CRISPR-Cas12a Biosensor Array for Ultrasensitive Detection of Unamplified DNA with Single-Nucleotide Polymorphic Discrimination.

Quantitative polymerase chain reaction as a powerful tool for DNA detection has been pivotal to a vast range of applications, including disease screening, food safety assessment, environmental monitoring, and many others. However, the essential target amplification step in combination with fluorescence readout poses a significant challenge to rapid and streamlined analysis. The discovery and engineering of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) technology have recently paved the way for a novel approach to nucleic acid detection, but the majority of current CRISPR-mediated DNA detection platforms are limited by insufficient sensitivity and still require target preamplification. Herein, we report a CRISPR-Cas12a-mediated graphene field-effect transistor (gFET) array, named CRISPR Cas12a-gFET, for amplification-free, ultrasensitive, and reliable detection of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) targets. CRISPR Cas12a-gFET leverages the multiturnover trans-cleavage activity of CRISPR Cas12a for intrinsic signal amplification and ultrasensitivity of gFET. As demonstrated, CRISPR Cas12a-gFET achieves a limit of detection of 1 aM for the ssDNA human papillomavirus 16 synthetic target and 10 aM for the dsDNA Escherichia coli plasmid target without target preamplification. In addition, an array of 48 sensors on a single 1.5 cm × 1.5 cm chip is employed to improve data reliability. Finally, Cas12a-gFET demonstrates the capability to discriminate single-nucleotide polymorphisms. Together, the CRISPR Cas12a-gFET biosensor array provides a detection tool for amplification-free, ultrasensitive, reliable, and highly specific DNA detections.