A novel and sensitive trefoil-structured biosensor based on nanoporous gold for simultaneous determination of microRNA-21 and microRNA-16.

Although electrochemical biosensors have been developed to detect multiple microRNAs (miRNAs) simultaneously through loading different capture probes, high surface-induced perturbation and competition among probes have reduced the detection sensitivity. To address these challenges, a trefoil DNA capture probe (TDCP) was designed for microRNA-21 (miR-21) and microRNA-16 (miR-16) detection simultaneously. The TDCP exhibits a stable structure, low spatial resistance, and integral rigidity, which decreases high surface-induced perturbations and competition to improve the accessibility of the target miRNA. Meanwhile, the two capture domains in the TDCP are positioned apart, which increases the local concentration of target reactions and enhances hybridization efficiency. Two catalytic hairpin assembly (CHA) footholds were incorporated into the TDCP design to simplify the experimental process and reduce incubation time. As a result, the linear ranges of miR-21 and miR-16 were from 1 fM to 10 nM, with a limit of detection (LOD) of 0.059 fM and 0.084 fM, respectively. Additionally, miR-16 could also be used as an endogenous standard in biological samples, which corrects the bias in miR-21 detection arising from sample pre-processing and miRNA extraction. This strategy enhanced the accuracy and reproducibility of the trefoil biosensor. In tests of six A549 samples, the relative standard deviation (RSD) decreased from 40.13% to 14.38%, meeting clinical detection requirements. Overall, the trefoil biosensor provides novel possibilities for highly sensitive, reproducible, and accurate miRNA detection, with potential applications in early cancer diagnosis.