Functionalized carbon nanotube-assisted dual-mode CRISPR/Cas12a detection of hepatitis C virus via catalytic assembly circuit-driven Y-shaped dsDNA activators.

Hepatitis C virus (HCV) is a major etiological agent of liver diseases and remains a serious global health threat. Herein, we report a dual-modal HCV biosensing platform for ultrasensitive HCV RNA detection by integrating near-infrared fluorescence and colorimetric readouts. In this system, the presence of HCV RNA initiates a catalytic assembly circuit (CAC) that forms a Y-shaped DNA structure, exposing two double-stranded DNA activators with complete protospacer adjacent motif (PAM) to trigger CRISPR/Cas12a nuclease activity. The combination of CRISPR/Cas12a-driven signal amplification and hemin-binding aptamer-functionalized single-walled carbon nanotubes (HeApt-SWCNTs) enables highly sensitive target quantification. Upon exposure to hydrogen peroxide (H2O2), ferric ion in hemin catalyzes a Fenton-like reaction, generating hydroxyl radicals (·OH) that quench SWCNT fluorescence and oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to its blue oxidized form (oxTMB). In the presence of target HCV RNA, CRISPR/Cas12a-mediated HeApt cleavage inhibits ·OH generation, resulting in SWCNT fluorescence recovery and suppresses TMB oxidation. Under optimal conditions, detection limits of 0.23 fM and 4.1 fM are achieved for the fluorescence and colorimetric modes, respectively. This integrated CAC-Cas12a-HeApt-SWCNTs (CCHS) biosensing strategy offers high specificity, dual-mode signal reliability, and broad potential for early diagnosis of HCV and other RNA viruses.