A G4-nanotweezer/DNA tetrahedron-confined dual-mode ECL/SERS biosensor for ultrasensitive rabies viral RNA detection via CRISPR-Cas12a-regulated hemin switching.

Reliable detection of low-abundance rabies viral RNA (RABV RNA) requires sensitive readouts that tolerate interfacial and matrix-associated signal variation. We developed a DNA tetrahedron-confined G4-nanotweezer biosensor combining electrochemiluminescence (ECL) and surface-enhanced Raman scattering (SERS). A Ti3C2/CsPbBr3@PDA@AuNPs nanocomposite (PAM) provided the dual-mode transducing interface, with hemin serving as both an ECL-modulating species and a SERS reporter. CsPbBr3 served as the ECL emitter, PDA improved interfacial stability, AuNPs provided SERS hotspots and DNA anchoring sites, and Ti3C2 acted as a conductive scaffold. Without target RNA, the bridging strand (ST) promoted a closed G4/hemin configuration, producing high SERS and low ECL signals. RABV RNA-triggered binding-induced isothermal amplification generated single-stranded DNA activators for CRISPR-Cas12a. Subsequent ST cleavage prevented nanotweezer closure and reduced interfacial hemin enrichment, producing an ECL-on/SERS-off response. Control experiments linked hemin-associated ECL modulation to the interfacial configuration without establishing a unique microscopic quenching pathway. The ratiometric signal R = IECL/ISERS enabled quantification over 1 aM to 50 fM. Additional low-concentration calibration yielded an estimated detection limit of 0.0675 aM by blank-threshold inverse prediction, extrapolated below the lowest measured nonzero standard of 0.10 aM. At 10 fM, matched-channel comparisons gave ratiometric relative standard deviations of 2.49% for ten successive readings and 2.87% across six independently prepared electrodes. In tenfold-diluted human serum at 10 fM, the ratio showed an 8.20% absolute deviation from phosphate-buffered saline, indicating partial matrix-effect compensation. This shared-reporter architecture links programmable DNA switching to complementary optical readouts for sensitive ratiometric viral RNA analysis.