Anti-miR oligo-mediated detection of human salivary microRNAs for mild traumatic brain injury

Mild traumatic brain injury (mTBI), often resulting from traffic accidents, workplace incidents, sports, or recreational activities, is a neurological condition that significantly impacts the daily lives of many individuals. The absence of reliable biomarkers and the non-specific nature of mTBI symptoms pose challenges for accurate diagnosis, leading to undetected cases and potential long-term consequences. Current diagnostic approaches, including neuroimaging, serum biomarkers, and cognitive assessments, suffer from cost, invasiveness, and sensitivity limitations. To address this, we developed a novel electrochemical detection platform for salivary microRNAs (miRNAs), offering a rapid, non-invasive, and cost-effective alternative for mTBI diagnosis. Key challenges in point-of-care miRNA detection lie in low abundance, short length, sequence complementarity, degradation, and amplification-free detection with high sensitivity and specificity. This platform technology introduces a de novo-synthesized, conductive carboxyl-functionalized thiophene polymer (AAOT:PSS)-coated gold electrode, enabling the covalent attachment of streptavidin-linked, biotinylated anti-miRNAs with methylene blue as the electrochemical reporter. This system successfully detected picomolar concentrations of mTBI-associated miRNAs (miR-let7a, miR-30e, miR-21) in saliva, outperforming traditional methods and establishing salivary miRNAs as highly reliable biomarkers for mTBI. Our approach leverages the mTBI-induced upregulation of miR-let7a, miR-30e, miR-21 as proof-of-concept targets with scope of multiplexing while achieving 100% sensitivity and specificity in patient-derived samples validated via PCR and clinical assessments.