Washing-Free Electrochemiluminescence Biosensor for the Simultaneous Determination of N6 Methyladenosines Incorporating a Tri-Double Resolution Strategy

We propose a novel washing-free electrochemiluminescence (ECL) biosensor for the simultaneous detection of two types of N6 methyladenosines-RNAs (m⁶A-RNAs), which are potential cancer biomarkers, on the basis of binding-induced DNA strand displacement (BINSD). The biosensor integrated a tri-double resolution strategy that combined spatial and potential resolution, hybridization and antibody recognition, and ECL luminescence and quenching. The biosensor was fabricated by separately immobilizing two ECL reagents (gold nanoparticles/g-C₃N₄ nanosheets and ruthenium bipyridine derivative/gold nanoparticles/Nafion) and the capture DNA probe on the two sections of glassy carbon electrode. As a proof of concept, m⁶A-Let-7a-5p and m⁶A-miR-17-5p were chosen as model analytes, while m⁶A antibody-DNA3/ferrocene-DNA4/ferrocene-DNA5 was designed as an m⁶A-binding probe and DNA6/DNA7 was designed as a hybridization probe with DNA3 to release the quenching probes ferrocene-DNA4/ferrocene-DNA5. The recognition process led to the quenching of the ECL signals from both probes via BINSD. The proposed biosensor has the advantage of being washing-free. The ECL methods using the fabricated ECL biosensor with the designed probes exhibited a low detection limit of 0.03 pM for two m⁶A-RNAs and high selectivity. This work reveals that this strategy is promising for developing an ECL method for the simultaneous detection of two m⁶A-RNAs. The proposed strategy could be expanded to develop the analytical methods for the simultaneous detection of other RNA modifications by changing the antibody and hybridization probe sequences.