Electrochemical Biosensors Containing Fe-Metal Organic Framework Doped Polyaniline Nanocomposites for Sensitive Detection of miR-574-5P Based on DNA Walker Amplification

Electrochemical tracers with high signal input are essential for electrochemical sensors to overcome the limitations of imprecise detection outcomes. Herein, Fe-metal organic framework doped polyaniline (Fe-MOF/PANI) was developed for the first time as a high signal input tracer for electrochemical signal generation and amplification. Together with a three-dimensional (3D) DNA walker machine, it enabled the sensitive detection of the target microRNA (miRNA). First, the DNA walkers were initiated by target miRNA-574-5p and powered by the process of Nb.BtsI-mediated digestion. Fe3O4 magnetic beads (MBs) were modified with substrate DNA (S-DNA) to generate 3D DNA tracks. DNA walkers moved independently along 3D DNA tracks powered by the nicking endonuclease. Second, output DNA fragments were produced away from the MBs' surface because S-DNA was cleaved during the movement. Finally, the released output DNA was dropped onto the modified electrode surface of the capture probe and hybridized with high signal input tracers to form a sandwich structure, resulting in a stable and robust current response. The proposed electrochemical biosensor demonstrated exceptional efficacy for amplified detection of miR-574-5P in a wide linear range from 1 fM to 1 μM, with a limit of detection (LOD) of 0.237 fM under optimum conditions. Moreover, the inherent selectivity of the DNA walker allows the biosensor to accurately identify targets in human serum. Therefore, our work paves the way for the fabrication and design of high signal electrochemical biosensor tracers, which exhibit immense potential for bioanalytical applications.