A magnetic separation-assisted auto-cyclic primer extension for OSCC-associated salivary miRNA detection.

It has attracted considerable attention in the detection of salivary miRNAs for the non-invasive diagnosis of oral squamous cell carcinoma (OSCC). Herein, we report an innovative magnetic separation-assisted auto-cyclic primer extension (MS-ACPE) for label-free and sensitive detection of miRNA-31 in human saliva. In this work, low-abundance miRNA-31 is initially transduced into primers that can be selectively separated and concentrated using a simple magnetic separation technology. By leveraging the high local concentration, the recognition and capture events between the primers and the hairpin can be greatly enhanced. Consequently, a "bind-copy-release" cycle can be effectively initiated, generating a user-prescribed, ultra-long single-stranded DNA with numerous repetitive quadruplex sequences. This allows for directly lighting up the fluorescence of thioflavin T, enabling amplified detection of miRNA-31. We experimentally demonstrate that MS-ACPE exhibits high specificity and sensitivity for miRNA-31, with a limit of detection as low as 0.31 pM. Furthermore, its reliability and applicability for the detection of miRNA-31 in saliva samples have been explored. More importantly, this novel MS-ACPE can effectively discriminate cancer patients from clinical samples with high accuracy (AUC = 1), potentially opening new avenues for the non-invasive diagnosis of OSCC in clinical applications.