Disposable laser-induced graphene-based biosensor strip for the detection of N-protein using CRISPR activation and ratiometric electrochemical readout

Pathogen detection is important for infectious diseases prevention and control. This study presents a novel positive-response, ratiometric electrochemical biosensor constructed on disposable laser-induced graphene (LIG) electrodes. The platform provides dual electrochemical signals for ultrasensitive detection of the SARS-CoV-2 N-protein by harnessing target-activated CRISPR-Cas12a trans-cleavage activity. The developed sensor incorporates a hairpin oligonucleotide (ON) functionalized on gold nanoparticles-deposited LIG electrode surface. Methylene blue (MB) molecules bind to guanine (G) bases of the hairpin ON through inherent affinity, generating the first electrochemical signal. Upon target-induced CRISPR-Cas12a activation, ferrocene (Fc)-labelled indicator ON hybridises with the hairpin ON, displacing the MB molecules and concurrently introduces the Fc as a secondary electrochemical signal reporter. Such displacement triggers a quantifiable decrease in MB electrochemical current and an increase in Fc electrochemical current, generating an Fc/MB ratiometric signal that enhances with increasing target levels and serves as the robust sensor response. The developed ratiometric biosensor achieves a linear response from 0.01 pM to 100 pM of N-protein with a detection limit of 1.3 × 10^-3 pM and with excellent selectivity. The clinical feasibility of the developed ratiometric electrochemical biosensor was confirmed by detecting N-protein in the inactivated cell-cultured SARS-CoV-2. This work demonstrates significant potential in rapid pathogen detection and point-of-care diagnostics.