Finger-actuated microfluidic device for point-of-care circulating cell-free nucleic acid extraction.

Abstract

Timely and accurate nucleic acid extraction is a critical bottleneck in point-of-care (POC) molecular diagnostics, particularly for low-abundance cell-free nucleic acids (DNA, RNA, miRNA) in plasma. We present a fully finger-actuated microfluidic device for power-free, pipette-free, nucleic acid extraction. The prototype device integrates finger-actuated valves, pump and blister reagent storage, enabling a complete, intuitive, "sample-to-eluate" workflow, including lysis, binding, washing and elution <i>via</i> manual actuation in approximately 35 minutes. The device operation was benchmarked against a commercial magnetic bead-based manual benchtop and an automated robotic workflow. In DNA-spiked aqueous samples (5-20 ng mL^-1), the device demonstrated recoveries of 84.6%, against 90.7% on the bench. Optimisation of magnetic bead volume identified 50 μL as the threshold for maximal recovery while minimising reagent consumption. Recovery from human pooled plasma samples were found to be on average 18, 62, and 552% higher than that obtained with the control bench extraction on initial sample volumes of 100, 75 and 50 μL, respectively. Coefficient of variation (CV%) for all techniques remained within reported laboratory ranges (20-80%). The device's clinical utility was piloted using patient samples positive for cytomegalovirus (CMV), monkeypox virus (MPXV) or chikungunya (CHIKV). The device achieved successful qPCR amplification for all targets, with viral DNA Cq values within as little as 0.3 cycles of the standard protocol. This instrument-free, manual circulating nucleic acid extraction tool could help bridge the gap between lateral flow based nucleic acid amplification technologies and real-world decentralised diagnostics, offering a scalable solution for infectious disease management in resource-limited settings.