Multiple on-line active valves based centrifugal microfluidics for dynamic solid-phase enrichment and purification of viral nucleic acid.

Abstract

Point of care testing (POCT) of nucleic acids holds significant importance in the realm of infectious disease prevention and control, as well as the advancement of personalized precision medicine. Nevertheless, conventional nucleic acid testing methods continue to face challenges such as prolonged detection times and dependence on extensive specialized equipment and personnel, rendering them unsuitable for point of care applications. Here, we proposed an innovative active centrifugal microfluidic system (ACMS) for automatic nucleic acid extraction, encompassing modules for active valve control and magnetic control. An on-chip centrifugal puncture valve (PV) was devised based on the elastic tolerance differences between silicone membranes and tinfoils to release pre-embedded liquid reagents on demand. Furthermore, we have utilized the returnable valve (RV) technology to accurately control the retention and release of liquids, leveraging the high elastic tolerance of the silicone membrane. By incorporating an online controllable magnetic valve, we have achieved controlled and rapid aggregation and dispersion of magnetic beads. The final chip encapsulates multiple reagents and magnetic beads necessary for nucleic acid extraction. Upon sample addition and loading into the instrument, automated on-chip sample loading and nucleic acid extraction, purification, and collection can be accomplished within 30 minutes, halving the overall operation time and even increasing the efficiency of pseudovirus extraction by three orders of magnitude. Consequently, real-time fluorescence quantitative PCR amplification has successfully detected multiple targets of the SARS-CoV-2 virus (with an impressive detection limit as low as 10 copies per μL), along with targeted sequencing analysis yielding a conformity rate of 99%.