Nanobody-based nanoarchitectonics of ultra-sensitive indirect competitive chemiluminescent enzyme immunoassay for aflatoxin B1 in cereals

Aflatoxin B 1 (AFB 1 ), the most dangerous mycotoxins found in grains, is a powerful carcinogen. Consequently, creating immunoassay techniques which are rapid, robust, sensitive, and user-friendly for its detection is crucial to ensuring food safety. In this study, a nanobody (variable domain of heavy chain of heavy chain antibody, VHH) against AFB 1 was prepared and purified successfully based on recombinant plasmid pET22b(+)-VHH-AFB 1 obtained in previous research work. A novel nanobody-based nanoarchitectonics of indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) for the ultra-sensitive detection of AFB 1 was constructed under the optimal experimental conditions. The icCLEIA method in the study showed high sensitivity to AFB 1 with a 50 % inhibitory concentration (IC 50 ) of 0.63 ng/mL, and a limit of detection (LOD) of 0.066 ng/mL, and a linear range (IC 20  ~ IC 80 ) of 0.15–2.90 ng/mL. The icCLEIA method based on the purified nanobody had litter cross-reactivity (CR% < 0.1) with other structural analogues, which indicated that the method had high specificity for AFB 1. The average recoveries of AFB 1 in spiked millet samples ranged from 85.6 % to 103.4 % and the coefficient variation (CV) values were less than 3.14 %. In the experiment of recovery test, a strong linear correlation (R 2  = 0.9991) was observed by comparing the results from the nanobody-based icCLEIA with the UPLC-MS/MS reference method. The above results indicated that the nanobody-based nanoarchitectonics of icCLEIA is a reliable and effective alternative for quantifying AFB 1 in various grains.