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

Abstract

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.