Two-Dimensional Nanozyme-Catalyzed Colorimetric CRISPR Assay for the Microfluidic Detection of Monkeypox Virus.

The recent monkeypox epidemic outbreaks worldwide highlight the urgent need for fast and precise diagnostic solutions, especially in resource-limited settings. Here, a two-dimensional nanozyme-catalyzed colorimetric CRISPR assay for the microfluidic detection of the monkeypox virus (MPXV) was established. We utilized graphene oxide as a substrate for the adsorption of gold seeds and the deposition of a porous Pt shell to prepare high-performance two-dimensional GO@Pt nanomaterials. The viral nucleic acids released from clinical samples initiated a single-step recombinase polymerase amplification-CRISPR/Cas13a for the trans-cleavage of ssRNA reporters labeled with FAM and biotin. These reporters can be recognized by FAM antibody-conjugated GO@Pt nanozymes and streptavidin-coated magnetic beads. The formed sandwich immunocomplexes can catalyze the oxidation of a colorless 3,3',5,5'-tetramethylbenzidine substrate with a distinct color change. The proposed GO@Pt-catalyzed colorimetric CRISPR assay exhibited a limit of detection of 1 copy/μL of MPXV in 60 min. Forty clinical samples, including rash fluid swabs and oral swabs, were tested with 100% agreement with the real-time PCR. These results indicate the excellent potential of GO@Pt-catalyzed colorimetric CRISPR for the sensitive and accurate testing of MPXV under resource-constrained conditions.