CRISPR/Cas12a regulated preassembled bulb-shaped G-quadruplex signal unit for FL/CM dual-mode ultrasensitive detection of miRNA-155.

High sensitivity and specificity in microRNA detection are of great significance for early cancer screening. This study employed a pre-assembled bulb-shaped G-quadruplex signal unit (G4MB) as a novel and efficient label-free probe. The products amplified by the miRNA-155-targeted exponential amplification reaction (EXPAR) activated the trans-cleavage activity of CRISPR/Cas12a, disrupting the G4MB structure to achieve dual-channel fluorescence/colorimetric (FL/CM) inverse signal output. Due to the strong signal amplification of EXPAR, the highly efficient cleavage by CRISPR/Cas12a, and the ultra-high response signal of the structurally stable G4MB probe, the FL mode achieved a high signal-to-noise ratio (S/N) of approximately 12.5. The CM mode, combined with smart devices for RGB curve adjustment, successfully corrected the background and provided precise and objective image data support while allowing results to be observed with the naked eye. Additionally, the sensor system exhibited high accuracy in complex human serum environments and RNA extracted from three different types of cells. Moreover, the G4MB probe required no complicated labeling, demonstrated structural stability, and had a rapid response. Most importantly, this study analyzed the advantages of the G4MB and applied it to miRNA detection for the first time, providing practical insights for biosensor construction, molecular diagnostics, and clinical applications.