Coupling smartphone and CRISPR-Cas12a for digital and multiplexed nucleic acid detection

Accurate, rapid, and multiplexed nucleic acid detection is of great value for applications in biomedicine and agriculture. Here, we demonstrated a one-step Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic method for the digital and multiplexed detection of both single-stranded (ss) and double-stranded (ds) DNA on a handheld smartphone device. ssDNA targets exhibited faster reaction kinetics of this single-step CRISPR-Cas12a assay than dsDNA counterparts. Under optimized conditions, picomolar levels of ssDNA targets can be detected in 96-well plates by using a benchtop plate reader. The detection sensitivity can be further improved to 5 fM when running the reaction in a microwell-based digital assay chip. Multiplexed detection of hepatitis B virus and human papillomavirus DNA markers was demonstrated on the smartphone-based platform. Finally, the one-step CRISPR-Cas12a assay performed robustly in human serum samples with a recovery rate of ssDNA detection between 96% and 105.6%, suggesting a high potential for clinical diagnostic applications in point-of-care settings.