Light-up RNA aptamer-based T-NASBA: a one-pot, label-free strategy for miRNA detection

MicroRNAs (miRNAs) are pivotal regulators of cellular processes, with dysregulation linked to diverse diseases, particularly cancer. Current methods, while effective, are often expensive, complex, and require sophisticated equipment. To address these limitations, we developed a label-free, one-pot miRNA detection platform integrating toehold-mediated strand displacement (TMSD) with nucleic acid sequence-based amplification (NASBA). This method utilizes a TMSD-regulated hairpin Primer1 that selectively responds to target miRNA, triggering NASBA to generate a light-Up RNA aptamer for real-time, target-dependent fluorescence signaling-eliminating the need for fluorophore-labeled probes or additional modifications. By systematic optimization of the reaction conditions, the platform achieved a detection limit of 4.31 pM, with a strong linear correlation between fluorescence intensity and miRNA concentration over a range from 10 pM to 1 nM. The platform also demonstrated excellent reproducibility and precision in spike-and-recovery tests with human serum, with recovery rates between 99.85 $\%$ and 108.28 $\%$ . T-NASBA provides a novel, cost-effective, and label-free platform for miRNA detection, offering a simple alternative that requires no complex instrumentation. This makes T-NASBA ideal for a wide range of applications, including point-of-care diagnostics and research, further advancing the field of miRNA-based diagnostics and monitoring.