| Oxyfluorfen[2-chloro-a,a,a-trifluoro-p-tolyl-(3-ethoxy-4-nitro-phenyl)]is a kind of fluorinated diphenyl ether herbicide that developed by the Rohm and Hass.It is mainly used to inhibit weed photosynthesis to achieve herbicidal purpose.The drug is used in a wide range,broad weed control spectrum,long duration,high activity and a variety of combination with other herbicides.And there is no residual toxicity to the succeeding crops,it has the low toxicity to humans and animals,but highly toxic to aquatic and fishes,and this issue has gradually attracted people's attention.At present,the detection method of oxyfluorfen is limited to the method of instrumental analysis(such as high performance liquid chromatography,gas chromatography,HPLC-MS,etc.),however,there are some problems by instrument detection such as high consumption(the instrument is very expensive),high pollution(dosage of organic solvent,not environmentally friendly),complex operation program and difficulties of realizing high-throughput detection.The immune analysis is a simple,rapid,sensitive,low-cost,high-throughput detection technology,which has become one of the hotspots and development trends in the field of agricultural product quality safety testing.In this paper,a high-quality murine monoclonal antibody was prepared by using the oxyfluorfen,and the indirect enzyme-linked immunosorbent assay(ic-ELISA),chemiluminescent enzyme immunoassay(CLEIA),direct competition and indirect competition time-resolved fluorescence immunoassay(dc-TRFIA and ic-TRFIA),fluorescence polarization immunoassay(FPIA)were established by using this monoclonal antibody.The results showed that the five kinds of immunoassay methods could be applied to the rapid detection of oxyfluorfen in agricultural products and environmental samples.It also could provide the reliable theoretical basis and data support for the development of the subsequent immunization kit and test strip,and provid a new research idea for the rapid detection of pesticide small molecules.The main research contents were as follows:1.The preparation of monoclonal antibody for oxyfluorfenIn this study,the nitro group of the oxyfluorfen was reduced to amino group by Zn powder and acetic acid-hydrochloric acid,in this way to get the oxyfluorfen hapten.And the hapten was synthesized by the method of mixed acid anhydride method with the ovalbumin(OVA)to get the coating antigen,and the immune antigen was prepared by coupling glutaraldehyde with bovine serum albumin(BSA).With the way of rapid and adjuvant immunization,the BALB/c female mice were immunized by the immune antigen.After obtained the immunization mouse spleen cells,these spleen cells and Sp2/0 myeloma cells were fused,positive clones were selected and subcloned.The results showed that the positive clones obtained by conventional immunization was much more competitive of oxyfluorfen than rapid immunization.Therefore,the hybridoma cell strain(1A7D6F5),which had the highest sensitivity to the monoclonal antibody,was selected to produce monoclonal antibody(McAb).The McAb subtype was IgG M and the titer was 5.73×105.And the McAb was sequenced by light and heavy chain.It showed that the McAb was highly effective antibodies,which can be used as an antibody material to establish different oxyfluorfen immunoassay methods.2.The development of enzyme-linked immunosorbent assays for oxyfluorfenThe enzyme-linked immunosorbent assays(ic-ELISA)was applied to the preparation of monoclonal antibody.And the concentration of the coating antigen,the working concentration of the antibody and the working buffer solution were optimized.Under the optimum conditions,the standard curve of the ic-ELISA was established.It was calculated that the inhibition concentration(IC50)was 0.065 mg/L,the lowest detection limit(LOD,IC20)was 0.0048 mg/L,the linear range(IC20-IC80)was 0.0048-0.63 mg/L.Except with benzofluorfen(CR=13.54%)and bifenox(CR=13.54%)interaction,the cross reactivities(CRs)with some analogues of oxyfluorfen were lower than 0.02%.The average recoveries were tested by adding 0.005-0.5mg/kg oxyfluorfen standard solution to five kinds of substrates,such as soil,apple,peach,pear and grape.The average recoveries were 74.1-107.2%and the relative standard deviations(RSDs)were 2.7-9.7%.The ic-ELISA method was validated with real samples by using GC-ECD method.The line equation and correlation coefficient of two methods were y=1.0001x+0.0081,R2=0.986,which showed that the established ic-ELISA was accurate detection results and reliable.It also proved the method can be applied to the actual production of environmental and agricultural products in the oxyfluorfen residue detection.3.The development of chemiluminescent enzyme immunoassay for oxyfluorfenIn the chemiluminescence system,the chemiluminescent enzyme immunoassay(CLEIA)was established by using the prepared oxyfluorfen McAb,The emission spectrum is determined for this mode.And the luminescence time,the working concentration of the antibody and the working buffer conditions were optimized.Under optimal operating conditions,the IC50 of this method was 0.021 mg/L,the lowest detection limit IC20 was 0.0016 mg/L,and the IC20-IC90 was 0016-0.28 mg/L.In addition to the presence of a certain cross reaction with the benzofluorfen(CR=13.12%)and bifenox(CR=9.52%)interaction,the CRs with other analogues of oxyfluorfen were less than 0.02%.The recoveries were obtained in five kinds of substrates,such as soil,apple,peach,pear and grape.The average recoveries were as follows:77.2-106.4,the RSDs were 2.4-7.9%.After the addition of the recovery test,the GC-ECD was used to test the correlation of the real samples.The results showed that the correlation equation between the two tests was highly correlated(y=0.9239x+0.0119,R2=0.9931),which proved that the CLEIA method established in this study had high reliability and accuracy and could be used in the environment and the of agricultural products rapid detection.4.The development of time-resolved fluorescence immunoassay for oxyfluorfenThe direct and indirect competition time-resolved fluorescence immunoassay(dc-TRFIA,ic-TRFIA)were established by combining the autofluorescence properties of lanthanide europium(Eu)with the monoclonal antibody of oxyfluorfen.The purified Europium antibody was optimized and the conditions such as the working concentration of europium antibody,monoclonal antibody and working buffer were optimized.In the optimal condition,the IC50 of dc-TRFIA was 10.27 ng/mL,the lowest detection limit IC10 was 0.071 ng/mL,the detection range(IC10-IC90)was 0.071-1074.3 ng/mL,the detection range(IC10-IC90)and IC50 of ic-TRFIA was 0.024-504.6 ng/mL,2.76 ng/mL.The comparison showed that the sensitivity and detection limit of ic-TRFIA were superior to dc-TRFIA,so the ic-TRFIA was used for further analysis.The CRs tests showed that the CR with other oxyfluprfen structure analogues was less than 0.02%,except that there was a certain CR with the benzofluorfen(CR=11.58)and the bifenox(CR=8.23%).The average recoveries of ic-TRFIA were 74.6-108.3%,and the RSDs were between 2.1%and 10.9%,in the addition recovery test with five substrates.The results of the correlation test with the real samples of GC-ECD showed that they were highly correlated(y=0.975x-0.4446,R2=0.9901),which proved that the TRFIA method established in this study had high reliability and accuracy and could be used in environment and agricultural products for rapid detection of oxyfluorfen residues.5.The development of fluorescence polarization immunoassay for oxyfluorfenThe study put forward Fluorescence polarization immunoassay(FPIA)with oxyfluorfen monoclonal antibody and the working conditions of fluorescent tracer,antibody working concentration and working buffer were optimized.The optimal working conditions was pH 7.5,including 20%methanol borate saline buffer in 0.4 mol/LNa+.Under optimal conditions,the lowest detection limit IC10 was 1.15ng/mL the linear range(IC10-IC90)was 1.15 ng/mL-312.72 ng/mL,IC50 was 5.71 ng/mL according to the standard curves that were obtained.The cross-reactivity experiments indicated this method was highly specific to oxyfluorfen,and the cross-reactivity rate with analogues was lower than 0.02%except that there was a certain CR with the benzofluorfen(CR=14.57%)and the bifenox(CR=9.49%).Four substrates of soil,grape,pear,and peach were spiked with oxyfluorfen standards as the recovery test.The average recoveries were 79.2-107.1%and the relative standard deviations(RSDs)were 2.2-10.3%.The correlation equation between the measured results of FPIA and GC-ECD was y=0.9113x+0.0611 and R2=0.972,which was highly correlated with the results of the study.The FPIA method has high reliability and accuracy and can be used for the rapid detection of oxyfluorfen in agricultural products and environmental samples. |
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