Self-primer exponential Cas12a tandem reaction-amplified and [Cu(tz)] nanosheets based neoteric biosensing platform for quantifying multiple mTBI-related miRNAs

Mild traumatic brain injury (mTBI) may cause increasing risk of long-term sequelae during brain development among children and adolescents. Recent investigations have declared that salivary microRNAs (miRNAs) in mTBI were reliable diagnostic markers. Herein, a versatile biosensor, namely, self-primer exponential amplified Cas12a tandem (SP-ExACT) reaction for quantifying mTBI-related miRNAs was established by integrating CRISPR-Cas12a with copper(I) 1,2,4-triazolate coordination polymer ([Cu(tz)]) nanosheets. Unlike conventional strand displacement amplification, we designed DNA hairpins with integrated primer and template sequences to generate multiple single-stranded DNA (ssDNA) identical to targets. They could trigger the following rolling circle amplification together and then activate Cas12a to indiscriminately cleave the intact fluorophores-labeled ssDNA that absorbed on [Cu(tz)] nanosheets. The corresponding fluorescence signal would recover for the desorption of cleaved short ssDNA from nanosheets. Owing to the remarkable performance of SP-ExACT-[Cu(tz)] reaction, this biosensor could detect let-7a, miR-30e and miR-21 with a LOD of 11.6, 5.4, 7.4 fM, respectively. This study explored a novel fluorescence biosensor based on [Cu(tz)] nanosheets combined with CRISPR-Cas12a to reduce initial background signal, and combined multiple signal amplification strategies to achieve ultra-sensitive detection. Moreover, multiple mTBI-related miRNAs detection could avoid false positive/negative results, and a universal detection platform was constructed through changing corresponding sequence.