A Novel “On–Off–On” Electrochemiluminescence Biosensor from Multifunctional AuPt Triangular Nanoprisms/Carbon Nitride Nanosheets

Abstract In this study, a novel electrochemiluminescence (ECL) biosensor is constructed by employing multifunctional gold–platinum triangular nanoprisms decorated on carbon nitride nanosheets (AuPt/CN) and an exonuclease III (Exo III)-assisted target recycling amplification strategy for the ultrasensitive detection of miRNA-499 via an “on–off-on” readout. CN nanosheets work as an excellent ECL luminophore, while AuPt TNPs enhance catalytic activity and electron-transfer capability and boost ECL emission. The AuPt/CN-modified glassy carbon electrode (AuPt/CN/GCE) produces a strong initial ECL signal, i.e., “on” status. Subsequent binding of ferrocene (Fc)-labeled hairpin DNA (H-Fc) via Au–S bonds, brings the -Fc group close to AuPt/CN/GCE, resulting in the quenching of the ECL signal of H-Fc/AuPt/CN/GCE, i.e., “off” status. When miRNA-499 is present, the Exo III-assisted recycling amplification strategy is activated: miRNA-499 hybridizes with H-Fc to form miRNA-499/H-Fc/AuPt/CN/GCE, and Exo III selectively cleaves the blunt 3′-terminus of H-Fc. As a result, the Fc group is removed from the AuPt/CN/GCE surface, while miRNA-499 is released to trigger the next amplification cycle and restore the ECL signal, i.e., “on” status. The as-designed ECL biosensor can ultrasensitively detect miRNA-499 with a detection limit (LOD) of 0.07 fM (S/N = 3) in the range of 0.1 fM to 10 nM.