Photoswitchable CRISPR/Cas12a-Amplified and Co3O4@Au Nanoemitter Based Triple-Amplified Diagnostic Electrochemiluminescence Biosensor for Detection of miRNA-141

In this work, a CRISPR/Cas12a initiated switchable ternary electrochemiluminescence (ECL) biosensor combined with a Co₃O₄@Au nanoemitter is presented for the in vitro monitoring of miRNA-141. Benefiting from the advantages of high-throughput cargo payload capability and superconductivity, three-dimensional reduced graphene oxide (3D-rGO) was designated as an introductory conducting stratum of a paper working electrode (PWE). With the collaborative participation of Co₃O₄@Au NPs, the transmutation of TPrA in the Ru(bpy)₃²⁺/TPrA system can be riotously expedited into exorbitant free radical ions TPrA^•, which provoked the exaggeration of the ECL signal. Moreover, the programmable enzyme-free hybrid chain reaction (HCR) amplifier on the PWE surface accurately anchored the assembly of nucleic acid tandem and accomplished the secondary recursion of the signal. Impressively, the multifunctional CRISPR/Cas12a with nonspecific cis/trans-splitting decomposition manipulated the photoswitch of the "on-off" signal state that avoided the false-positive diagnosis. The presented multistrategy cooperative biosensor demonstrated extraordinary sensitivity and specificity, with a low detection limit of 3.3 fM (S/N = 3) in the concentration scope from 10 fM to 100 nM, which fully corresponded to the expectation. Overall, this innovative methodology paved a generous avenue for evaluating multifarious biotransformations and provided a tremendous impetus to the development of real-time diagnosis and clinical detection of other biomarkers.