Novel Sensing Strategy for MicroRNA via DSN–RCA Dual Amplification and Optical Tweezer-Assisted Suspension Bead Arrays

Traditional microRNA detection methods have some limitations that cannot be ignored. Enzyme-catalyzed nucleic acid amplification has gradually become an important method in bioanalysis and is expected to be a substitute for traditional methods. Combining enzymes with different functions provides a wide range of analytical design possibilities to create high-performance sensing strategies. Meanwhile, the optical tweezers that produce the tightly focused laser beam are integrated with a suspension bead array that fully concentrates the signal labels, contributing to stable signal output and improved detection efficiency. Herein, we have developed a novel "DSN-RCA dual amplification and optical tweezer-assisted suspension bead array imaging" sensing strategy for highly efficient detection of microRNA-21 (miRNA-21). The conversion of low abundance targets into bulk fluorescence signal probes was achieved by duplex-specific nuclease (DSN), and signal enrichment on bead supports rather than dispersion in solution was realized by rolling circle amplification (RCA) in one step. The synergistic integration of dual amplification and the application of bead supports effectively improved the sensitivity and specificity. Finally, the suspension bead array was created by the modified optical tweezer system, which realized the high-quality imaging analysis and improved the detection efficiency. The proposed sensing strategy achieved a limit of detection (LOD) as low as 5.90 pM with excellent specificity, stability, and reproducibility and was successfully applied to human serum samples, which has great potential for application in clinical serological research.