| Along with the development of agriculture, pesticides are more widely used in agricultural production, which not only benefit to agriculture but also shown some infects to environment and human beings. Imazalil, one of the systemic pesticides imidazole, has been used widely in antisepsis and storage of fruits and vegetables, which exists in agriculture pruducts, owing to the excessive or unreasonable use. In order to ensure the shealth of consumers, imazalil monitoring in agricultural and sideline products should be strengthend.At presents, instrumental analysis is the dominant residue detection method of imazalil, which have some disadvantages, such as pre-processing comples, expensive equipment and skilled technical personnel need, all of this made the fast detection impracticable. Therefore, an effective and fast detection method for imazalil residues is very necessary. In this paper, two immunoassays (IA), enzyme linked immunosorbent assay (ELISA) and electrochemical immunoassay (ECIA), had been developed for imazalil detection. The methods are simple, rapid and sensitive detection of low-cost and available on-line testing, which has a certain practical significane.In this paper, the degradation of imazalil had been used to synthesis hapten Imazalil, then polyclonal antibody had been prepared for ELISA kit and electrochemical sensor for imazalil detection. The details and results are as follows:1,Hapten Imazalil, after been synthesized with imidazole ethanol by nuclephilic substitution reaction, identified by thin-layer chromatography (TLC) and'H-NMR, hapten was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to get artificial immune antigen and artificial coating antigen respectively. The binding ratio of artificial antigens were dectcted by UV spectroscopy, the results,13:1 and 7:1, respectively. Preliminary suggested that the artificial antigens were synthesised successfully. The titers of positive serum were up to 64000 in animal experiments.2,6The best condition for ELISA was concluded by optimizing influencing factors, coating and blocking agent, reaction time, organic solvent content and so on, which shown as, coating with CBS(0.01 mol/L,PH=9.6) at 4℃for 12 h, blocking with 2% gelatine for 2 h, reacting with TMB for 15 min, the the best react content for antigen and antibody were 5μg.mL-1 and 32000 times' dilution, PBS(0.01 mol/L,pH7.4) as diluent in this course. Based on those conditions, the affinity and specificity of antibody had been tested by Indirect competitor ELISA. And the result turned out that the antibody of imazalil showed high affinity, with the linear regression equation y=0.1653x+0.4596, R=0.9972 and the standard curve showed good linear relationship at the range of 0.01～50μg.mL-1, IC50 and IC10 was 1.75μg.mL"1, respectively. Simultaneity, the coss-reaction rate to imazalil analogue were less then 2%, which means the antibody, produced in this paper, showed good specificity to imazalil.3,With the polycolonal antibody prepared in this paper, indirect ELISA kit for imazalil detecting in citrus fruits had been developed, with lowest detectable limit at 0.0136μg.mL-1. Comparing to the GC analyzing, the average recovery were 96.99% and 97.73%, respectively, and had correspond ratio of 99.24%. The precision, accuracy and sensitivity tests estimate turned out that the sample average recovery was 97.38%, the coefficient of variation of infra and inner plate were 6.18%-6.87% and 3.42%-4.02%, the sensitivity was 0.0136μg.mL-1,which would meet the imazalil test requirement of citrus fruits. The ELISA kit is stable at 37℃for at least 15 days and for greater than 6 month when kept at 4℃. tolerate repeated freeze cycles twice.4,In this paper, A electrochemical immunosenser was designed for imazalil testing, with the activator of EDC and NHS, imazalil polycolonal antibody was based on the ordered, stable self-assembled monolayer formed on gold surface with covalent bond. The quantitative analysis of the electrochemical immunosenser was based on the antigen-antibody reaction, with the testing scope and LDL for 0.01-5μg.mL-1 and 0.162μg.mL-1, respectively. It can reuse for 5 times with the Gly-HCl buffer solution, and be stable for 2 months at 4℃.
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