Bioinspired Compartmentalization Coupled with DNA-Gated Magnetic Nanoprobes Enables Simultaneous Electrochemical Detection of Dual MicroRNAs

Abstract

Simultaneous analysis of multiple microRNAs (miRNAs) is highly desirable for reliable molecular diagnosis, yet multiplexed amplification assays are often compromised by nonspecific amplification, signal crosstalk, and background leakage. Inspired by biological compartmentalization, we developed a spatially isolated and DNA-gated electrochemical sensing platform for the simultaneous detection of miRNA-21 and miRNA-155. A custom dual-chamber polymerase chain reaction (PCR) tube was employed to physically separate two target-triggered exponential amplification reactions, allowing each miRNA to generate its corresponding single-stranded DNA product independently. The amplified products subsequently acted as molecular keys to open sequence-programmed DNA gates on mesoporous Fe3O4 magnetic nanoprobes loaded with electroactive reporters. Through toehold-mediated strand displacement, miRNA-21-derived products triggered ferrocene release, while miRNA-155-derived products induced methylene blue release, producing two distinguishable differential pulse voltammetry signals on screen-printed electrodes. By integrating spatially orthogonal amplification with programmable gated release, the proposed strategy effectively suppressed inter-reaction interference and converted trace miRNA inputs into amplified electrochemical outputs. Under optimized conditions, the sensor exhibited a wide linear range from 1 fM to 10 nM, with detection limits of 0.96 fM for miRNA-21 and 0.83 fM for miRNA-155. The platform also showed excellent selectivity, satisfactory reproducibility, good storage stability, and reliable recoveries in diluted human serum. Furthermore, it successfully differentiated MCF-7 breast cancer cells from LO2 control cells, with expression trends consistent with reverse transcription quantitative PCR (RT-qPCR). This compartmentalized DNA-gated sensing strategy provides a versatile and interference-resistant route for multiplexed nucleic acid analysis and holds promise for early cancer-related molecular diagnostics.