A novel nucleic acid extraction system integrating a switching valve-assisted microfluidic cartridge and a constant pressure pump

Background: Nucleic acid extraction (NAE) is essential in molecular diagnostics, genetic engineering, and DNA sequencing. While advances in switching valve-assisted, self-contained microfluidic (S²M) cartridges have improved the NAE processes, challenges persist in streamlining workflows, reducing processing times, and enhancing multi-target detection. Achieving these objectives requires precise control of on-chip fluid dynamics and efficient transfer of nucleic acid solutions (NAS) to detection areas. However, research on novel S²M cartridge designs and compatible fluid control systems is still limited.

Result: This study presented an innovative NAE system that integrated an S²M cartridge, a constant pressure (CP) pump, a metal heating bath, a rotating lever, and a magnet. The S²M cartridge contained the reagents for both NAE and detection, while the CP pump provided stable, precise, and rapid fluid delivery, outperforming traditional peristaltic and injection pumps, especially for handling small volumes and reducing pulsation. A custom rotating lever connects reagent chambers, facilitating mixing, cleaning, elution, and NAS transfer. This system showed advantages over manual methods, achieving all processes in 20 min. The CP pump-assisted S²M cartridge enabled rapid MBs re-suspension with a recovery rate of 80 %. The system's sensitivity, repeatability, and ease of use were also confirmed. The NAE system can extract Escherichia coli DNA (50 CFU mL^-1) from food and serum samples and viral DNA (HBV, HCV, HIV) at 200 copies mL^-1 from serum samples.

Significance: This study highlighted the need for an efficient fluid system in the S²M cartridge for stable NAE. By streamlining NAE and facilitating on-chip reagent preparation, the system enhanced point-of-care diagnostics. Although manual steps remain, the CP pump effectively managed fluid processes, paving the way for automated pathogen detection. Moreover, when combined with visual detection reagents, the NAE system showed promise as a direct molecular diagnostic tool.