On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection

Abstract

We developed a microfluidic chip integrated with nucleic acid purification and droplet-based digital polymerase chain reaction (ddPCR) modules to realize a ‘sample-in, result-out’ infectious virus diagnosis. The whole process involved pulling magnetic beads through drops in an oil-enclosed environment. The purified nucleic acids were dispensed into microdroplets by a concentric-ring, oil–water-mixing, flow-focusing droplets generator driven under negative pressure conditions. Microdroplets were generated with good uniformity (CV = 5.8%), adjustable diameters (50–200 μm), and controllable flow rates (0–0.3 μL/s). Further verification was provided by quantitative detection of plasmids. We observed a linear correlation of R2 = 0.9998 in the concentration range from 10 to 105 copies/μL. Finally, this chip was applied to quantify the nucleic acid concentrations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The measured nucleic acid recovery rate of 75 ± 8.8% and detection limit of 10 copies/μL proved its on-chip purification and accurate detection abilities. This chip can potentially be a valuable tool in point-of-care testing.

The development of droplet-based microfluidic virus detection technology for human infectious diseases

“…Following the individual droplet analysis, the target gene fragment-containing droplets are given the labels 1 and 0, and when paired with the Poisson distribution, the specific target gene fragments are determined. 18 For “in-sample, out-of-result” infectious virus diagnostics, Ma Cong et al 19 developed microfluidic chips coupled with nucleic acid purification and ddPCR modules. Throughout the entire procedure, magnetic beads were pulled through the oil-enclosed droplets.…”

“…A concentric ring, oil–water mixing, and flow-focused droplet generator are used to disperse purified nucleic acids into microdroplets while being powered by negative pressure. 19 Chen, Liao et al have also reported a ddPCR platform that achieves high sensitivity and rapidity without the need for bulky laboratory equipment. 20…”

Establishment and Validation of an Integrated Microfluidic Step Emulsification Chip Supporting Droplet Digital Nucleic Acid Analysis

“…Based on the insolubility between oil and water, droplet dNAA (ddNAA) is one of the dNAA methods that disperses sample solutions into micro-droplets in an oil phase fluid, using microfluidic approaches such as flow focusing, coaxial flow, step emulsification, ink-jetting, ultrasound excitation, oscillating, etc. [4][5][6][7][8][9]. For a certain volume of sample Biosensors 2023, 13, 888 2 of 12 solution, generating droplets with smaller sizes means larger numbers of droplets would be obtained, which leads to a higher detection sensibility.…”