A simple method for point-of-need extraction, concentration and rapid multi-mycotoxin immunodetection in feeds using aqueous two-phase systems.

The rapid detection of mycotoxins in feed samples is becoming an increasingly relevant challenge for the food production sector, in order to effectively enforce current regulations and assure food and feed safety. To achieve rapid mycotoxin detection, several biosensing strategies have been published, many reaching assay times of the order of a few minutes. However, the vast majority of these rely on sample preparation based on volatile organic solvents, often comprising complex multi-step procedures and devoid of clean-up and/or concentration effects. Here, a novel sample preparation methodology based on a green, non-toxic and inexpensive polyethylene glycol-sodium citrate aqueous two-phase system is reported, providing single-step extraction and concentration of three target mycotoxins within 20min: aflatoxin B1 (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON). With point-of-need applications in mind, the extraction procedure was optimized and validated using a rapid multi-toxin microfluidic competitive immunoassay. The assay was successfully tested with spiked complex solid matrices including corn, soy, chickpea and sunflower-based feeds and limits of detection of 4.6ngg±15.8%, 24.1ngg±8.1% and 129.7ngg±53.1% (±CV) were obtained in corn for AFB1, OTA and DON, respectively. These sensitivities are fit-for-purpose at the required regulatory and recommended limits for animal feed, providing an effective and safe semi-quantitative mycotoxin analysis that can be performed in the field.