Colorimetric ELISA based on urease catalysis curcumin as a ratiometric indicator for the sensitive determination of aflatoxin B1 in grain products

There is an urgent need to measure aflatoxin B1 (AFB1) in food to prevent contaminated food consumption. In this work, a novel colorimetric enzyme-linked immunosorbent assay (ELISA) was developed for the detection of AFB1 using curcumin as a colorimetric indicator. An indirect competitive enzyme-label immunoassay was developed using urease and rabbit anti-mouse immunoglobulin G labeled with gold nanoparticles as the signal-transduction tag. Urease catalyzed the hydrolysis of urea to produce ammonia, which increased the pH of the solution. The phenolic hydroxyl group of curcumin ionized into phenolic oxygen anions under alkaline conditions, which strengthened the synergistic effect of electron supply and absorption in curcumin. As a result, the color of curcumin changed from yellow to reddish-brown, producing a visible color change. Under optimal conditions, AFB1 could be qualitatively determined with the naked eye, and quantitatively assessed by measuring the ratio of absorbance at wavelengths of 550 and 428 nm. The change in the ratio of absorbance Δ550/Δ428 decreased linearly in a range of 0.01–5 ng mL−1, and the limit of detection was 67 pg mL−1. Therefore, the selectivity and reliability of this proposed method were well validated. This method was also successfully used for the quantitation of AFB1 in spiked rice flour and wheat flour samples. This approach may broaden the application field of colorimetric ELISA for aflatoxin, providing a promising platform for the rapid screening of aflatoxin in food.