An electrochemical aptamer-based sensor for detection of aminoglycosides in milk: addressing practical challenges

Abstract

This study investigated the practical application of an electrochemical aptamer-based (E-AB) sensor for aminoglycoside detection in milk using a previously reported aminoglycoside-binding aptamer. We investigated key factors that influence sensor performance and shelf-life, including electrode cleaning protocols, electrode type and substrate, sample pretreatment, and sensor storage conditions. Our findings demonstrate that sensors fabricated on laser-ablated electrodes on flexible poly(ethylene terephthalate) (PET) substrate exhibited a superior response to gentamicin-spiked solutions relative to screen-printed electrodes on ceramic substrate, with over 84% improvement in limit of detection (LOD). Electrochemical cleaning with H₂SO₄ potential cycling proved more effective for sensor fabrication than oxidation cleaning using a mixture of KOH and H₂O₂. Various milk sample pretreatment methods were tested, including solid-phase extraction, MeCN-treatment, acid-treatment and Carrez-reagent treatment that could potentially be applied for on-site detection. While these methods offered fast separation of milk components, they suffered from drawbacks such as long drying times, additional processing steps, or difficulty scaling up, making them unsuitable for on-site use. Diluting the milk sample up to 50% with PBS binding buffer was a more straightforward approach for gentamicin detection, giving an estimated LOD of 100 μM (100–240 μM linear dynamic range). The fabricated sensors stored in water at 4 °C, maintained stable performance for up to 5 days, evaluated against 50% diluted milk spiked with 500 μM gentamicin. Finally, we demonstrated the potential for on-site application using a miniaturised potentiostat connected to a mobile phone.