Cas12a-based direct visualization of nanoparticle-stabilized fluorescence signal for multiplex detection of DNA methylation biomarkers

The CRISPR-Cas12a RNA-guided complexes hold immense promise for nucleic acid detection. However, limitations arise from their specificity in detecting off-targets and the stability of the signal molecules. Here, we have developed a platform that integrates multiplex amplification and nanomolecular-reporting signals, allowing us to detect various clinically relevant nucleic acid targets with enhanced stability, sensitivity, and visual interpretation. Through the electrostatic co-assembly of the Oligo reporter with oppositely charged nanoparticles, we observed a significant enhancement in its stability in low-pollution environments, reaching up to a threefold increase compared to the original version. Additionally, the fluorescence efficiency was expanded by three orders of magnitude, broadening the detection range considerably. Utilizing a multiplex strategy, this assay can accomplish simultaneous detection of multiple targets and single-point indication detection of nine specific targets. This significant advancement heightened the sensitivity of disease screening and improved the accuracy of diagnosing disease-related changes. We tested this assay in a colorectal cancer model, demonstrating that it can identify DNA methylation features at the aM-level within 40–60 min. Validation using clinical samples yielded consistent results with qPCR and bisulfite sequencing, affirming the assay's reliability and potential for clinical applications.