Fabrication of a fully integrated paper microdevice for point-of-care testing of infectious disease using Safranin O dye coupled with loop-mediated isothermal amplification.

In this study, we introduce a paper microdevice fully integrating DNA extraction, loop-mediated isothermal amplification (LAMP), and Safranin O-based colorimetric detection of two major infectious pathogens, namely SARS-CoV-2 and Enterococcus faecium. The paper microdevice is composed of two parts: sample and reaction chambers. A sealing film acted as a bottom layer to allow foldable motion for transferring DNA from sample chamber to reaction chamber in a seamless manner. An FTA card was employed in the sample chamber for DNA extraction and purification from bacteria-spiked milk. After LAMP reaction at 65 °C for 30 min, a novel aggregation-based DNA detection was obtained by Safranin O polymerization in the reaction chamber. Specifically, Safranin O underwent polymerization by addition of oxidant to form Safranin O oligomers. The electrostatic interaction between the positively charged Safranin O oligomers and the negatively charged DNA comprising LAMP amplicons resulted in the aggregation with a dark red color. Meanwhile, in the absence of LAMP amplicons, Safranin O oligomers were well dispersed and displayed their original red color. By using Safranin O-based detection, SARS-CoV-2 and E. faecium were successfully identified by naked eye within 60 min, and the limits of detection were 10 ng/μL and 10 CFU/mL, respectively. These results indicate that a fully integrated paper microdevice plays an important role in sample-in-answer-out format in the genetic analyses of infectious disease and serves as a rapid tool for controlling the spread of diseases.