MnO ₂ @Mn ₃ O ₄ Heterojunction as a Coreactant Catalyst Collaborated with T-Shaped DNA Cycling–CRISPR/Cas12a Cascade Amplification for Locus-Specific N6-Methyladenosine RNA Modification Detection

As the predominant RNA modification, N6-methyladenosine (m⁶A) is recognized to play pivotal regulatory roles in fundamental cellular functions and oncogenic processes. However, the precise analysis of site-specific m⁶A modifications continues to present significant challenges. In this work, a unique electrochemiluminescence (ECL) biosensor for the locus-specific detection of m⁶A in RNA was developed in the first utilization of a MnO₂@Mn₃O₄ heterojunction as a coreaction catalyst in collaboration with a T-shaped DNA cycling-CRISPR/Cas12a cascade amplification strategy. The MnO₂@Mn₃O₄ heterojunction was observed to significantly enhance coreactant catalytic activity, yielding a 7.3-fold increase in the ECL intensity of the gold nanoparticles (AuNPs)/MnO₂@Mn₃O₄/(2,2'-bipyridine) dichlororuthenium(II) (Ru(bpy)₃²⁺)/Nafion/GCE compared to the AuNPs/Ru(bpy)₃²⁺/Nafion/GCE. Sequentially, the T-shaped DNA cycling amplification strategy effectively converted the target m⁶A RNA into an amplified biosignal, further enhanced by a CRISPR/Cas12a signal amplification system mediated by framework nucleic acid (FNA)-based photocontrollable nucleic acid protection, ensuring the sensitivity and specificity of m⁶A RNA. The integration of the triple signal amplification strategy achieved detection limits as low as 0.05 pM (S/N = 3) for a linearity spanning from 100 fM to 100 nM. The proposed ECL biosensor has been applied in detecting site-specific m⁶A modifications in total real RNA samples extracted from HeLa cells, demonstrating its promising applications for clinical diagnosis.