera la competitivitat de l'anàlisi POC del ADN. S'ha demostrat que les tecnologies desenvolupades en aquesta tesi podrien donar suport als assajos de genotipat en àrees de recursos escassos, com a centres d'atenció primària i països emergents. A través d'aquesta democratització de les proves genètiques i realització estudis d'associació adequats, el diagnòstic molecular i les pràctiques en medicina personalitzada podrien estendre la seua aplicació a la rutina clínica.[PT] A determinação de biomarcadores genéticos está tornando-se cada vez mais extensa e popular, sendo comercializada até em kits para medicina personalizada. O estabelecimento de variações específicas de genotipo para cada paciente, tais como os polimorfismo de nucleotídeo único, pode ser uma ferramenta fundamental no campo do diagnóstico, prognóstico e seleção de terapias. No entanto, o uso de testes de DNA ainda não encontra-se totalmente implementado na área de saúde geral, principalmente devido às barreiras técnicas e econômicas associadas às tecnologias atuais, limitadas apenas a centros especializados e grandes hospitais. Nesta tese, o principal objetivo foi superar esses obstáculos desenvolvendo sistemas de genotipagem point-of-care (POC) de DNA, mais simples, rápidos e acessíveis. A discriminação de alelos foi alcançada empregando reações enzimáticas isotérmicas, como amplificação por recombinase polimerase (RPA), ligação de oligonucleotídeos e amplificação isotérmica mediada por loop (LAMP). Tais processos foram integrados a indicadores colorimétricos e ensaios imunoenzimáticos, em formato micromatriz. Usando discos compactos e chips de policarbonato como plataforma de ensaio, os analitos foram detectados através de dispositivos eletrônicos de consumo, como leitor de disco, scanner de mesa e smartphone. Para demonstrar suas capacidades, os sistemas resultantes foram aplicados para identificação de polimorfismos em amostras de DNA humano, associados a terapias antitabagismo, para depressão e doenças relacionadas à coagulação do sangue. Após a seleção das condições adequadas, todas as estratégias estudadas foram capazes de discriminar SNPs em amostras contendo até 100 cópias de DNA genômico, com uma taxa de erro inferior a 15%. Mais importante, os métodos desenvolvidos reduziram o tempo de ensaio a valores entre 70 e 140 minutos, com um custo similar a um método análogo baseado em reação em cadeia da polimerase (PCR), mas mantendo ou aumentando a eficiência da amplificação e eliminando a necessidade de cicladores de temperatura e scanners de fluorescência especializados. Em conclusão, os biosensores baseados em reações enzimáticas isotérmicas e dispositivos eletrônicos de consumo incrementam grandemente a competitividade da análise POC de DNA. Foi demonstrado que as tecnologias desenvolvidas nesta tese poderiam dar suporte a ensaios de genotipagem em lugares com poucos recursos, como centros de atenção primária e países emergentes. Através desta democratização dos testes genéticos e com a realização de estudos de associação adequados, o diagnóstico molecular e as práticas de medicina personalizada poderiam ter sua aplicação extendida à rotina clínica.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 Project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project)Yamanaka, ES. (2020). Isothermal-based DNA biosensors for application in pharmacogenetics [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/148366TESISCompendi

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

An update on novel approaches for diagnosis and treatment of SARS-CoV-2 infection

The ongoing pandemic of coronavirus disease 2019 (COVID-19) has made a serious public health and economic crisis worldwide which united global efforts to develop rapid, precise, and cost-efficient diagnostics, vaccines, and therapeutics. Numerous multi-disciplinary studies and techniques have been designed to investigate and develop various approaches to help frontline health workers, policymakers, and populations to overcome the disease. While these techniques have been reviewed within individual disciplines, it is now timely to provide a cross-disciplinary overview of novel diagnostic and therapeutic approaches summarizing complementary efforts across multiple fields of research and technology. Accordingly, we reviewed and summarized various advanced novel approaches used for diagnosis and treatment of COVID-19 to help researchers across diverse disciplines on their prioritization of resources for research and development and to give them better a picture of the latest techniques. These include artificial intelligence, nano-based, CRISPR-based, and mass spectrometry technologies as well as neutralizing factors and traditional medicines. We also reviewed new approaches for vaccine development and developed a dashboard to provide frequent updates on the current and future approved vaccines

Microfabricated Tools and Engineering Methods for Sensing Bioanalytes

There is a convergence between the needs of the medical community and the capabilities of the engineering community. For example, the scale of biomedical devices and sensors allow for finer, more cost-effective quantification of biological and chemical targets. By using micro-fabrication techniques, we design and demonstrate a variety of microfluidic sensors and actuators that allow us to interact with a biochemical environment. We demonstrate the performance of microfluidic blood-filtrations chips, immune-diagnostic assays, and evaporative coolers. Furthermore, we show how micro-fabricated platinum filaments can be used for highly localized heating and temperature measurement. We demonstrate that these filaments can be used as miniature IR spectroscopic sources. Finally, we describe and demonstrate novel combinatorial coding methods for increasing the information extracted from biochemical reactions. We show proof-principle of these techniques in the context of Taqman PCR as well as persistence length PCR

Identification and Characterization of Microbial Contaminants and Associated Bacterial Viruses in Bioethanol Production Facilities to Suggest a Potential Alternative to Antibiotic Treatment

In recent years, bioethanol has received worldwide interest as a bioenergy source. This interest has stimulated the production of substantial quantities of ethanol annually. However, the inability to produce bioethanol under sterile conditions plagues the industry, resulting in frequent microbial contamination. Bacterial contamination is one of the more challenging problems facing the bioethanol industry because contaminants drastically lower ethanol yield. Conventional methods of antibiotic application to eradicate bacterial contaminants are expensive and prohibitive. A more sustainable approach to control bacterial contamination of industrial ethanol fermentation systems is to use bacteriophages (phage). The goal of this research was to create a cocktail of phages capable of infecting and eliminating bacterial contaminants that hinder the production of bioethanol. I isolated, purified, and characterized the common bacterial contaminants in an industrial bioethanol fermenter and beerwell and demonstrated that bacteriophage could be induced from some of these cultures. Further research is needed to determine if virulent mutants of these phages can be generated

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

Computational and Experimental Approaches to Reveal the Effects of Single Nucleotide Polymorphisms with Respect to Disease Diagnostics

DNA mutations are the cause of many human diseases and they are the reason for natural differences among individuals by affecting the structure, function, interactions, and other properties of DNA and expressed proteins. The ability to predict whether a given mutation is disease-causing or harmless is of great importance for the early detection of patients with a high risk of developing a particular disease and would pave the way for personalized medicine and diagnostics. Here we review existing methods and techniques to study and predict the effects of DNA mutations from three different perspectives: in silico, in vitro and in vivo. It is emphasized that the problem is complicated and successful detection of a pathogenic mutation frequently requires a combination of several methods and a knowledge of the biological phenomena associated with the corresponding macromolecules

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

PCR assays for detection of human astroviruses: In silico evaluation and design, and in vitro application to samples collected from patients in the Netherlands

Molecular basis of virus replication, viral pathogenesis and antiviral strategie