Double-layer NiCo heterostructure and enzyme-free dual-circle DNA signal amplification for simultaneous detection of dual target microRNAs of lung cancer using microfluidic electrochemical sensor

Detection of microRNAs (miRNAs) reveals tumor information at the transcriptional level, which is crucial for early diagnosis and prognosis of tumors. However, the rapid detection of miRNAs in biological samples without relying on large equipment remains a significant challenge. In this work, a double-layer NiCo heterostructure (DL-H) for enhancing the reaction efficiency of DNA labels, and dual-target cycling reaction system were developed to improve detection sensitivity and shorten the detection time within 30 min. The DL-H and catechol provide favorable condition for methylene blue (MB) oxidation and single-electron transfer of ferrocene (Fc). The target cycling reaction allows one target to release multiple Fc/MB labels from the electrode surface, enhancing the signal differentiation triggered by the target. The obtained sensor possesses a linear detection range of 10 fM-900 pM for miRNA-574-5p and miRNA-1254. The detection limit is 302 aM for miRNA-574-5p, while 85 aM for miRNA-1254. And the detection results of clinical samples present a strong correlation with the PCR. More importantly, a sensing automatic preparation-detection system was developed based on microfluidic chip, to resolve the reliance on highly trained technicians for the electrochemical sensing preparation. This development provides a valuable foundation for the advancement of portable detection tool of tumor biomarker.