Sample-to-answer nucleic acid detection using a fully integrated microdevice for nucleic acid extraction and smartphone-based droplet digital RPA/CRISPR.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has advanced rapidly due to its simplicity, high specificity, and high sensitivity. However, most conventional CRISPR-based detection methods lack quantitative capability. Although the strategy of digital PCR technology enhances the sensitivity of CRISPR detection and enables quantification analysis through sample dispersion, integration remains limited. In this study, we developed a one-pot digital recombinase polymerase amplification (RPA)/CRISPR nucleic acid detection platform by integrating RPA-assisted CRISPR nucleic acid detection with droplet microfluidics. A microfluidic chip was designed to integrate nucleic acid extraction, reagent mixing, droplet generation and collection, and on-chip isothermal reaction. The one-pot RPA/CRISPR reaction system and droplet preparation conditions were systematically optimized. In addition, a portable smartphone-based temperature control and fluorescence imaging device was developed to enable convenient "sample-to-answer" nucleic acid quantification with high sensitivity. This platform allows digital quantitative detection of SARS-CoV-2 samples within 50 min, achieving a detection limit as low as 1 copy/μL, with results comparable to commercial instruments. By integrating the immiscible filtration assisted by surface tension (IFAST)-based nucleic acid extraction with droplet digital RPA/CRISPR and utilizing smartphones and cloud processing for real-time result analysis, this study offers a novel "sample-to-answer" and highly sensitive nucleic acid quantification solution for point-of-care testing (POCT).