Dendritic Mesoporous UiO-66 as Nanoreactors for Dual-Mode Detection of the Rabies Virus Nucleoprotein Gene

Rabies, a zoonotic infectious disease of the central nervous system caused by the rabies virus (RABV), remains an acute infectious disease with the highest human mortality rate to date. Nonetheless, the detection of RABV has continued to present significant challenges. Herein, we developed a dual-mode electrochemiluminescence (ECL) and fluorescence (FL) biosensor utilizing dendritic mesoporous UiO-66 (DMAUiO) as an ECL nanoreactor. This system integrated a CRISPR/Cas12a-driven mimic-hybridization chain reaction (mimic-HCR) isothermal amplification strategy for the highly sensitive detection of the RABV nucleoprotein (RABV-N) gene. The prepared DMAUiO nanoreactor exhibited a rich pore structure that facilitated efficient loading of luminol (Lu) and enabled rapid diffusion of coreactants into its channels. In the presence of RABV-N, mimic-HCR-mediated isothermal amplification generated abundant output DNA, which activated the <i>trans-cleavage</i> activity of the Cas12a protein. Subsequently, activated Cas12a cleaved the magnetic DNA assembly (MDA), releasing 6-carboxyfluorescein (FAM) and dopamine (DA), where DA specifically functioned as an ECL quencher. This cleavage event led to FL signal recovery and ECL signal quenching, enabling dual-mode detection. The developed biosensor achieved linear detection ranges of 0.5 pM-8 nM (ECL) and 5 pM-10 nM (FL) for RABV-N, with detection limits of 0.17 and 4.1 pM, respectively. Our proposed dual-mode biosensor demonstrated excellent practicability in rat cerebrospinal fluid samples, providing a novel analytical platform for rabies diagnosis and biological analysis.