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

Abstract

Herein, a novel single-enzyme-assisted\ndual recycle amplification\nstrategy based on T7 exonuclease (T7 Exo) and a strand-displacement\nreaction (SDR) was designed to fabricate a photoelectrochemical (PEC)\nbiosensor for sensitive microRNA-141 (miRNA-141) detection with the\nuse of laminar bismuth tungstate (Bi₂WO₆) as\nphotoactive material. Compared with a traditional enzyme-assisted\ndual recycle amplification strategy, the presented method could effectively\nrefrain the enzyme interference reaction, reduce environmental sensitivity,\nand save cost. Here, hairpin DNA1 (H1) decorated on magnetic beads\n(MB) hybridized with target miRNA-141 to form an H1/miRNA-141 heteroduplex.\nWith 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\nand an H1/H2-SiO₂ duplex was formed, realizing the reuse\nof 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\nand cycled, which could initiate more T7 Exo digestion and free abundant\noutput DNA-SiO₂. Through such a process, a tiny miRNA-141\ncould induce substantial output DNA-SiO₂, effectively improving\nthe target amplification efficiency and detection sensitivity of a\nPEC biosensor. Furthermore, Bi₂WO₆ was modified\non an electrode to provide a superior initial PEC signal due to its\nexcellent electronic transformation capacity. With the introduction\nof output DNA-SiO₂, the hairpin structure of H3 on the\nelectrode was opened, making SiO₂ close to the electrode\nsurface, which significantly decreases the PEC signal. This work first\nestablished the PEC biosensor featuring a single-enzyme-assisted dual\nrecycle amplification process for sensitive detection of biomarkers.