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/Cas12abased 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. Recent studies indicated that miR-31 was validated as an upregulated biomarker in saliva for OSCC. 8 Accurate and sensitive quantification of salivary miR-31 could contribute to the easier medical screening of OSCC in underserved communities and modify the outcome of OSCC. 9,10 Toward these needs, a sensitive and portable sensor is urgently in demand to quantify salivary miR-31 for the early diagnosis and effective monitoring of OSCC in underserved communities.Recent advances of CRISPR-associated (CRISPR/Cas) systems were applied to construct detection methods. 11−14 The unique collateral trans-cleavage nuclease activity of the Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity and specificity. 15−17 The collateral DNase activity of Cas12a can be switched on by the target DNA, which usually helps ultrasensitive detection of DNA. 11,18,19 Unlike Cas13a, both the target and probe of Cas12a are DNA, which improves the stability of the detection assay and reduces costing significantly. 15,20 Therefore, utilizing Cas12a for the detection of miRNA would be more reliable

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Sensitive and portable quantification of biomarkers has particular significance in the monitoring and treatment of clinical diseases. Conventional immunoassays were accustomed to introducing or incorporating enzymes for signal amplification, which commonly suffered from poor stability and inferior tolerance. Herein, we constructed a novel nonenzyme amplification methodology based on fluorogenic Ag + -tetrazolate aggregation coupled with silver corrosion sensitization for biomarker determination. A significant cascade enhancement strategy was achieved by the valid aggregation luminescence caused by the potent disintegration of silver nanoparticles. Furthermore, efficient magnetic separation was also combined and performed for the rapidity and simplicity of operation. As the target, the detection limit of prostate-specific antigen was 15.66 pg/mL in our designed biosensor. Besides, a good linear relationship was obtained. The designed biosensor demonstrated good specificity and was successfully applied to clinical serum sample detection.