Detection of fluorescence by miniaturized in-chip- incorporated microscope for digital Polymerase Chain Reaction (dPCR)

Classic biopsies are largely used for cancer diagnosis, which are often an invasive procedure. Alternatively, circulating tumour DNA in the blood can be used as liquid biopsy. For DNA amplification, digital polymer- ase chain reaction (dPCR) is becoming the selected procedure for extremely sensitive quantification of nu- cleic acid molecules. Droplet-based microfluidic platforms are widely used in dPCR, since microfluidics allows the efficient partitioning of samples in nanolitre-sized droplets, using oil-water interfaces, which sta- tistically results in one or zero DNA strand target molecule per droplet prior to amplification, showing a positive or negative fluorescent signal during amplification. In the present thesis, it is proposed a novel dPCR detection technique using a miniaturized fluorescence microscope, UCLA Miniscope, integrated in a droplet generator microfluidic device. The Miniscope was developed for neuroscience research due to its portability and lightweight, to be carried by small animals. This fluorescence detection technique is straight- forward, miniaturized, and inexpensive. To assess fluorescence detection sensitivity by the Miniscope, sev- eral fluorescein isothiocyanate (FITC) solutions were tested, with concentrations ranging from 1 nM to 100 μM, and the lowest concentration to present a detectable fluorescence intensity was 1 μM. Moreover, posi- tive and non-template-control PCR mixes of the cancer biomarker c-Myc after end-point amplification con- taining fluorescent DNA-binding dye EvaGreen were tested, and it was possible to discriminate the positive droplets and the non-template-control droplets. This research opens new prospects for the advancement of improved lab-on-a-chip methods designed for dPCR analysis and for further biological and medical applica- tions.As biópsias clássicas são amplamente utilizadas para o diagnóstico do cancro, o que é muitas vezes um pro- cedimento invasivo. Alternativamente, o DNA tumoral circulante no sangue pode ser utilizado como biópsia líquida. Para a amplificação de DNA, a reação em cadeia da polimerase digital (dPCR) está a tornar-se o procedimento escolhido para a quantificação ultrassensível de moléculas de ácidos nucleicos. Plataformas de microfluídica baseadas em gotículas são muito utilizadas em dPCR, uma vez que a microfluídica permite a compartimentação eficiente de amostras em gotículas nanométricas, usando interfaces óleo-água, o que re- sulta estatisticamente em uma ou zero moléculas alvo de cadeias de DNA por gota antes da amplificação, mostrando um sinal fluorescente positivo ou negativo durante a amplificação. Nesta tese, é proposta uma nova técnica de deteção de dPCR usando um microscópio de fluorescência miniaturizado, o UCLA Minisco- pe, integrado num dispositivo microfluídico gerador de gotículas. O Miniscope foi desenvolvido para inves- tigação em neurociência devido à sua portabilidade e leveza, para ser transportado por pequenos animais. Este sistema de deteção de fluorescência é simples, miniaturizado e económico. Para avaliar a sensibilidade de deteção de fluorescência do Miniscope, foram testadas várias soluções de isotiocianato de fluoresceína (FITC), com concentrações entre 1 nM e 100 μM, sendo que a menor concentração a apresentar uma intensi- dade de fluorescência detetável foi 1 μM. Além disso, foram testadas misturas amplificadas por PCR do biomarcador de cancro c-Myc positivo e de controlo (sem DNA) contendo o corante fluorescente intercalan- te de DNA EvaGreen, e foi possível discriminar as gotículas positivas e as de controlo. Este trabalho abre novas perspectivas para o desenvolvimento de métodos melhorados de lab-on-chip para análise de dPCR e para outras aplicações médicas e biológicas

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

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

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted

THE FUTURE AND PROSPECTS OF BIO-CHIPS

Nanotechnology deals with manipulation of matter at atomic and molecular level. A general description for nanotechnology is provided by National Nanotechnology Initiative, that defines nanotechnology as the manipulation of matter with at least one dimension is  around 1-100 nanometer. The major application of modern nanotechnology in future would be creation of biological chips which include organ-on-chip, human-on-chip, and lab-on-chip. The world needs easy, readily accessible health care for which one of the solutions would be lab-on-chip. A lab-on-chip is a device that consists of several laboratory functions on single chip of only few millimeters to centimeter in size. Apart from health care a major problem to be addressed is the drug development for out raging diseases where many drugs fail on human trial, for such problems the boon given by nanotechnology is the organ-on-chip and human-on-chip. These chips are multichannel 3D micro fluidic cell culture chips which mimics the organs environment and its interaction within the cells. This would help us to understand the human physiology with respect to each organ thus avoiding the testing of new drugs on animals and the drugs toxicity tests on them. Although the concept is still in infancy stage many initiative are taken to improve this technology as this could replace the already existing traditional technology that is time and cost consuming

Microfluidics for Biosensing and Diagnostics

Efforts to miniaturize sensing and diagnostic devices and to integrate multiple functions into one device have caused massive growth in the field of microfluidics and this integration is now recognized as an important feature of most new diagnostic approaches. These approaches have and continue to change the field of biosensing and diagnostics. In this Special Issue, we present a small collection of works describing microfluidics with applications in biosensing and diagnostics

Application of microfluidics in biosensors

This chapter reviews the up-to-date researches in the field of biosensors integrated with microfluidic techniques, most of which are research works published within the last five years. The trend of integrating microfluidics into biosensing technologies is analyzed, as the features of microfluidics fulfills the technical requirements recently raised in the field of healthcare. The current research works are discussed in details based on the biological recognition events and the transduction mechanism. Their pros and cons are discussed. The potential of microfluidics-integrated biosensors are prospective and their future applications in the field of healthcare is revolutionary, for example in the field of portable and wearable biosensor, which can be fabricated at extremely low cost ad require no professional operation, and this can widen the application of biosensor especially for remote districts and extreme poverty populations. The possible future research interests in this field are proposed in this chapter

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

System Integration - A Major Step toward Lab on a Chip

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications