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

Point-of-Care Devices for Viral Detection: COVID-19 Pandemic and Beyond

The pandemic of COVID-19 and its widespread transmission have made us realize the importance of early, quick diagnostic tests for facilitating effective cure and management. The primary obstacles encountered were accurately distinguishing COVID-19 from other illnesses including the flu, common cold, etc. While the polymerase chain reaction technique is a robust technique for the determination of SARS-CoV-2 in patients of COVID-19, there arises a high demand for affordable, quick, user-friendly, and precise point-of-care (POC) diagnostic in therapeutic settings. The necessity for available tests with rapid outcomes spurred the advancement of POC tests that are characterized by speed, automation, and high precision and accuracy. Paper-based POC devices have gained increasing interest in recent years because of rapid, low-cost detection without requiring external instruments. At present, microfluidic paper-based analysis devices have garnered public attention and accelerated the development of such POCT for efficient multistep assays. In the current review, our focus will be on the fabrication of detection modules for SARS-CoV-2. Here, we have included a discussion on various strategies for the detection of viral moieties. The compilation of these strategies would offer comprehensive insight into the detection of the causative agent preparedness for future pandemics. We also provide a descriptive outline for paper-based diagnostic platforms, involving the determination mechanisms, as well as a commercial kit for COVID-19 as well as their outlook

Progress in fluorescence biosensing and food safety towards point-of-detection (PoD) system

The detection of pathogens in food substances is of crucial concern for public health and for the safety of the natural environment. Nanomaterials, with their high sensitivity and selectivity have an edge over conventional organic dyes in fluorescent-based detection methods. Advances in microfluidic technology in biosensors have taken place to meet the user criteria of sensitive, inexpensive, user-friendly, and quick detection. In this review, we have summarized the use of fluorescence-based nanomaterials and the latest research approaches towards integrated biosensors, including microsystems containing fluorescence-based detection, various model systems with nano materials, DNA probes, and antibodies. Paper-based lateral-flow test strips and microchips as well as the most-used trapping components are also reviewed, and the possibility of their performance in portable devices evaluated. We also present a current market-available portable system which was developed for food screening and highlight the future direction for the development of fluorescence-based systems for on-site detection and stratification of common foodborne pathogens

MakerFluidics: low cost microfluidics for synthetic biology

Recent advancements in multilayer, multicellular, genetic logic circuits often rely on manual intervention throughout the computation cycle and orthogonal signals for each chemical “wire”. These constraints can prevent genetic circuits from scaling. Microfluidic devices can be used to mitigate these constraints. However, continuous-flow microfluidics are largely designed through artisanal processes involving hand-drawing features and accomplishing design rule checks visually: processes that are also inextensible. Additionally, continuous-flow microfluidic routing is only a consideration during chip design and, once built, the routing structure becomes “frozen in silicon,” or for many microfluidic chips “frozen in polydimethylsiloxane (PDMS)”; any changes to fluid routing often require an entirely new device and control infrastructure. The cost of fabricating and controlling a new device is high in terms of time and money; attempts to reduce one cost measure are, generally, paid through increases in the other. This work has three main thrusts: to create a microfluidic fabrication framework, called MakerFluidics, that lowers the barrier to entry for designing and fabricating microfluidics in a manner amenable to automation; to prove this methodology can design, fabricate, and control complex and novel microfluidic devices; and to demonstrate the methodology can be used to solve biologically-relevant problems. Utilizing accessible technologies, rapid prototyping, and scalable design practices, the MakerFluidics framework has demonstrated its ability to design, fabricate and control novel, complex and scalable microfludic devices. This was proven through the development of a reconfigurable, continuous-flow routing fabric driven by a modular, scalable primitive called a transposer. In addition to creating complex microfluidic networks, MakerFluidics was deployed in support of cutting-edge, application-focused research at the Charles Stark Draper Laboratory. Informed by a design of experiments approach using the parametric rapid prototyping capabilities made possible by MakerFluidics, a plastic blood--bacteria separation device was optimized, demonstrating that the new device geometry can separate bacteria from blood while operating at 275% greater flow rate as well as reduce the power requirement by 82% for equivalent separation performance when compared to the state of the art. Ultimately, MakerFluidics demonstrated the ability to design, fabricate, and control complex and practical microfluidic devices while lowering the barrier to entry to continuous-flow microfluidics, thus democratizing cutting edge technology beyond a handful of well-resourced and specialized labs

Microfabrication and Applications of Opto-Microfluidic Sensors

A review of research activities on opto-microfluidic sensors carried out by the research groups in Canada is presented. After a brief introduction of this exciting research field, detailed discussion is focused on different techniques for the fabrication of opto-microfluidic sensors, and various applications of these devices for bioanalysis, chemical detection, and optical measurement. Our current research on femtosecond laser microfabrication of optofluidic devices is introduced and some experimental results are elaborated. The research on opto-microfluidics provides highly sensitive opto-microfluidic sensors for practical applications with significant advantages of portability, efficiency, sensitivity, versatility, and low cost

Miniaturizing High Throughput Droplet Assays For Ultrasensitive Molecular Detection On A Portable Platform

Digital droplet assays – in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually – have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are mainly confined to laboratory settings due to the instrumentation necessary to serially generate, control, and measure millions of compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology has three key innovations: 1. the integration and parallel operation of hundred droplet generators onto a single chip that operates \u3e100x faster than a single droplet generator. 2. the fluorescence detection of droplets at \u3e100x faster than conventional in-flow detection using time-domain encoded mobile-phone imaging, and 3. the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation. By using this time-domain modulation with cloud computing, we overcome the low framerate of digital imaging, and achieve throughputs of one million droplets per second. To demonstrate the power of this approach, we performed a duplex digital enzyme-linked immunosorbent assay (ELISA) in serum to show a 1000x improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system. This work has broad potential for ultrasensitive, highly multiplexed detection, in a mobile format. Building on our initial demonstration, we explored the following: (i) we demonstrated that the platform can be extended to \u3e100x multiplexing by using time-domain encoded light sources to detect color-coded beads that each correspond to a unique assay, (ii) we demonstrated that the platform can be extended to the detection of nucleic acid by implementing polymerase chain reaction, and (iii) we demonstrated that sensitivity can be improved with a nanoparticle-enhanced ELISA. Clinical applications can be expanded to measure numerous biomarkers simultaneously such as surface markers, proteins, and nucleic acids. Ultimately, by building a robust device, suitable for low-cost implementation with ultrasensitive capabilities, this platform can be used as a tool to quantify numerous medical conditions and help physicians choose optimal treatment strategies to enable personalized medicine in a cost-effective manner

레이저 활성 세포 분리 기기를 이용한 조직 내 세포 분리 및 전장 유전체 및 전사체 분석 기술 개발

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2020. 8. 권성훈.In this dissertation, Spatially-resolved Laser Activated Cell Sorting (SLACS) technique is introduced, and its applications in genomics and transcriptomics are demonstrated. All biological mass is comprised of biological cells, each of which contain its own multi-billion bytes worth of data from genetic molecules, such as DNA or RNA. After the Human Genome Project sequenced one persons genome in ten years, the massively parallel sequencing technologies that are referred to the next generation sequencing (NGS) sprouted innovations in biology, providing further insights into biology and generating revolutions in diagnostics and therapeutics. However, these technologies were only applicable to pools of heterogeneous genetic molecules, hindering thorough explorations of genetic landscapes in the different cells within a biospecimen. Therefore, efforts to separate each and every cell from the pool of cells have generated numerous single cell isolation methodologies, which can be categorized into three: those that separate cell using microfluidics, microarrays, and optics. Advancement in micro-technologies particularly provided advantages in manipulating single cells because biological cell sizes that usually range from microns to tens of microns. State-of-art cell separation technologies that utilize microfluidic properties were rapidly commercialized, enabling high throughput single cell analysis that can process hundreds to thousands of single cells at a time. These utilize cell dissociation and compartmentalization in a microfluidic chambers or a pico-liter droplets, in which biomolecular techniques can amplify the desired genetic molecules. The amplified products such as the genomes or the transcriptomes of the single cells are sequenced through NGS, providing insights into how the dissociated cells were functioning in the biospecimen. However, the dissociation process of the cells that are originally adhered to each other can be harsh and requires the surface proteins that interact with another to be degraded. This process has raised many doubts on whether the cell state is the same before it is dissociated within a solvent. Therefore, microarrays of chemically synthesized oligonucleotides that can capture the poly adenosine tail, or poly (A) tail, were developed to capture the messenger RNAs (mRNAs) directly from the biological specimens. These technologies, however, require large resolution of the oligonucleotide spots because of the technical limitations in chemical DNA synthesis technologies and cross-contaminations between the spots. Optical separation of the cells from biospecimen has been extensively investigated with conventional laser capture microdissection (LCM) devices that utilize laser to transfer target area of interest to the desired receiver. However, these utilize either ultraviolet (UV) lasers to catapult the desired areas that can be highly damaging to the biomolecules within, or thermoplastics that can be melt down using near-infrared (IR) lasers and transfer the desired region of interest for further biological assays. However the thermoplastic approach often cause cross-contamination and has low throughput because the specimen has to be isolated in a contact manner. In this dissertation, the development of an optical cell sorter, or spatially-resolved laser activated cell sorter (SLACS) that uses pulsed near-IR laser that can optomechanically isolate the cells with low damage and high throughput is described. The engineering process of this novel device and two softwares and their applications in NGS technologies are described. Furthermore, the applications of SLACS for genomics and transcriptomics are demonstrated. Proof-of-concept studies for future applications of SLACS are also described.본 학위 논문에서는 이 논문에서는 SLACS (Spatially-resolved Laser Activated Cell Sorting) 기술이 도입되었으며 유전체학 및 전사체학에 대한 응용이 시연되었다. 모든 생물학적 덩어리는 생물학적 세포로 구성되며, 각각의 세포는 DNA 또는 RNA와 같은 유전자 분자로부터 얻은 수십억 바이트의 데이터를 포함한다. 휴먼 게놈 프로젝트가 10 년 안에 한 사람의 게놈을 시퀀싱 한 후, 차세대 시퀀싱 (NGS)과 관련되는 대규모 병렬 시퀀싱 기술은 생물학의 혁신을 불러 일으켜 생물학에 대한 통찰력을 제공하고 진단 및 치료에서 혁명을 일으켰다. 그러나 이 기술들은 이종 유전자 분자의 풀에만 적용 할 수 있으며, 생물 표본 내의 다른 세포에서 유전자 지형의 철저한 탐색을 방해하였다. 따라서, 세포 풀에서 각각의 모든 세포를 따로 분리하려는 노력은 수많은 단일 세포 분리 방법론을 생성하였으며, 이는 미세유체학, 마이크로 어레이 및 광학을 사용하여 세포를 분리하는 것의 세 가지로 분류 될 수 있다. 일반적으로 수 마이크로미터에서 수십 마이크로미터에 이르는 생물학적 세포 크기 때문에 단일 기술의 진보는 단일 세포 조작에 이점을 제공 하였다. 미세 유체 특성을 이용하는 최첨단 세포 분리 기술이 빠르게 상용화되어 한 번에 수백에서 수천 개의 단일 세포를 처리 할 수 있는 고 처리량 단일 세포 분석이 가능해졌습니다. 이들은 미세 분자 챔버 또는 피코 리터 액적에서 세포 해리 및 구획화를 이용하며, 여기서 생체 분자 기술은 원하는 유전자 분자를 증폭시킬 수 있다. 단일 세포의 게놈 또는 전 사체와 같은 증폭 된 생성물은 NGS를 통해 시퀀싱되어, 해리 된 세포가 생체 시편에서 어떻게 기능하는지에 대한 통찰력을 제공한다. 그러나, 원래 서로 부착 된 세포의 해리 과정은 가혹할 수 있으며, 서로 상호 작용하는 표면 단백질이 분해 될 것을 요구한다. 이 공정은 전지 상태가 용매 내에서 해리되기 전에 동일한 지에 대해 많은 의문을 제기했다. 따라서, 폴리아데노신 꼬리 또는 폴리 (A) 꼬리를 포획 할 수 있는 화학적으로 합성 된 올리고 뉴클레오티드의 마이크로 어레이는 생물학적 표본으로부터 메신저 RNA (mRNA)를 직접 포획하기 위해 개발되었다. 그러나, 이들 기술은 화학적 DNA 합성 기술의 기술적 한계 및 스폿 간의 교차 오염으로 인해 올리고 뉴클레오티드 스폿의 큰 해상도를 요구한다. 생체 시료로부터 세포의 광학적 분리는 관심 대상 영역을 원하는 수신기로 전달하기 위해 레이저를 이용하는 종래의 레이저 캡처 미세 해부 (LCM) 장치로 광범위하게 조사되었다. 그러나, 이들은 자외선 (UV) 레이저를 사용하여 생체 내 분자에 크게 손상을 줄 수있는 원하는 영역을 만들거나 근적외선 (IR) 레이저를 사용하여 녹일 수 있고 추가 생물학적 물질을 위해 원하는 관심 영역을 전달할 수있는 열가소성 수지를 사용한다. 분석. 그러나 열가소성 방식은 종종 교차 오염을 유발하고 시편을 접촉 방식으로 분리해야하기 때문에 처리량이 낮다. 이 논문에서는 광학 셀 분류기 또는 낮은 손상과 높은 처리량으로 셀을 광학적으로 분리 할 수 있는 펄스 형 근적외선 레이저를 사용하는 SLACS (공간적으로 해결 된 레이저 활성화 셀 분류기)의 개발에 대해 설명하였다. 이 새로운 장치의 엔지니어링 프로세스와 NGS 기술의 두 소프트웨어 및 응용 프로그램에 대해 설명하였다. 또한, 게놈 및 전 사체에 대한 SLACS의 적용이 입증되었다. SLACS의 향후 응용에 대한 개념 증명 연구도 설명하였다.CHAPTER 1. INTRODUCTION １ 1.1. Spatially resolved omics for atlasing human cells in the biological circuitry ２ 1.1.1. The emergence of single cell sequencing technologies ３ 1.1.2. Spatially resolved omics technologies and needs for development ７ 1.2. Main Concept: Development of spatially-resolved laser activated cell sorter (SLACS) and compatible omics technologies １４ 1.3. Outline of the dissertation １５ CHAPTER 2. BACKGROUND １６ 2.1. Previous spatial omics technologies １７ 2.1.1. In situ spatial omics technologies １７ 2.1.2. Isolate-and-transfer technologies for spatial omics ２０ 2.2. Commercialized spatial omics technologies ２３ 2.3. Previous research in the group ２５ CHAPTER 3. PLATFORM DEVELOPMENT ２９ 3.1. Development of SLACS and remote selection system ３０ 3.2. Whole genome sequencing strategies for SLACS ３３ 3.3. Whole transcriptome sequencing strategies for SLACS ４２ CHAPTER 4. PLATFORM APPLICATION ４８ 4.1. Applications of SLACS to spatial genomics ４９ 4.2. Applications of SLACS to spatial transcriptomics ６２ 4.3. Applications of OPENchip and future perspectives with SLACS ６５ CHAPTER 5. CONCLUSION AND DISCUSSION ７９ 5.1. Summary of dissertation ８０ 5.2. Comparison with previous technology ８３ 5.3. Limit of the platform ８４ 5.4. Future work ８６ BIBLIOGRAPHY ８８ 국문 초록 ９５Docto

Optimization of Continuous Flow Polymerase Chain Reaction with Microfluidic Reactors

The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific ~gion ofDNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to cont,amination due its inherent efficiency and high sensitivity. By employing microfluidic' systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR . in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and ,' temperature control/determination are the main focus within this work. Microchannels and microreactors are char:acterized by extremely high surface-tovolume ratios. This dictates that surfaces play a dominant role in defining the efficiency ofPCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within??????? this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing Copper oxide and Zinc oxide' nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to singlestranded DNA and the catalytic extension of this primer to fonn the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton e'xcitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.Imperial Users onl

Desarrollo de métodos integrados para la determinación de biomarcadores genéticos

Tesis por compendio[EN] The selective and sensitive detection of single nucleotide variations is essential for early diagnosis, individualized therapy, and disease prognosis. The SARS-CoV-2 pandemic has highlighted the pressing need to develop reliable, rapid and simple detection methods for mass diagnosis. Likewise, there is a growing demand for genomic biosensors that are selective, multi-analyte and low-cost and allow the detection and identification of certain oncological biomarkers. This doctoral thesis has focused on the development of integrated systems of isothermal amplification, selective hybridization and optical biosensing for detection of point mutations in the PIK3CA, KRAS and BRAF genes associated with colorectal cancer. These predictive biomarkers are related to increased cell proliferation, apoptosis, and resistance to monoclonal antibody treatments (Cetuximab and Panitumumab). Isothermal DNA amplification methods have been explored, as they are particularly suitable for the development of a new generation of diagnostic devices aimed at supporting precision medicine. Specifically, isothermal amplification by recombinase-polymerase (RPA) has been selected as an alternative to detection methods that require unique infrastructures. In addition, its integration with bioanalytical platforms has been investigated, which represents a great advance for the simplification of biorecognition processes and their optical detection. This methodology has presented advantages over other DNA detection systems, in terms of portability and equipment, as well as reduction of test times and the possibility of performing diagnostic tests outside the laboratory. This doctoral thesis, framed in this context, is structured in 4 chapters: In chapter 1 a new variant of recombinase-polymerase amplification is presented, called RPA-blocked, based on the enrichment of minority alleles by introducing a blocking agent. In addition, this research has developed an analytical support consisting of a polycarbonate chip with covalently anchored allele-specific probes.The integration of the method has allowed the development of a portable system for the simultaneous genotyping of mutations in exons 9 and 20 of the PIK3CA gene. in cell lines and tumor tissues of cancer patients. In Chapter 2, the development of a genosensor that incorporates magnetic particles conjugated to allele-specific probes for the concentration and detection of the selective amplification product derived from RPA-blocked is described. With this genosensor, hybridization times and reaction volumes have been reduced. This approach has resulted in a portable and low-cost system for genotyping the KRAS gene applicable to solid tumor samples. Chapters 3 and 4 focus on the development of thermoplastic surfaces for the covalent anchoring of allele-specific probes mediated by dendrimeric molecules, with the aim of increasing the immobilization density. Thus, the covalent anchoring to activated polycarbonate and cycloolefin thermoplastic surfaces of allele-specific probes has been studied, mediated by carboxylic dendrimers through carbodiimide chemistry and by dendrons through the chemoselective reaction of the thiol-ino group. These multiplexed genosensors have allowed the genotyping of the V600 codon of the BRAF gene and the H1047 codon of the PIK3CA gene, in biopsied tissue samples. The research carried out in this thesis has given rise to new methodological contributions of interest based on obtaining integrated biosensor systems. These platforms will contribute to the development of massive diagnostic tools.[ES] La detección selectiva y sensible de variaciones de nucleótido único es fundamental para el diagnóstico precoz, la terapia individualizada y el pronóstico de enfermedades. La pandemia SARS-CoV-2 ha puesto de manifiesto la necesidad acuciante de desarrollar métodos de detección fiables, rápidos y sencillos para el diagnóstico masivo. Asimismo, existe una demanda creciente de biosensores genómicos que sean selectivos, multianalito y de bajo coste y permitan detectar e identificar ciertos biomarcadores oncológicos.La presente tesis doctoral se ha centrado en el desarrollo de sistemas integrados de amplificación isoterma, hibridación selectiva y biosensado óptico para la detección de mutaciones puntuales en los genes PIK3CA, KRAS y BRAF asociadas al cáncer colorrectal. Estos biomarcadores predictivos se relacionan con un incremento de la proliferación celular, apoptosis y resistencia a tratamientos con anticuerpos monoclonales (Cetuximab y Panitumumab). En este contexto, se han explorado los métodos isotermos de amplificación de ADN, dado que son particularmente adecuados para el desarrollo de una nueva generación de dispositivos de diagnóstico dirigidos a apoyar la medicina de precisión. En concreto, se ha seleccionado la amplificación isoterma por recombinasa-polimerasa (RPA) como una alternativa a los métodos de detección que requieren de infraestructuras singulares. Además, se ha investigado su integración con plataformas bioanalíticas, lo que supone un gran avance para la simplificación de los procesos de biorreconocimiento y su detección óptica. Esta metodología ha presentado ventajas frente a otros sistemas de detección de ADN, en términos de portabilidad y equipos, así como reducción de tiempos de ensayo y la posibilidad de realizar las pruebas de diagnóstico fuera del laboratorio. La presente tesis doctoral, enmarcada en este contexto, se estructura en 4 capítulos: En el capítulo 1 se presenta una nueva variante de la amplificación por recombinasa-polimerasa, denominada RPA-bloqueada, basado en el enriquecimiento de alelos minoritarios mediante de la introducción de un agente bloqueante. Además, en esta investigación se ha desarrollado un soporte analítico formado por un chip de policarbonato con sondas alelo-específicas ancladas covalentemente.La integración del método ha permitido desarrollar un sistema portátil para el genotipado simultáneo de mutaciones en los exones 9 y 20 del gen PIK3CA en líneas celulares y en tejidos tumorales de pacientes oncológicos. En el capítulo 2, se describe el desarrollo de un genosensor que incorpora partículas magnéticas conjugadas a sondas alelo-específicas para la concentración y detección del producto de amplificación selectivo derivado de la RPA-bloqueada. Con este genosensor, se han reducido los tiempos de hibridación y los volúmenes de reacción. Esta aproximación se ha concretado en un sistema portátil y de bajo coste para el genotipado del gen KRAS aplicable a muestras de tumores sólidos. Los capítulos 3 y 4 se centran en el desarrollo de superficies termoplásticas para el anclaje covalente de sondas alelo-específicas mediado por moléculas dendriméricas, con el objetivo de incrementar la densidad de inmovilización. Así, se ha estudiado el anclaje covalente a superficies termoplásticas de policarbonato y cicloolefina activadas, de sondas alelo-específicas, mediado por dendrímeros carboxílicos mediante la química de la carbodiimida y por dendrones mediante la reacción quimioselectiva del grupo tiol-ino. Dichos genosensores multiplexados han permitido el genotipado del codón V600 del gen BRAF y el codón H1047 del gen PIK3CA, en muestras de tejido biopsiado. Las investigaciones desarrolladas en la presente tesis han dado lugar a nuevas aportaciones metodológicas de interés basadas en la obtención de sistemas biosensores integrados. Estas plataformas, contribuirán al desarrollo de herramientas de diag[CAT] La detecció selectiva i sensible de variacions de nucleòtid únic és fonamental per al diagnòstic precoç, la teràpia individualitzada i el pronòstic de malalties. La pandèmia SARS-CoV-2 ha posat de manifest la necessitat apressant de desenvolupar mètodes de detecció fiables, ràpids i senzills per al diagnòstic massiu. Així mateix, existeix una demanda creixent de biosensors genòmics que siguen selectius, multianàlit i de baix cost i permeten detectar i identificar uns certs biomarcadors oncológics. La present tesi doctoral s'ha centrat en el desenvolupament de sistemes integrats d'amplificació isoterma, hibridació selectiva i biosensat òptic per a la detecció de mutacions puntuals en els gens PIK3CA, KRAS i BRAF associades al càncer colorectal. Aquests biomarcadors predictius es relacionen amb un increment de la proliferació cel·lular, apoptosi i resistència a tractaments amb anticossos monoclonals (Cetuximab i Panitumumab). S'han explorat els mètodes isoterms d'amplificació d'ADN, atés que són particularment adequats per al desenvolupament d'una nova generació de dispositius de diagnòstic dirigits a donar suport a la medicina de precisió. En concret, s'ha seleccionat l'amplificació isoterma per recombinasa-polimerasa (RPA) com una alternativa als mètodes de detecció que requereixen d'infraestructures singulars. A més, s'ha investigat la seua integració amb plataformes bioanalítiques, la qual cosa suposa un gran avanç per a la simplificació dels processos de biorreconeiximent i la seua detecció òptica. Aquesta metodologia ha presentat avantatges enfront d'altres sistemes de detecció d'ADN, en termes de portabilitat i equips, així com reducció de temps d'assaig i la possibilitat de realitzar les proves de diagnòstic fora del laboratori. La present tesi doctoral, emmarcada en aquest context, s'estructura en 4 capítols: En el capítol 1 es presenta una nova variant de l'amplificació per recombinasa-polimerasa, denominada RPA-bloquejada, basat en l'enriquiment d'al·lels minoritaris mitjançant de la introducció d'un agent bloquejant. A més, en aquesta investigació s'ha desenvolupat un suport analític format per un xip de policarbonat amb sondes al·lel-específiques ancorades covalentemente.la integració del mètode ha permés desenvolupar un sistema portàtil per al genotipat simultani de mutacions en els exons 9 i 20 del gen PIK3CA en línies cel·lulars i en teixits tumorals de pacients oncològics. En el capítol 2, es descriu el desenvolupament d'un genosensor que incorpora partícules magnètiques conjugades a sondes al·lel-específiques per a la concentració i detecció del producte d'amplificació selectiu derivat de la RPA-bloquejada. Amb aquest genosensor, s'han reduït els temps d'hibridació i els volums de reacció. Aquesta aproximació s'ha concretat en un sistema portàtil i de baix cost per al genotipat del gen KRAS aplicable a mostres de tumors sòlids. Els capítols 3 i 4 se centren en el desenvolupament de superfícies termoplàstiques per a l'ancoratge covalent de sondes al·lel-específiques mediat per molècules dendrimériques, amb l'objectiu d'incrementar la densitat d'immobilització. Així, s'ha estudiat l'ancoratge covalent a superfícies termoplàstiques de policarbonat i cicloolefina activades, de sondes al·lel-específiques, mediat per dendrimers carboxílics mitjançant la química de la carbodiimida i per dendrones mitjançant la reacció quimioselectiva del grup tiol-ino. Dits genosensors multiplexats han permés el genotipat del codó V600 del gen BRAF i el codó H1047 del gen PIK3CA, en mostres de teixit biopsiat Les investigacions desenvolupades en la present tesi han donat lloc a noves aportacions metodològiques d'interés basades en l'obtenció de sistemes biosensors integrats. Aquestes plataformes, contribuiran al desenvolupament d'eines de diagnòstic massiu.Martorell Tejedor, S. (2021). Desarrollo de métodos integrados para la determinación de biomarcadores genéticos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/176006TESISCompendi