CRISPR/Cas13a-driven lateral flow assay for preamplification-free and ultrasensitive miRNA-21 detection.

Developing a preamplification-free and sensitive clustered regularly interspaced short palindromic repeats (CRISPR)-based method is significant but still extremely challenging for microRNA (miRNA) detection. Here we present a combination of a CRISPR/Cas13a-based reaction with a lateral flow biosensor, which enables the quantitative and colorimetric readout of preamplification-free miRNA detection at room temperature. In this work, the reaction principle and the structure of the lateral flow strip are well-designed to achieve surface-enhanced Raman scattering (SERS)/colorimetric dual-signal "turn-on" response of target miRNA. The CRISPR/Cas13a Reporter is engineered with a DNA-RNA splicing structure to generate DNA cleavage products and reduce nonspecific collateral cleavage. Without the need for nucleic acid preamplification strategy, the developed CRISPR/Cas13a-driven lateral flow biosensor enables the microRNA-21 (miR-21) detection at room temperature with a readout time of 10 min and a total process time of less than 45 min, achieving an impressive limit of detection of 8.96 aM by SERS and 1 fM by visualization, respectively. Moreover, the platform demonstrated excellent recovery rates in spiked human serum samples. The proposed CRISPR/Cas13a-driven, dual-signal "turn-on"-responded lateral flow platform has the potential to simultaneously meet the requirements of convenient point-of-care visualization detection and more accurate and sensitive SERS detection of miR-21, offering a cost-effective, rapid, and reliable tool for early cancer diagnosis.