A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification of DNA

Digital Microfluidics (DMF) is an innovative technology for liquid manipulation at microliter- to picoliter-scale, with tremendous potential of application in biosensing. DMF allows maneuvering single droplets over an electrode array, by means of electrowetting-on-dielectric (EWOD), that allows changing the contact angle of a droplet over a dielectric. Each droplet is thus considered a microreactor, with an unparalleled potential to perform chemical and biological reactions. Several aspects inherent to DMF platforms, such as multiplex assay capability and integration capability, make them promising for lab-on-chip and point-of-care (PoC) applications, e.g. DNA amplification assays or disease detection. DNA detection strategies for PoC have been profiting from recent development of isothermal amplification schemes, of which Loop-mediated Isothermal Amplification (LAMP) is a major methodology, allowing a 109-fold amplification efficiency in one hour. Here, I demonstrate for the first time the effective coupling of DMF and LAMP, resulting in a DMF device capable of performing LAMP reactions. This novel DMF platform has been developed and characterised, which allows successful amplification of a c-Myc gene fragment by LAMP. Precise temperature control is achieved by using a transparent heating element, connected to a looping feedback control system. This platform is able to amplify just 0.5 ng/μL of the target DNA, in only 45 minutes, for a device temperature of 65 °C and a reaction volume of 1.62 μL, one of the lowest volumes ever reported. Moreover, the electrophoretic analysis indicates that the amplification efficiency of the on-chip LAMP is considerably higher than that from the bench-top reaction

World-to-digital microfluidics for transformation and enzymatic assays

Digital microfluidics (DMF) is a technique for the manipulation of discrete droplets on an array of electrodes, which allows the controlled movement of fluids and represents an alternative from the conventional microfluidic paradigm of transporting fluids in enclosed channels. One of the major benefits of DMF is that fluid motion and control is achieved without external pumps and fabricated valves – it only requires the use of electric fields. The automation component of DMF has pushed the barriers of this ‘lab-on-chip’ technology; however, integration with external components (i.e. world-to-chip) interfaces has been a challenge. For example, the delivering of the biological fluids to the chip and integrating temperature control on a single platform are considered as two world-to-chip challenges in DMF. To address these two challenges, my thesis describes two world-to-chip components that are integrated with the DMF device: reagent delivery and temperature control. This new platform enables us to perform a variety of biological or chemical experiments on a chip with reduced manual intervention. Specifically, the new platform enabled an increase in reservoir volume on the chip by 40-fold from ~10 µL to 400 µL which allowed more reproducible dispensing and eliminated the need to refill the reservoirs during the biological assay. In addition, we integrated a closed-loop temperature control system that enabled fast and rapid changes in temperature on the chip. To show the utility of the world-to-chip interfaces, we validated the system by automating bacterial transformation and enzymatic assay procedures, which show that both procedures require world-to-chip interfaces for accurate and precise implementation. Overall, we propose that this system has the potential to be integrated for other types of biological assays and experiments which require fluidic control, automation, and temperature control

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

Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device

Cell-surface carbohydrates—or glycans—influence growth, differentiation, and immune response mechanisms. Alterations to the glycome can be markers for diseases including diabetes, neurodegenerative disorders, and cancer. Fucosyltransferases catalyze the addition of a fucose sugar residue to specific cell-surface glycans, which are involved in intercellular cell rolling/adhesion interactions such as white blood cells homing to inflammation sites and sperm-egg binding in fertilization. Fucosylated glycans are also implicated in inflammatory disease and cancers. In viral and microbial infections, fucosyltransferases can play a role in the adhesion and colonization of the host organism, as in the case of Helicobacter pylori α(1,3)-fucosyltransferase (FucT). To better our understanding of glycome alterations and improve medical diagnostics and treatments, screens for glycosyltransferase activity and inhibition are needed. Efficient screens for specific glycosylations tend toward costly materials, instrumentation, and specialized skillsets- here, we present a novel inhibition assay for FucT using the fluorogenically labeled disaccharide, MU-β-LacNAc. The assay shows good potential for high throughput (Z’=0.78 in 384-well plate), though such an application is not shown here. It was also implemented on a digital microfluidic (DMF) platform, where inhibition curves of FucT by GDP, a product of the glycosyltransferase reaction that exhibits an inhibitory feed-back loop, were generated on-device. Results of the assay on DMF (IC50 = 0.093 mM ± 0.037) were shown to be comparable to results in a 384-well plate (IC50 = 0.114 mM ± 0.086), achieving a 87.5% reduction in reaction volume and setting the groundwork for future fully automated screens for potential inhibitors of glycosyltransferases