CRISPR/Cas12a-Triggered Dual-Mode Electrochemical and Raman-Silent Surface-Enhanced Raman Scattering Biosensing for Sensitive HBV DNA Detection

Sensitive and reliable quantification of hepatitis B virus (HBV) DNA is important for monitoring viral replication and therapeutic response. Electrochemical (EC) sensing offers simple quantitative readout, but resolving current changes at low target concentrations remains challenging. Surface-enhanced Raman scattering (SERS), in contrast, provides signal amplification but generally requires specialized optical instrumentation and may suffer from matrix interference. To accommodate different analytical requirements, we developed a CRISPR/Cas12a-triggered dual-mode EC/SERS biosensing platform. This platform integrates a conductive/plasmonic gold nanoparticles (Au NPs)/copper(II)-2,3,6,7,10,11-hexahydroxytriphenylene (Cu3(HHTP)2)/indium tin oxide (ITO) interface with a CRISPR-responsive DNA hydrogel. The hydrogel serves as a target-responsive interfacial gate, with Cas12a-mediated cleavage regulating electrode accessibility to facilitate EC transduction and promote plasmonic coupling between SERS tags and Au NPs. Ag@Au nanocubes functionalized with 4-mercaptobenzonitrile were employed as SERS tags, providing a characteristic CN signal at 2228 cm−1 in the Raman-silent region. The EC mode exhibited a linear response to the logarithm of HBV DNA concentration from 100 fM to 10 nM, with a limit of detection (LOD) of 17.1 fM. The SERS mode was evaluated in parallel on a separate, identically prepared hydrogel-modified electrode, yielding a linear range of 5 fM to 1 nM and an LOD of 0.143 fM. This platform combines the operational simplicity and quantitative capability of EC sensing with the high sensitivity of SERS detection, providing complementary readout options for different HBV DNA testing requirements.