One-shot high-resolution melting curve analysis for KRAS point-mutation discrimination on a digital microfluidics platform

Abstract

Proposed high-accuracy one-shot melting curve analysis on a digital microfluidics platform, enabling KRAS gene discrimination with single-nucleotide mutations in 3 seconds.

Combining sensors and actuators with electrowetting-on-dielectric (EWOD): advanced digital microfluidic systems for biomedical applications

“…100 Temperature control units are typically composed of microheaters/coolers, thermal sensors, and microcontrollers to form a close-looped control. Distinct strategies are adopted for integrating temperature control capability on the EWOD platform, namely, (1) placing heaters and sensors externally in the chip holding structures (system integration), 33,88,[97][98][99][100][101][102][103][104][105][106] (2) fabricating heaters and/or sensors on EWOD substrates (chip-scale integration), 57,89,[92][93][94]96,[107][108][109] and (3) using EWOD electrodes for multifunctional heating/sensing. 14,[110][111][112][113][114] For strategy (1), Shen et al 100 patterned a resistive microheater on a separate substrate (on the right) stacked and aligned with the EWOD bottom plate (on the left) (Fig.…”

“…Specifically for assays requiring cycling temperatures (i.e., PCR), another two different approaches are developed and validated: (1) continuously adjust the set value of a sole heater on the anchored target droplet, 35,36,44,103,104 as performed in commercial thermal cyclers; and (2) shuttle the target droplet between different temperature regions on the chip by electrowetting. 88,89,101,102,115 For approach (1), Rival et al 44 realized total messenger RNA (mRNA) extraction from a human cell line and reverse transcription quantitative PCR (RT-qPCR) assay, in which parallel PCR blocks are designed on the EWOD chip for 20 individual droplets to settle (Fig. 3D).…”

“…3E). Li et al 101 demonstrated EWOD-based DNA melt curve analysis comparable to the traditional qPCR method, by dispensing the columns of picoliter-scale microdroplets from EWOD-driven mother droplets along a temperature gradient generated by a spatially gapped heat source and a heat sink (Fig. 3F).…”

“…Witters et al 123 used the Teflon patterning technique to create a microwell array for trapping of single magnetic particles (MPs) for fluorescence detection. Li et al 101 replaced conventional Teflon coating on the surface of their EWOD chips with novel superhydrophobic coating and patterned superhydrophilic microwells to maximize the wettability contrast between the microwell and the surrounding, thus realizing efficient dispensing of an array of satellite droplets for DNA melt curve analysis (Fig. 3F).…”

Digital microfluidics for biological analysis and applications

“…Over the past ten years, the tremendous development of DMF has facilitated biomolecule analysis and applications, including nucleic acids, 16,17 proteins, [18][19][20] and hormones. 21 The tiny reaction volume in DMF can increase the biomolecule concentration to maximize the reaction efficiency, while the confined reaction space can exclude exogenous contaminants.…”