Ultrasensitive on-site detection of aflatoxin M1 in milk using a chitosan-MWCNT-graphene nanocomposite aptasensor with sub-regulatory limit capability

This study presents a novel electrochemical aptasensor for ultrasensitive detection of aflatoxin M1 (AFM1) in milk samples, utilizing a gold electrode modified with chitosan-functionalized multiwalled carbon nanotube/graphene nanocomposite (CS/f-MWCNTs-Gr). The platform was fabricated through covalent immobilization of an amino-modified aptamer onto the nanocomposite surface, enhancing electronic transmission and biorecognition efficiency. Cyclic voltammetry (CV) confirmed stepwise electrode modification, while square wave voltammetry (SWV) quantified AFM1 via suppression of the [Fe(CN)6]3−/4− redox signal upon target binding. Under optimized conditions, the sensor demonstrated a wide linear range (1–1000 nM) covering EU/US regulatory limits, an ultra-low detection limit (0.03 nM, 9.8 ng/L, below EU regulatory limit of 25 ng/kg), and exceptional specificity (> 90% signal suppression against interferents at 10-fold higher concentrations). The aptasensor exhibited high reproducibility (RSD = 5.4%, n = 5) and retained 92% signal after 14-day storage. Validated in commercial milk, it achieved 96–106% recoveries with RSD < 4.9% (n = 5), outperforming reference methods in precision and practicality. This cost-effective platform shows significant potential for on-site monitoring of mycotoxins in dairy products.