Construction of a Cytosine-Adjusted Electrochemiluminescence Resonance Energy Transfer System for MicroRNA Detection.

The cytosines in cluster-nucleation sequences play a vital role in the formation of silver nanoclusters (Ag NCs). Here, an innovative electrochemiluminescence (ECL) resonance energy transfer (RET) sensing system was developed using CdS quantum dots (QDs) as ECL donor and Ag NCs as ECL acceptor. Modulation of the number of cytosines in the cluster-nucleation sequences allowed tuning of Ag NCs absorption bands to match with the ECL emission spectrum of CdS QDs, yielding effective ECL-RET. The sensitivity of detection was improved by dual-target recycling amplification based on duplex-specific nuclease (DSN) and catalytic hairpin assembly. In the presence of target microRNA-21 (miRNA-21), DSN selectively cleaved the complementary DNA section (S1), resulting in the release of the transduction section (S2) and the reuse of miRNA-21 in the next recycling amplification. Interaction of the stem-loop structure of the DNA1 segment (H1) on CdS QDs-modified electrode with S2 led to the opening of the hairpin structure of H1 and the formation of H1:S2 duplex. Then, hairpin DNA2 encapsulated Ag NCs hybridized with the remaining single-stranded DNA segment of H1, and the S2 strand was replaced. Finally, the dissociated S2 participated in subsequent reaction cycles, introducing Ag NCs to the electrode surface and leading to ECL signal quenching of the CdS QDs. The proposed sensor showed excellent performance in detecting miRNA-21 at a wide linear range from 1 fM to 100 pM. The practical application ability of the strategy was tested in HeLa cells with acceptable results, suggesting that the detection platform is a promising approach for disease diagnosis and molecular biology research.