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A heavy-chain antibody (VHH) library against procymidone (PRM) was constructed via immunizing Bactrian camels. Through careful biopanning, seven nanobodies (Nbs) with different sequences were obtained. The variability in their performance was primarily attributed to the amino acid differences in complementarity-determining region 3 (CDR3), as analyzed by molecular docking. The Nb exhibiting the highest sensitivity, named NbFM5, was biotinylated and conjugated to streptavidin-labeled gold nanoparticles to preserve the epitope's activity and prevent a decrease in sensitivity due to traditional random electrostatic adsorption. Subsequently, a simple and sensitive immunochromatographic assay (ICA) was developed for rapid detection of PRM based on biotinylated Nb (btNb). The developed btNb-ICA showed a cut-off value of 200 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 6.04 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.88 ng/mL. The recoveries in actual samples of crops ranged from 82.2 to 117.3%, aligning well with the results obtained from GC-MS/MS (R2 = 0.995). In summary, the developed btNb-ICA demonstrated high specificity and good accuracy for the rapid detection of PRM residues in vegetables. The total analysis time from preparing the sample to obtaining the result was less than 25 min.

Food-borne biotoxins from microbes, plants, or animals contaminate unclean, spoiled, and rotten foods, posing significant health risks. Neutralizing such toxins is vital for human health, especially after food poisoning. Nanobodies (Nbs), a type of single-domain antibodies derived from the genetic cloning of a variable domain of heavy chain antibodies (VHHs) in camels, offer unique advantages in toxin neutralization. Their small size, high stability, and precise binding enable effective neutralization. The use of Nbs in neutralizing food-borne biotoxins offers numerous benefits, and their genetic malleability allows tailored optimization for diverse toxins. As nanotechnology continues to evolve and improve, Nbs are poised to become increasingly efficient and safer tools for toxin neutralization, playing a pivotal role in safeguarding human health and environmental safety. This review not only highlights the efficacy of these agents in neutralizing toxins but also proposes innovative solutions to address their current challenges. It lays a solid foundation for their further development in this crucial field and propels their commercial application, thereby contributing significantly to advancements in this domain.

Emetic Bacillus cereus ( B. cereus ), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene ( ces B). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10 -2 ng μL -1 and 10 2 CFU mL -1 , respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.

Herein, the UiO-66-NH 2 @quantum dot (NU66@QD) was synthesized with excellent fluorescence intensity and biocompatibility, which was used to develop a multiple immunochromatographic assay (ICA) for the detection of aflatoxin B 1 (AFB 1 ), fumonisin B 1 (FB 1 ), deoxynivalenol (DON), T-2 toxins (T-2), and zearalenone (ZEN) in cereals and feed. Five monoclonal antibodies and NU66@QD were efficiently labeled by a one-step mixed method to form a multiple detection probe. The limits of detection of the proposed NU66@QD-ICA for AFB 1 /FB 1 /DON/T-2/ZEN were 0.04/0.28/0.25/0.09/0.08 μg/kg. The recoveries ranged from 82.83-117.44%, with the coefficient of variation from 2.88-11.80%. A parallel analysis in 35 naturally contaminated cereal and feed samples was confirmed by LC-MS/MS, and the results showed a good correlation ( R 2 > 0.9), indicating the practical reliability of the multiple NU66@QD-ICA. Overall, the introduction of the novel nanomaterial NU66@QD provides a highly sensitive and efficient multiplex detection strategy for the development of ICA.

Aflatoxin B 1 (AFB 1 ), a potent food-borne hepatocarcinogen, is the most toxic aflatoxin that induces liver injury in humans and animals. Species-specific sensitivities of aflatoxins cannot be fully explained by differences in the metabolism of AFB 1 between animal species. The gut microbiota are critical in inflammatory liver injury, but it remains to reveal the role of gut microbiota in AFB 1 -induced liver injury. Here, mice were gavaged with AFB 1 for 28 days. Then, the modulation of gut microbiota, colonic barrier, and liver pyroptosis and inflammation were analyzed. To further verify the direct role of gut microbiota in AFB 1 -induced liver injury, mice were treated with antibiotic mixtures (ABXs) to deplete the microbiota, and fecal microbiota transplantation (FMT) was conducted. The treatment of AFB 1 in mice altered gut microbiota composition, such as increasing the relative abundance of Bacteroides , Parabacteroides , and Lactobacillus , inducing colonic barrier dysfunction and promoting liver pyroptosis. In ABX-treated mice, AFB 1 had little effect on the colonic barrier and liver pyroptosis. Notably, after FMT, in which the mice were colonized with gut microbiota from AFB 1 -treated mice, colonic barrier dysfunction, and liver pyroptosis and inflammation were obliviously identified. We proposed that the gut microbiota directly participated in AFB 1 -induced liver pyroptosis and inflammation. These results provide new insights into the mechanisms of AFB 1 hepatotoxicity and pave a window for new targeted interventions to prevent or reduce AFB 1 hepatotoxicity.

Screening for ″zero tolerance″ β-agonists requires broad-specificity and sensitivity methods. Herein, R -(-)-salbutamol (SAL) is chirally separated and designed as a hapten, and a monoclonal antibody (mAb) was first prepared with an IC 50 of 0.27 ng/mL, which can recognize 38 β-agonists simultaneously. The broad-specificity of chiral mAb was explored by molecular simulation technology. Magnetic nanoparticles (MNPs) were then synthesized and applied as a signal tracer to develop a lateral flow immunoassay (LFIA). The limits of detection of MNPs-LFIA for SAL in swine urine and pork were 0.05 and 0.09 μg/kg, which was (2-125)-fold lower than that of the reported LFIAs. The recoveries were between 95.8 and 116.7%, with the coefficient of variation from 2.7 to 15.4%. Parallel analysis of 44 samples by commercial ELISA kits confirmed the reliability. Therefore, our work not only provides a broad-specificity and ultrasensitive method for β-agonists but also suggests that chirality is the new general theory that guided the rational hapten design.

Egg yolk immunoglobulins (IgY), as nutraceutical supplement for therapeutic or prophylactic intervention, have been extensively studied. The effects of IgY on small molecular toxin-induced toxicity in animals are unclear. In the present study, the protection of highly purified and specific anti-AFB 1 IgY against AFB 1 -induced genotoxicity and oxidative damage on the rat liver model were investigated. Our results revealed that AFB 1 induced significant oxidative damage markers, as well as AFB 1 -induced protein expression in antioxidant, pro- and antiapoptosis processes in rat liver. These effects could be significantly inhibited by cogavage with anti-AFB 1 IgY in a dose-dependent manner. However, anti-AFB 1 IgY did not significantly induce hepatic CAT and SOD1. To explore mechanisms, metabolite experiments were established to evaluate the influence of anti-AFB 1 IgY on the absorption of AFB 1 in rats. Middle and high doses of anti-AFB 1 IgY reduced hepatic AFB 1 -DNA adducts by 43.3% and 52.9%, AFB 1 - N 7 -guanine urinary adducts by 19.6% and 34.4%, and AFB 1 -albumin adducts by 10.5% and 21.1%, respectively. The feces of high dose anti-AFB 1 IgY cogavaged rats contained approximately 2-fold higher AFB 1 equivalents at 3-6 h after ingestion than AFB 1 group feces, indicating IgY inhibited AFB 1 uptake. These results had provided insight that anti-AFB 1 IgY could prevent animal organs from damage caused by AFB 1 and will be beneficial for the application of detoxification antibody as a supplement in food.

Aflatoxin B 1 (AFB 1 ) causes hepatotoxic, genotoxic, and immunotoxic effects in a variety of species. Although various neutralizing agents of AFB 1 toxicity have been studied, the egg yolk immunoglobulin (IgY) detoxification of small molecular toxins and the mechanisms underlying such effects have not yet been reported. In this investigation, anti-AFB 1 IgY against AFB 1 was successfully raised, and a competitive indirect enzyme-linked immunosorbent assay was established with a sensitive half-maximal inhibitory concentration (IC 50, 2.4 ng/mL) and dynamic working range (0.13-43.0 ng/mL). The anti-AFB 1 IgY obtained reduced AFB 1 -induced cytotoxicity, cellular dysfunction, and genotoxicity by protecting cells against apoptotic body formation and DNA strand breaks, preventing G2/M phase cell cycle arrest, reducing AFB 1 -DNA adduct and reactive oxygen species production and maintaining cell migration and invasion and the mitochondrial membrane potential. Anti-AFB 1 IgY significantly inhibited the AFB 1 -induced expression of proteins related to antioxidative, pro-apoptotic, and antiapoptotic processes in a strong dose-dependent manner. These experiments demonstrated that the anti-AFB 1 IgY-bound AFB 1 could not enter cells. This is the first time that IgY has been found to reduce the effects of small molecular toxins, which will be beneficial for the development of antibodies as detoxication agents.