Rapid and ultrasensitive detection of mpox virus using CRISPR/Cas12b-empowered graphene field-effect transistors

Mpox is an infectious viral disease, and, as of January 12, 2023, about 84 560 distinct cases have been detected, involving about 110 countries since May 2022. Most clustered regularly interspaced short palindromic repeat (CRISPR)-based detection methods require long assay time because of the pre-amplification to target nucleic acids. Herein, we designed a new prototypal mpox biosensor that allows amplification-free nucleic acid detection by combining CRISPR/Cas12b system and ultrasensitive graphene field-effect transistor (gFET). The CRISPR/Cas12b-gFET enabled the detection of the mpox virus (MPXV) DNA targets at a sensitivity of ∼1 aM within about 20 min. We designed several sgRNAs to recognize the target genes of MPXV and then Cas12b protein cleaved the target genes to form a double-strand break. The specificity of sgRNAs of MPXV designed was verified among other homologous Orthopoxviruses, and the sgRNA could distinguish between recent 2022 MPXV and West African MPXV with the mutation site, showing its potential application in MPXV detection. Thus, the demonstrated CRISPR-gFET detection technology could serve as a sensitive and rapid diagnostic tool for MPXV and other DNA viruses in the future.