Ultrasensitive detection of monkeypox virus: harnessing synergistic CRISPR-driven signal amplification on a DNA...

Rapid and ultrasensitive detection of emerging infectious diseases is critical for public health security. Herein, an electrochemical biosensor was developed for ultrasensitive detection of monkeypox virus (MPXV) by integrating CRISPR/Cas12a-driven signal amplification strategy with tetrahedral DNA nanostructure (TDN)-based sensing interface. The added MPXV DNA can efficiently activate the cleavage activity of Cas12a protein, thereby mediating the CRISPR-driven hybridization chain reaction (HCR) on TDN sensing interface. The horseradish peroxidase (HRP)-labeled HCR product can catalyze 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) reaction to generate an amplified electrochemical signal. Based on the signal change, the CRISPR-driven electrochemical biosensor exhibited better detection performance comparable to those of pre-amplification CRISPR-based biosensors for MPXV detection, including wide linear range, an ultralow detection limit, exceptional selectivity against non-target viruses (CPXV, ETCV, VZV, HSV), high reproducibility and accepted stability. Integrated with a smartphone-based portable device, the designed point-of-care testing (POCT) electrochemical biosensor can accurately detect MPXV in 10 % human saliva. This work provides a promising sensing platform for rapid, accurate and on-site detection of infectious diseases.