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This work was funded by FEDER funds through the COMPETE 2020 Program (POCI-01-0145-FEDER-007728). BV thanks SFRH/BPD/124311/2016. PVB thanksWater JPI Proj TRACE (Ref 281715). Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings. publishersversion published

Digital Microfluidics (DMF) is a technology capable of maneuvering picoliter to microliter droplets in an independent and individual manner, with a wide variety of uses for bioassays and biosensing. These systems are advantageous for their small volumes, higher portability and multiplex assay capabilities, proving to be very capable of lab-on-chip and point-of-care applications. One of these applications are DNA amplification assays, of which, Loop Mediated Isothermal Amplification (LAMP), that has received increased interest from the scientific community. This method is a sensitive and simple diagnostic tool for fast detection and identification of molecular biomarkers enabling real-time monitoring. Nevertheless, sensing methods coupled with DMF devices are still uncapable of measuring the progress of said reaction in real-time. This work explores two real-time LAMP measurement approaches to be coupled with a DMF system. The first approach uses an H-shaped device, where human c-Myc proto-oncogene and human 18S housekeeping gene are amplified and measured in real-time through fluorescence methods. The second approach uses interdigitated electrodes, where human c-Myc proto-oncogene is amplified and measured in real-time through Electrochemical Impedance Spectroscopy (EIS). Following development and characterization of both techniques, fluorescence measuring devices show 49% fluorescence signal difference between positive and negative controls end-points. EIS measuring devices indicate significant differences between commercial solutions with pH 4, 7 and 10, by Ciclic Voltammetry. This suggests that such devices could be used for real-time, label free, LAMP monitoring, since significant pH changes occur during a LAMP reaction

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.

This thesis is one of the first reports of digital microfluidics on paper and the first in which the chip’s circuit was screen printed unto the paper. The use of the screen printing technique, being a low cost and fast method for electrodes deposition, makes the all chip processing much more aligned with the low cost choice of paper as a substrate. Functioning chips were developed that were capable of working at as low as 50 V, performing all the digital microfluidics operations: movement, dispensing, merging and splitting of the droplets. Silver ink electrodes were screen printed unto paper substrates, covered by Parylene-C (through vapor deposition) as dielectric and Teflon AF 1600 (through spin coating) as hydrophobic layer. The morphology of different paper substrates, silver inks (with different annealing conditions) and Parylene deposition conditions were studied by optical microscopy, AFM, SEM and 3D profilometry. Resolution tests for the printing process and electrical characterization of the silver electrodes were also made. As a showcase of the applications potential of these chips as a biosensing device, a colorimetric peroxidase detection test was successfully done on chip, using 200 nL to 350 nL droplets dispensed from 1 μL drops.

Digital Microfluidics (DMF) is a second generation technique, derived from the conventional microfluidics that instead of using continuous liquid fluxes, it uses only individual droplets driven by external electric signals. In this thesis a new DMF control/sensing system for visualization, droplet control (movement, dispensing, merging and splitting) and real time impedance measurement have been developed. The software for the proposed system was implemented in MATLAB with a graphical user interface. An Arduino was used as control board and dedicated circuits for voltage switching and contacts were designed and implemented in printed circuit boards. A high resolution camera was integrated for visualization. In our new approach, the DMF chips are driven by a dual-tone signal where the sum of two independent ac signals (one for droplet operations and the other for impedance sensing) is applied to the electrodes, and afterwards independently evaluated by a lock-in amplifier. With this new approach we were able to choose the appropriated amplitudes and frequencies for the different proposes (actuation and sensing). The measurements made were used to evaluate the real time droplet impedance enabling the knowledge of its position and velocity. This new approach opens new possibilities for impedance sensing and feedback control in DMF devices.

Digital microfluidics (DMF) is a field which has emerged in the last decade as a re-liable and versatile tool for sensing applications based on liquid reactions. DMF allows the discrete displacement of droplets, over an array of electrodes, by the application of voltage, and also the dispensing from a reservoir, mixing, merging and splitting fluidic operations. The main drawback of these devices is due to the need of high driving volt-ages for droplet operations. In this work, alternative dielectric layers combinations were studied aiming the reduction of these driving voltages. DMF chips were designed, pro-duced and optimized according to the theory of electrowetting-on-dielectric, adopting different combinations of parylene-C and tantalum pentoxide (Ta2O5) as dielectric ma-terials, and Teflon as hydrophobic layer. With both devices’ configurations, i.e., Parylene as single dielectric, and multilayer chips combining Parylene and Ta2O5, it was possible to perform all the fluidic opera-tions in the microliter down to hundreds of nanoliters range. Multilayer chips presented significant reduction on driving voltages for droplet op-erations in silicone oil filler medium: from 70 V (parylene only) down to 30 V (parylene/Ta2O5) for dispensing; and from 50 V (parylene only) down to 15 V (parylene/Ta2O5) for movement. Peroxidase colorimetric reactions were successfully performed as proof-of-concept, using multilayer configuration devices.