Novel Single-Enzyme-Assisted Dual Recycle Amplification Strategy for Sensitive Photoelectrochemical MicroRNA Assay

Herein, a novel single-enzyme-assisted dual recycle amplification strategy based on T7 exonuclease (T7 Exo) and a strand-displacement reaction (SDR) was designed to fabricate a photoelectrochemical (PEC) biosensor for sensitive microRNA-141 (miRNA-141) detection with the use of laminar bismuth tungstate (Bi₂WO₆) as photoactive material. Compared with a traditional enzyme-assisted dual recycle amplification strategy, the presented method could effectively refrain the enzyme interference reaction, reduce environmental sensitivity, and save cost. Here, hairpin DNA1 (H1) decorated on magnetic beads (MB) hybridized with target miRNA-141 to form an H1/miRNA-141 heteroduplex. With the introduction of hairpin DNA2 (H2)-labeled SiO₂ (H2-SiO₂), SDR was triggered between H2-SiO₂ and H1, thus miRNA-141 was displaced from the H1/miRNA-141 heteroduplex and an H1/H2-SiO₂ duplex was formed, realizing the reuse of the target. In the presence of T7 Exo, the H1/H2-SiO₂ duplex was digested with the release of output DNA-SiO₂. To enhance the target conversion rate, H1-MB was intactly released and cycled, which could initiate more T7 Exo digestion and free abundant output DNA-SiO₂. Through such a process, a tiny miRNA-141 could induce substantial output DNA-SiO₂, effectively improving the target amplification efficiency and detection sensitivity of a PEC biosensor. Furthermore, Bi₂WO₆ was modified on an electrode to provide a superior initial PEC signal due to its excellent electronic transformation capacity. With the introduction of output DNA-SiO₂, the hairpin structure of H3 on the electrode was opened, making SiO₂ close to the electrode surface, which significantly decreases the PEC signal. This work first established the PEC biosensor featuring a single-enzyme-assisted dual recycle amplification process for sensitive detection of biomarkers.