Paper-Based Strip for Ultrasensitive Detection of OSCC-Associated Salivary MicroRNA via CRISPR/Cas12a Coupling with IS-Primer Amplification Reaction

As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people’s health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of “invading stacking primer” (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5′ and 3′ termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.