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

Abstract 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.