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The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens. Microfluidics, which involves the manipulation of small volumes of fluidic samples, has been shown to be an ideal approach for POC analysis. Among the various microfluidic platforms available, digital microfluidics (DMF) offers high degree of configurability in manipulating μL/nL-scale liquid and achieving automation. However, the successful implementation of ultrafast PCR on DMF platforms presents challenges due to inherent system instability. In this study, we developed a robust and ultrafast PCR in 3.7–5 min with a detection sensitivity comparable to conventional PCR. Specifically, the implementation of the pincer heating scheme homogenises the temperature within a drop. The utilization of a μm-scale porous hydrophobic membrane suppresses the formation of bubbles under high temperatures. The design of a groove around the high-temperature zone effectively mitigates the temperature interference. The integration of a soluble sensor into the droplets provides an accurate and instant in-drop temperature sensing. We envision that the fast, robust, sensitive, and automatic DMF system will empower the POC testing for infectious diseases. [ABSTRACT FROM AUTHOR]

Highlights • Achieving the reference hybridization equilibrium with >13 times faster reaction rate. • Rapid mixing with wide range of DNA concentrations (from 1 to 10 μM). • On-chip isothermal DNA hybridization within ±0.1°C. Abstract DNA hybridization kinetics has been playing a critical role in molecular diagnostics for binding discrimination, but its study on digital microfluidic (DMF) systems is ultimately restrained by the laminar flow condition. The kinetic mixing technique is widely employed to ensure a fast reaction rate, but poses intrinsic risk in cross contamination and exhibits instable fluorescence intensity during the droplet transportation. While the electrothermal technique can provide stationary droplet mixing through the established thermal gradient within the hybridization solution, the significant increase in the droplet temperature will inevitably undermine the hybridization equilibrium and jeopardize the binding discrimination. To enhance the hybridization efficiency while ensuring a stable droplet temperature (within ±0.1℃), this paper presents a DMF platform that can perform isothermal hydrodynamic-flow-enhanced droplet mixing. Specifically, with a single electrode, droplet-boundary oscillation under a slow AC actuation is studied for improving the reaction rate. The dependencies between the mixing efficiency and the actuation voltage, actuation frequency and the spacer thickness are also systematically studied. Reliable mixing efficiency improvement is further validated over a wide range of solute concentrations. The results from real-time on-chip DNA hybridization kinetics with stationary droplets using the complete sandwiched DMF system shows that the proposed rapid mixer can achieve the same hybridization equilibrium with >13 times faster reaction rate when compared to the reference one through pure diffusion, while preventing biased hybridization kinetics as demonstrated in the electrothermal technique. [ABSTRACT FROM AUTHOR]

Digital microfluidics (DMF) shows great potential in the manipulation of individual droplets. However, the limited number of electrode control wirings for droplet control intrinsically restrains the massive generation of pL/nL individual droplets, fundamentally hindering the realization of high throughput applications on DMF such as digital nucleic acid amplification and analysis. This work demonstrates on-chip high throughput digital loop-mediated isothermal amplification (dLAMP) on a DMF platform based on a functional substrate with super-wettability contrast. Exploiting the passive dispensing technique as empowered by electrowetting on dielectric (EWOD) effect, a total of 1818 individual droplets with high uniformity are generated on the super-hydrophilic microarray within 1 min. Separation within the oil phase without tubes and valves can avoid evaporation, ensuring a precise total reaction volume. Absolute quantification of λDNA with a dynamic range of 1–1000 copies/μL is achieved by counting the amount of positive microdroplets after on-chip incubation, demonstrating the strong potential for rapid, low-cost, reliable, and quantitative nucleic acid analysis with high accuracy. • High-throughput digital microfluidics platform. • High uniformity among microdroplets. • No dead volume for ensuring a precise total reaction volume. • On-chip high precision absolute quantification of nucleic acid. [ABSTRACT FROM AUTHOR]