Validation of a gold nanoparticle-based biosensor using RT-qPCR for screening pasture-fed and organic milk based on differential miRNA levels

Differentiating milk obtained from organic and conventional dairy production systems remains a major challenge for food authentication and traceability. MicroRNAs (miRNAs) have emerged as promising biomarkers of production systems because their expression levels are influenced by factors such as diet, grazing management, and environmental conditions. Among them, bta-miR-103 and bta-miR-155 have been associated with pasture-based dairy systems. However, their determination in milk samples is limited by the complexity of analytical techniques such as RT-qPCR and sequencing. In this study, a gold nanoparticle AuNPs-based colorimetric biosensor previously developed for synthetic miRNA detection was evaluated using endogenous miRNAs extracted directly from raw milk fat. A total of 57 bulk tank milk samples were collected from organic and conventional dairy farms under two grazing periods (6 hours and 18 hours of pasture access). The biosensor targeted bta-miR-103 and bta-miR-155 through analyte-induced AuNPs aggregation, producing a visible color change that was further confirmed by spectrophotometric measurements. Sensor performance was validated by RT-qPCR analysis of a representative set of samples. Molecular analysis revealed contrasting behaviors between the candidate biomarkers: bta-miR-103 displayed variable expression and substantial overlap among treatments, limiting its reliability as an isolated marker. In contrast, bta-miR-155 was primarily modulated by the production system, exhibiting elevated abundance in organic milk under standard pasture access. Nevertheless, both molecular quantification and biosensor response revealed comparable profiles between conventional milk produced under extended grazing and organic milk under moderate grazing, demonstrating that bta-miR-155 primarily mirrors fresh forage intake rather than providing an exclusive organic distinction across all management regimes. Overall, the AuNP biosensor offers a rapid, isothermal, and instrument-free first-line screening tool for triaging milk samples according to pasture exposure, providing an accessible strategy for preliminary screening within dairy traceability and quality control frameworks.