An "on-off-on" self-powered biosensor based on enzyme-free amplification for detection of three biomarkers

MicroRNAs (miRNAs), a type of small non-coding RNA sequences, are very important biomarkers and are involved in various physiological processes, such as cell proliferation, growth, differentiation, and apoptosis. Many reports have shown that miRNAs are closely associated with a variety of diseases, including neurodegenerative diseases and cancer. Currently, researchers have developed various methods for miRNAs detection, such as fluorescence, surface-enhanced Raman scattering, electrochemiluminescence, and electrochemical sensing. The detection of multiple miRNAs is significant for the diagnosis of diseases. However, it is rare for a single biosensing system to ultra-sensitively detect multiple miRNAs.

Based on enzyme biofuel cells (EBFCs) and catalytic hairpin assembly (CHA), a novel self-powered biosensor is designed for the sequential ultra-sensitive detection of miRNA-21, miRNA-146a, miRNA-155. First, miRNA-21 initiates CHA, and hairpin-tetrahedral DNA nanostructure (H-TDN) is captured on the biocathode. As a result, the open-circuit voltage 1 (E₁^OCV) increases, i.e., a signal "on" state. Next, in the presence of miRNA-146a, another CHA process is activated, and DNA1-glucose oxidase (DNA1-GOD) is replaced by hairpin 2 (H2) on the bioanode. This causes the open-circuit voltage (E₂^OCV) to decrease, i.e., a signal "off" state. Finally, when miRNA-155 is present, DNA2-GOD is captured by miRNA-155. And E₃^OCV increases, i.e., a signal "on" state. Moreover, the self-powered biosensor possesses good selectivity, high reproducibility, and excellent stability for miRNAs assay.

Based on the variation of the open-circuit voltage, this novel self-powered biosensor exhibits ultra-highly sensitive detection for miRNA-21 with the limit of detection (LOD) of 0.36 fM, miRNA-146a with the LOD of 0.16 fM, and miRNA-155 with the LOD of 0.23 fM. This novel self-powered biosensor provides a feasible solution to explore ultra-sensitive sequential biosensors with multi-target detection.