Single tube cell-free transcription and autocatalytic signal amplification based fluorescent biosensor for microRNA detection.

Cell-free biosensing systems offer programmable, rapid, and low-cost molecular detection, which have been applied in nucleic acid assays frequently, but mostly rely on multiple enzymes and separate amplification steps which limit their application. In this study, using miRNA-21 as a model target, a single-tube, isothermal, cell-free biosensing platform that integrates SplintR ligase-mediated template assembly with T7 RNA polymerase-driven, product-assisted autocatalytic transcriptional cycling has been developed. The presence of target miRNA triggers the ligation of two DNA probes to form a functional transcription template, initiating the synthesis of light-up RNA aptamers through the cell-free system. The transcribed aptamer also re-enters the reaction as an RNA "splint", regenerating new templates and triggering additional transcription rounds, thereby establishing an autocatalytic signal amplification under constant conditions. The one-pot workflow does not require reverse transcription, thermocycling, or separate pre-amplification, minimizing hands-on steps and reducing contamination risk. The produced RNA aptamer can bind with a cognate fluorogenic dye to produce fluorescence signal and this can be used for target quantification. The platform achieves a detection limit of 0.52 pM with a linear range of 1 pM-1 μM, and demonstrates high specificity against closely related miRNA sequences. The proposed system was successfully applied to detect target in clinical serum samples, yielding satisfactory results. This work presents a rapid, sensitive, and easy-to-use strategy for miRNA quantification, offering a robust tool for point-of-care testing and liquid biopsy applications.