MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging. This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an "on to off" SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.

Department of Precision Medicine University of Campania Luigi Vanvitelli 80138 Naples Italy

Department of Precision Medicine University of Campania Luigi Vanvitelli 80138 Naples Italy; Institute of Applied Sciences and Intelligent Systems Eduardo Caianiello Unit of Naples National Research Council 80131 Naples Italy

Department of Precision Medicine University of Campania Luigi Vanvitelli 80138 Naples Italy; Laboratory of Molecular and Precision Oncology Biogem Scarl Institute of Genetic Research 83031 Ariano Irpino Italy

Institute for Experimental Endocrinology and Oncology G Salvatore 2nd Unit National Research Council 80131 Naples Italy

Institute of Applied Physics Nello Carrara National Research Council 50019 Sesto Fiorentino FI Italy

Institute of Applied Sciences and Intelligent Systems Eduardo Caianiello Unit of Naples National Research Council 80131 Naples Italy

Institute of Biophysics 2nd Faculty of Medicine Charles University Prague 5 Úvalu 84 150 06 Prague Czech Republic; University Centre for Energy Efficient Buildings Czech Technical University Prague Trinecka 1024 27343 Bustehrad Czech Republic

Laboratory of Molecular and Precision Oncology Biogem Scarl Institute of Genetic Research 83031 Ariano Irpino Italy

U P Cytometric and Mutational Diagnostics AOU Policlinico University of Campania Luigi Vanvitelli Via Luciano Armanni 5 83031 Naples Italy

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