Marianna Rossetti, Ruslán Alvarez‐Diduk, Liming Hu, Chiara Capolungo, Gabriel Maroli, Enric Calucho, Arben Merkoçi
The detection of oligonucleotide biomarkers in biological fluids is central to modern diagnostics, particularly for minimally invasive liquid biopsy approaches. Among these, microRNAs (miRNAs) are highly informative due to their regulatory roles in gene expression and their remarkable stability in body fluids. However, established miRNA detection methods such as qRT-PCR and microarrays require complex, multi-step workflows, expensive instrumentation, and specialised laboratory infrastructure, limiting their use in point-of-care settings. Here, we present a flexible, dry-reagent paper-based biosensing platform for rapid and quantitative miRNA detection, based on fluorescent molecular beacon (MB)-functionalised gold nanoparticles (AuNPs) embedded within nitrocellulose membranes. Target-induced hybridisation of miRNAs to the MB probes generates a fluorescence signal that can be quantified using either a conventional microplate reader for high-throughput analysis or a fully portable, low-cost smartphone-based optical reader with LED excitation. The assay enables robust detection in the low-nanomolar concentration range directly on paper substrates, with improved apparent affinity compared to solution-based measurements. Although the current sensitivity does not yet reach the levels required for direct, amplification-free analysis of miRNAs in clinical liquid biopsies, the platform provides a scalable and versatile foundation compatible with upstream amplification strategies and decentralised testing workflows. Overall, this work establishes a user-friendly and adaptable diagnostic architecture with strong potential for translation into point-of-care nucleic acid testing in both clinical and resource-limited environments.