Rapid Detection Of Aflatoxin (M1、B1) In Food Based On UP-Converting Phosphor Technology

Objective Development and evaluation of an up-converting phosphor technology-based lateral flow assay (UPT-LF) for detection of AFM1 in milk power and milk and AFB1 in grain samples.Methods UPT-LF-AFM1 and UPT-LF-AFB1 were established with up-converting phosphor (UCP) nano-particles as the bio-label of competitive mode based LF assay. Sensitivity, quantitative ability and precision of UPT-LF-AFM1 and UPT-LF-AFB1 were evaluated by using simulated AFM1-postive samples, AFB1-postive samples with serial standard concentrations, respectively. The Specificity of UPT-LF-AFB1 was evaluated by using several homologous toxins including abrin, aflatoxin M1, ochratoxin, ricin, shiga toxin S1, shiga toxin S2, Staphylococcus aureus toxins and botulinum toxin. The qualitative and quantitative detection performance of UPT-LF-AFM1 was evaluated with liquid chromatograph-mass spectrometer (LC-MS) as the reference to detect samples of milk powder and milk simultaneously. Fifteen kinds of grain samples were used to evaluate the samples tolerance of UPT-LF-AFB1.Results1. Establishment of UPT-LF stripe:1) UPT-LF-AFM1:The suspension of UCP-AFM1-Ab and UCP- goat polyclonal antibody conjugates was sprayed on the glass fiber pad with a concentration of 1.5mg·mL-1.AFM1-BSA (0.5mg·mL-1) and rabbit polyclonal antibody against goat (0.3mg·mL-1) were sprayed at 2μL·cm-1 on the nitrocellulose membrane as test line and control line respectively.2) UPT-LF-AFB1: After systematically optimization, the suspension of UCP-AFBl-Ab and UCP-rabbit polyclonal antibody against goat conjugates was sprayed on the glass fiber pad with a concentration of 0.5mg·mL-1.AFB1-BSA (0.5mg· mL-1) and goat polyclonal antibody IgG (0.5mg·mL-1) were sprayed at 2μL ·cm-1 on the nitrocellulose membrane as test line and control line respectively.2. Evaluation of UPT-LF stripe:1) UPT-LF-AFM1:The detection limit of AFM1-UPT-LF reached 0.1ppb (μg·Kg-1) in milk powder and 0.3ppb (μg·L-1) in milk. There was a good linearity ranges from 0. lppb (μg·Kg-1) to 0.7ppb (μg·Kg-1) and 0.3ppb (μg·L-1) to 0.7ppb (μg·L-for milk powder and milk, respectively. The sensitivity, specificity and ROC area of UPT-LF-AFM1 for qualitative result could meet the need of national standard for AFM1 limit in dairy products. After statistical analysis, there was no significant difference (milk powder:t=0.66, P> 0.05; milk:t =1.01, P> 0.05) between UPT-LF-AFM1 and LC-MS for quantitative detection.2) UPT-LF-AFB1:The detection limit of UPT-LF-AFB1 reached 0.03ng·ml-1with a good linear (r=0.985) ranges from 0.03ng·mL-1 to 10000ng ·mL-1 between the signal of UPT-LF-AFB1 and the concentration of AFB1 with logistical of T/C as x and the logarithm of concentration as y. No false-positive result exist even at a very high concentration of toxins including abrin, ricin, OTA, Botulinum Toxin, Shiga Toxin(S1), Shiga Toxin(S2), Staphylococcus aureus toxins. As metabolites of AFB 1, AFM1’s similar structure leaded to cross-reactivity with the AFB1-UPT-LF. The maximum tolerance limit and detection limit of UPT-LF assay corresponding to each kind of real sample was determined by detected AFB1 in fifteen kinds of grain samples. The results showed that the UPT-LF assay has have good tolerance limit and detection limit which can satisfy the legal limit of China.Conclusion UPT-LF-AFM1 and UPT-LF-AFB1 were established in this study for detection of AFM1in milk powder and milk, AFB1 in grain samples. The high sensitivity, specialty, sample tolerance and user-friendly offered the possibility to realize on-site rapid detection of AFM1 in dairy products and AFB1 in grain samples quantitatively.