Near-infrared/glutathione-activated CRISPR/Cas13a sensing platform for the detection of multiple microRNAs

MicroRNAs (miRNAs) are important liquid biopsy biomarkers, and their multiplex detection is highly important for the early screening of hepatocellular carcinoma. This work developed a CRISPR/Cas13a biosensing platform based on near-infrared (NIR) light and glutathione (GSH) dual activation for highly sensitive and specific determination of miRNA-21, miRNA-155, and miRNA-224. The advantages of this platform lie in the introduction of GSH-responsive mesoporous silica carriers and photolytic linkers (pc-linkers), which transform the sensing process from "passive triggering" to "controllable activation", effectively suppressing nonspecific signals. Specifically, GSH can degrade mSiO₂ with tetrasulfide bonds, thereby degrading the carrier. It will release the CRISPR/Cas13a system loaded on the carrier, thereby achieving specific recognition and cleavage of miRNA-21/155; at the same time, 808 nm NIR light excites upconversion nanoparticles (UCNPs) to generate ultraviolet light, which cleaves the pc-linker to activate the detection of miRNA-224. The detection limits of the developed assay for detecting miRNA-21, miRNA-155, and miRNA-224 were 4.2 pM, 5.7 pM, and 0.205 nM, respectively. The recoveries in the serum spiked experiments were 98.97-109.24%, with a relative standard deviation of less than 4.613%, indicating that the method has good accuracy and reliability. This work proposes a novel and reliable strategy for detecting multiple miRNAs in complex biological samples.