High-transition-temperature paraffin integration in IFAST device for efficient and robust nucleic acid extraction and...

Infectious diseases are prevalent in resource-limited regions with restricted access to health care. Nucleic acid testing is the gold standard for pathogen diagnosis. However, traditional methods are resource-intensive, which limits their use in point-of-care settings. Microfluidic technologies, such as the immiscible phase filtration-assisted system (IFAST) using paramagnetic particles (PMPs), simplify nucleic acid extraction but face barrier stability issues. The interface between the aqueous and oil phases in current IFAST systems is destabilized under the conditions required for efficient RNA extraction. These conditions include the use of reagents containing high concentrations of surfactants and organic solvents, as well as thermal treatment, which reduces the operational stability, reproducibility, and compatibility of the current IFAST systems. We developed a high-transition-temperature (HTT) paraffin-embedded IFAST-based device to improve barrier stability and extraction efficiency. HTT paraffin remains semi-solid at 65 °C, providing a robust barrier during the thermal lysis and RT-LAMP processes. At 75 °C, the device maintained compartment integrity and reduced carryover during the nucleic acid-bound magnetic particle transfer. Testing with SARS-CoV-2 samples showed detection of as little as 1 copy/μL of the viral genome without false positives. By integrating RNA extraction and colorimetric RT-LAMP detection, this device provided rapid on-site testing, advancing accessible and effective disease management in regions that require rapid diagnostics.