Dual Paper-Based Electrochemical Device for Multiplex Detection of Triple-Negative Breast Cancer miRNA Signatures

In this technical note, we demonstrate how the merging of paper-based screen-printing, engineered probes, and multiplexing might offer a potent tool toward the analysis of cancer-related miRNA signatures. In fact, while single miRNA detection cannot be associated with a health condition, the detection of miRNA signatures (more than one sequence) offers a more comprehensive reflection of disease biology. The two working electrodes are engineered with rationally designed capture probes modified with two different redox mediators, namely methylene blue and ferrocene; this is necessary to discriminate against different miRNAs, i.e., miRNA-21 and miRNA-101, which are dysregulated in triple-negative breast cancer. The use of ferrocene and methylene blue as redox mediators enables independent, interference-free target detection within a single electrochemical system. The analytical characterization of the dual sensor for both analytes is consistent with the single systems, enabling their simultaneous detection in a wide concentration range (0.1-1000 nM). The platform achieves clinically relevant low-nanomolar limits of detection in both PBS and untreated human serum, while demonstrating excellent repeatability with a relative standard deviation of ∼4%, a rapid assay time of 30 min and straightforward operational procedures. Importantly, the proof-of-concept analysis performed in untreated spiked serum samples demonstrated reliable detection within a complex biological matrix. These findings highlight the potential of multiplex miRNA signature analysis in biofluids and warrant further evaluation in clinically relevant samples. Overall, this fast, low-cost paper-based platform enables multiplex analysis at the point of care and can be readily expanded to larger miRNA panels, providing a promising foundation for future diagnostic development.