Controlled Nanozyme-Substrate Accessibility for Ultrasensitive Electrochemical Detection of miRNA

MicroRNAs (miRNAs) are short noncoding RNAs that serve as key biomarkers in various diseases. Traditional detection methods face challenges due to their complexity and enzyme dependency. The one-step electrochemical hairpin probe strategy, while simple and enzyme-free, has limitations in miRNA detection as miRNA signal changes are not prominent. This is mainly because the small molecular size of miRNAs results in weak conformational changes, and the high catalytic activity of nanoenzymes enhances background signals, reducing the signal difference. To address this issue, we developed a new strategy based on controlled nanozyme-substrate accessibility. The introduction of a ferrocene (Fc)/Au/DNA composite as a signal-shielding layer blocked the access of H₂O₂ to the nanozyme graphene oxide/high-entropy alloys (GO/HEAs), which in turn suppressed the·OH generation pathway and markedly enhanced signal suppression after miRNA binding. This method enables highly sensitive and specific discrimination of miRNAs at different concentrations within a one-step detection system. When detecting miR-125b, the sensor achieved a detection limit of 1.67 fM, providing a new concept for the development of highly sensitive and specific biosensing methods.