Amplification-free colorimetric detection of exosomal miRNA using a CRISPR/Cas13a-activated HRP/Au/MoS₂ nanobiohybrid...

CRISPR-based, amplification-free colorimetric sensing has emerged as a powerful approach for exosomal miRNA (Exo-miR) analysis, offering high specificity, simple operation, and direct visual readout suitable for point-of-care diagnostics. However, the sensitivity of existing systems is limited because conventional colorimetric probes generate inherently weak signals, making the reliable detection of ultralow-abundance Exo-miRs difficult without amplification. Here, we firstly report an amplification-free colorimetric biosensor based on a horseradish peroxidase (HRP)/gold nanoparticle (Au)/molybdenum disulfide (MoS₂) nanobiohybrid catalyst (HRP/Au/MoS₂) that generates intense chromogenic signals via synergistic catalytic interactions. In this nanoparticle, MoS₂ provides a high-surface-area scaffold with intrinsic peroxidase-like activity, Au nanoparticles facilitate efficient electron transfer, and HRP contributes to high catalytic turnover. Together, these features markedly enhance the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and the resulting blue coloration. Using this signal-amplifying HRP/Au/MoS₂, a magnet-based sensing system was constructed by immobilizing the HRP/Au/MoS₂ onto magnetic nanobeads (MNBs) through biotinylated single-stranded RNA linkers (MNB-ssRNA-HRP/Au/MoS₂). This construction was then integrated into a CRISPR/Cas13a assay, in which the target miR-155 activates Cas13a, which cleaves the ssRNA linkers, releasing the HRP/Au/MoS₂ into solution for colorimetric readout. The resulting biosensor exhibits a detection range of 1 fM-1 nM (LOD = 0.82 fM), and single-nucleotide specificity without nucleic acid amplification. Furthermore, our system enables detection of Exo-miR-155 in both cell-derived exosomes and clinical samples, distinguishing cancer patients from healthy donors. Therefore, this platform represents a promising approach for highly sensitive, selective, simple, and time-efficient detection of Exo-miR, which can be used for liquid biopsy-based early cancer detection.