A Highly Sensitive Dual-Cathodic Ratiometric Electrochemiluminescence Biosensor Based on Functionalized Copper Nanoclusters and Nitrogen- and Sulfur-Codoped Carbon Dots for the Detection of miRNA-155

In most ratiometric electrochemiluminescence (ECL) sensors, the utilization of different co-reactants for anodic and cathodic ECL luminophores, along with a broad potential scanning range, restricts their practical applications. Herein, we first reported dual-cathodic potential-resolved ECL from nitrogen/sulfur-codoped carbon dots (N,S-CDs) and mercaptopropionic acid-functionalized copper nanoclusters (MPA-Cu NCs) using their common co-reactant K2S2O8 within a potential range of 0 to −2 V, and developed a ratiometric ECL biosensor for miRNA-155 analysis. Initially, the ECL peak of MPA-Cu NCs at approximately −2 V on the electrode was quenched through resonance energy transfer (RET) by methylene blue. Subsequently, trace target miRNA-155 was converted into abundant output DNA via a DNA walker mechanism. In the presence of Pb2+, partial DNA was cleaved to remove methylene blue, thereby restoring the ECL intensity of MPA-Cu NCs. Furthermore, the cleaved DNA fragments sparked rolling circle amplification (RCA), which ultimately facilitated the loading of N,S-CDs onto the electrode surface, generating an ECL peak at approximately −1 V. As the concentration of miRNA-155 increased, both ECL signals rose simultaneously but with different magnitudes. The fabricated ratiometric ECL sensor achieved a linear detection range for miRNA-155 from 10 aM to 0.1 nM, with a limit of detection of 2.91 aM. Overall, this study offers a new strategy for constructing dual-cathodic ratiometric ECL biosensors and provides a promising approach for early disease diagnosis.