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

There is an urgent need to measure aflatoxin B (AFB) in food to prevent contaminated food consumption. In this work, a novel colorimetric enzyme-linked immunosorbent assay (ELISA) was developed for the detection of AFB 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, AFB 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 Δ/Δ decreased linearly in a range of 0.01-5 ng mL, and the limit of detection was 67 pg mL. Therefore, the selectivity and reliability of this proposed method were well validated. This method was also successfully used for the quantitation of AFB 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.