Dual-Output Entropy-Driven Catalytic Amplification Coupled with G-Quadruplex Dimers for Ultrasensitive Label-Free Detection of miRNA-21

The connection between aberrant expression of microRNAs (miRNAs) and tumorigenesis positions miRNAs as promising targets for early cancer detection. However, the low abundance of miRNAs in biological fluids, such as blood, presents significant challenges that necessitate the development of highly sensitive detection platforms. In this study, we present a label-free fluorescent biosensor that utilizes a dual-output entropy-driven catalytic (EDC) amplification system in conjunction with G-quadruplex (G4) dimer-mediated signal enhancement for the ultrasensitive detection of miRNA-21. The synergistic effect of the EDC-based signal amplification and the high fluorescence quantum yield of G4 dimers resulted in a biosensor with outstanding sensitivity. The platform demonstrated a linear response over a concentration range from 0.1 pM to 100 pM (R2 = 0.998), with a limit of detection (LOD) calculated at 82 fM (S/N = 3). Specificity assays verified the biosensor's ability to accurately distinguish miRNA-21 from sequences with single, two, or three nucleotide mismatches, as well as from homologous miRNAs, underscoring its robust sequence selectivity. Furthermore, the biosensor's performance was assessed in human serum matrices, achieving recoveries between 96.2% and 104.8% and relative standard deviations below 2.4%. These results validate the biosensor's potential for clinical diagnostics, showcasing its efficacy and reliability in a complex biological environment.