Developing a Modular Platform for the Detection of <scp>microRNAs</scp> Using Rolling Circle Amplification and Multi‐Primed Chain Amplification

MicroRNAs (miRNAs) play critical regulatory roles in diverse biological processes and are key biomarkers in a wide range of physiological and pathological conditions, including cancer. However, their inherently low concentrations in biological samples pose a major challenge for reliable detection and quantification. To overcome this limitation, we developed a fluorescence-based biosensing platform that integrates rolling circle amplification (RCA) and multi-primed chain amplification (MCA) to enhance signal and detection sensitivity. The system is engineered to allow flexible reconfiguration for different miRNA targets by altering probe and primer sequences. In this modular system, miR-i, a miRNA commonly expressed in healthy and cancerous samples, serves as a universal initiator for RCA. Signal amplification was subsequently driven by hybridization with two randomly selected miRNAs (miR-A and miR-D), enabling evaluation of system performance under varied input conditions. Fluorescence emission was measured following the addition of a molecular beacon and subsequent spectrofluorometric analysis. The biosensor exhibited a strong linear correlation between miRNA concentration and fluorescence intensity, achieving a limit of detection (LOD), and limit of quantification (LOQ) below 10 pM in both buffer and human serum. These findings demonstrate the platform's high sensitivity and robustness. Importantly, modular architecture allows for easy reconfiguration to detect a wide array of miRNAs or other non-coding RNAs, positioning this platform as a broadly applicable tool for molecular diagnostics beyond any specific disease context.