Dual-signal-amplified electrochemiluminescence biosensor for microRNA detection by coupling cyclic enzyme with CdTe QDs aggregate as luminophor.

In this work, a dual-signal-amplified electrochemiluminescence (ECL) biosensor was proposed for the first time to detect microRNAs (miRNAs) based on cyclic enzyme and seeded-watermelon-like mesoporous nanospheres (mSiO2@CdTe@SiO2, mSQS NSs). mSQS NSs were successfully fabricated by inlaying the CdTe quantum dots (QDs) into the mesoporous silica (mSiO2) and future coating the surface with the silica layer. The obtained mSQS NSs contained tens of QDs and exhibited much stronger ECL signal than single QDs. The ECL biosensor achieved firstly signal amplification by using mSQS NSs to label the functional oligonucleotide probe (DNA-F) as enhanced ECL signal probes. Well-dispersed Fe3O4@Au nanoparticles were prepared as immobilization matrices to load hairpin-structured DNA probe (DNA-P). When the target miRNAs were present, hairpin DNA undertook conformation changes. Meanwhile, RNA/DNA duplexes was formed which cleaved by duplex-specific nuclease (DSN) to release miRNAs. Target miRNAs were cycled to hybridize with hairpin DNA, which achieved secondly signal amplification of the ECL biosensor. Thereafter, the complementarily parts between DNA-F and the rest DNA-P generated conjugates. The obtained conjugates would be collected on the surface of the electrode by effecting of magnet. Under the optimal conditions, the developed biosensor showed a wide linear range from 0.1 pM to 100 pM with a low detection limit of 33 fM (S/N = 3). The results of detection for the stability, specificity and reproducibility of ECL biosensor were outstanding. Simultaneously, the potential application of ECL biosensor was verified by using biosensor in serum sample.