Sub-7-second genotyping of single-nucleotide polymorphism by high-resolution melting curve analysis on a thermal digital microfluidic device

Abstract

We developed a thermal digital microfluidic (T-DMF) device enabling ultrafast DNA melting curve analysis (MCA). Within 7 seconds, the T-DMF device succeeded in differentiating a melting point difference down to 1.6 °C with a variation of 0.3 °C in a tiny droplet sample (1.2 μL), which was 300 times faster and with 20 times less sample spending than the standard MCA (35 minutes, 25 μL) run in a commercial qPCR machine. Such a performance makes it possible for a rapid discrimination of single-nucleotide mutation relevant to prompt clinical decision-making. Also, aided by electronic intelligent control, the T-DMF device facilitates sample handling and pipelining in an automatic serial manner. An optimized oval-shaped thermal electrode is introduced to achieve high thermal uniformity. A device-sealing technique averts sample contamination and permits uninterrupted chemical/biological reactions. Simple fabrication using a single chromium layer fulfills both the thermal and typical transport electrode requirements. Capable of thermally modulating DNA samples with ultrafast MCA, this T-DMF device has the potential for a wide variety of life science analyses, especially for disease diagnosis and prognosis.

A 3D microblade structure for precise and parallel droplet splitting on digital microfluidic chips

“…The fabrication of the DMF device used in this work was similar to that described previously. 39 Briefly, the device had a sandwiched structure in which an aqueous droplet of the sample was immersed in the medium oil (silicone oil, 1 Cst, Clearco, USA) between a bottom glass plate patterned with chromium (Cr) electrodes and a top plate made of indium tin oxide (ITO) glass. A 10 μm layer of SU8 photoresist (Microchem, Newton, Massachusetts, USA) was coated on the electrode array as the dielectric layer.…”

Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research

“…However, the rapid prototyping by inkjet printing facilitates flexible redesigning of the heater geometry for larger and smaller thermal masses (i.e., different-sized sample droplets), while meeting the required robustness in the range of physiologically relevant temperatures. Compared with previously reported approaches making use of Peltier element [ 34 ] or fully integrated, lithographically defined microheaters [ 35 , 36 ], our setup benefits from simplicity in terms of fabrication (rapid non-cleanroom method) and customization of the assay design (incl. the heated area and the heating power) by simply redesigning and printing new geometry on demand.…”

Closed-Tube Barcoding

“…We have already shown, however, that the simple LATE-PCR assays can be run in 10 nL volumes in under an hour and that all of the required reagents (including primers, probes, and Taq polymerase) can be dried down in advance; all that is needed to start the reaction is addition of a DNA template in water (Jia et al 2010). More recently, Jia and her colleagues have used electrodebased digital microfluidics to build chips that use mismatch-tolerant Molecular Beacons for high resolution melt curve analysis of single nucleotide polymorphisms in 1.2 L droplets, in an astoundingly short period of 7 s (Chen et al 2016). This is 300× faster than melt curve analysis of the same targets in 25 L reactions in standard real-time machines.…”