Preparation And Application Of Chitosan-based Immunoaffinity Column And Immunomagnetic Chitosan Beads For Analysis Of Zearalenone

Zearalenone（ZEN）is a potent estrogen metabolite produced by Fusarium spp.As one of the most widely distributed mycotoxins,ZEN has caused serious diseases in various grains around the world.ZEN can bind to estrogen receptors,disrupt the body’s hormone balance and cause many reproductive system diseases such as cervical cancer and breast cancer.Therefore,it is very necessary to develop pretreament methods for the determination of ZEN residues in agricultural products.In this paper,the generation of polyclonal antibody against ZEN,the preparation of chitosan-based immunoaffinity chromatography column and immunomagnetic chitosan beads were studied.Their potential application to the selective extraction of ZEN residues from food samples were also described.The main research contents and results are as follows:1.Polyclonal antibody（pAb）against ZEN was successfully generated by immunizing New Zealand white rabbit with self-made immunogen（ZEN-KLH）.The produced pAb exhibited good sensitivity to ZEN with an IC₅₀0 value of 1.82 ng/mL,and the cross-reactivity rates with its analogues（β-ZOL,α-ZAL,β-ZAL）and other three mycotoxins（DON,OTA,AFB₁）were less than 0.8%,indicating that the pAb had high specificity,which met the requirements for the preparation of immunoaffinity adsorbents;The cross-reactivity rate withα-ZOL is as high as 91.9%,indicating that the prepared antibody can be used for simultaneous purification and detection of ZEN andα-ZOL in samples.2.Chitosan microspheres with uniform particle size were successfully prepared by membrane emulsification,and the average particle size of the chitosan microspheres is 109μm,the particle size distribution span is 1.55;Compared with the commercially available Sepharose 4B with an average particle size of 86.6μm and a Span value of 1.50,the average particle size of the chitosan microspheres was slightly larger than Sepharose 4B,and the particle size distribution was almost the same as that of Sepharose 4B,indicating that the prepared chitosan microspheres had good particle size uniformity.3.Chitosan microspheres and Sepharose 4B were conjugated with pAb to prepare immunoaffinity columns respectively.The characterization results indicated that the binding capacity of chitosan-based immunoaffinity column and Sepharose4B-based immunoaffinity column were 6.54μg ZEN/mL gel and 3.06μg ZEN/mL gel,respectively;The recovery rates of chitosan-based and Sepharose 4B-based immunoaffinity columns were in the range of 84.9%⁹6.2%and 80.4%¹00.5%,and the relative standard deviations（RSDs）between columns were 4.5%and 8.6%,respectively;When challenged with ZEN-spiked samples（ZEN was spiked in corn flour at 50μg/kg and 100μg/kg）,recovery rates of ZEN by chitosan-based immunoaffinity columns were from 92.6%to 100.2%,with the RSD less than 4.4%,and recovery rates of ZEN by Sepharose 4B-based immunoaffinity columns were from 88.3%to 107.3%,with the RSD less than 7.9%.4.The magnetic chitosan beads were prepared by hydrothermal synthesis method（oil bath reflux）,and coupled with the antibody by epichlorohydrin to prepare the immunomagnetic chitosan beads,and their performance and applicaton were evaluated.The results showed that the maximum ZEN-binding capacity of immunomagnetic chitosan beads was 42.11μg ZEN/g;When the ZEN standard solutions of different concentrations were loaded,the recovery rates of ZEN were from 92.4%to 108.2%,the RSD was 5.8%,and the results showed that the repeatability was satisfied,the average recovery rate was higher than the above two immunoaffinity columns,and the rapid purification and separation of the target substance can be achieved within 3 minutes;When challenged with ZEN-spiked samples（ZEN was spiked in corn flour at 50μg/kg and 100μg/kg）,recovery rates of ZEN were from 91.7%to 104.3%and the RSD was less than 2.9%.