Dual-mode ECL/SERS ratiometric sensing of 3CLpro via enzyme-gated, entropy-driven DNA assembly on CsPbBr₃@PDA@Au

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease (3CLpro) is a key viral enzyme responsible for polyprotein processing and viral replication, making it an important functional biomarker for infection analysis and antiviral evaluation. Herein, we report a same-interface dual-mode electrochemiluminescence/surface-enhanced Raman scattering (ECL/SERS) biosensor for sensitive 3CLpro detection based on a CsPbBr3@PDA@Au nanocomposite and an entropy-driven DNA assembly strategy. In this design, 3CLpro specifically cleaves a DNA-peptide-DNA precursor to generate an intermediate DNA, which subsequently triggers interfacial strand-displacement assembly and recruits the Fc-labeled reporter strand to the electrode surface. This target-induced interfacial reconstruction leads to opposite signal changes, namely ECL attenuation and SERS enhancement, thereby enabling ratiometric quantification through I_SERS/I_ECL. The integrated dual-mode platform combines the high ECL activity of CsPbBr₃, the stabilizing and functional role of PDA, and the plasmonic/DNA-anchoring properties of Au nanoparticles, allowing 3CLpro activity to be transduced into a robust and self-validated analytical output. The proposed biosensor exhibited high sensitivity, good selectivity, satisfactory reproducibility, and favorable stability. According to the standard 3σ criterion, the limit of detection was calculated to be 2.44 aM. Moreover, the ratiometric readout improved analytical robustness by partially compensating for common-mode fluctuations associated with single-channel measurements. The method was further successfully applied to clinical samples, demonstrating its potential for reliable 3CLpro analysis in complex matrices. Overall, this work provides a dual-mode ratiometric biosensing strategy for 3CLpro that integrates activity-based target recognition, entropy-driven interfacial DNA reconstruction, and same-interface ECL/SERS readout, offering a promising platform for sensitive viral protease analysis and practical bioassay applications.