| Phthalate esters(PAEs) are widely used in the industrial production and plastic products as plasticizers, which have characteristics of persistent organic pollutants and widely exist in atmosphere, water, soil, biology and so on. PAEs, as one kind of important endocrine disrupting chemicals(EDCs), can enter into animal’s bodies and produce the effect of xenoestrogen. They prevent the hormones which are related with animal reproduction and development from synthesizing, secreting, storing, transporting,combining and removing, and interfere with normal levels of hormones in the blood.Some of PAEs are considered to be carcinogenic, teratogenic and mutagenic. In 1977,six kinds of PAEs compounds were listed key controled pollutants by U.S EPA, which contained dimethyl phthalate(DMP), diethyl phthalate(DEP), dibutyl phthalate(DBP),di-n-octyl phthalate(Dn OP), butyl benz phthalate(BBP) and diethylhexyl phthalate(DEHP), three kinds of PAEs(DMP, DBP, Dn OP) were also listed environmental priority pollutants blacklist by chinese environmental protection department in the1980 s.China is the largest country owning facility agricultural area in the world, the facility modes mainly contain plastic greenhouse and film mulching. PAEs can enter into the soil from agricultural film, and the broken and waste agricultural film can exist in the soil for a long time. They not only damage the soil structure and degrade the soil function, but also endanger human health by affecting the safety of agriculturalproducts through crop absorption. Therefore, the content of PAEs in soil was detected accurately have important significance for protecting agricultural ecological environment, ensuring the safety of agricultural products and protecting human health.In the early time, the determination methods of PAEs mainly contained colorimetric method, titration method and spectrophotometry. But these methods have low sensitivity and poor selectivity. At present, some instrumental analytical methods have been commonly used to detect PAEs in different matrices. Although being accurate and sensitive, these methods have some disadvantages, such as the complicated extraction and cleanup procedures, tedious and time consuming operation, expensive equipment and difficulties for on-line continuous analysis. Therefore, there is a growing demand for developing simple, rapid, sensitive and reliable analytical methods to determine PAEs.In recent years, immunoassay based on the specific binding between antigen and antibody becomes a research focus. The enzyme-linked immunosorbent assay(ELISA),based on solid phase carrier binding antigen-antibody, has many advantages of no demands for expensive instrument, high specificity and sensitivity, convenience and rapidness. ELISA shows unique advantages in field screening and rapid detection of massive samples, which is widely applied to clinical diagnosis, drug screening,environmental analysis and monitoring, and etc. At present, some researches on immune detection for PAEs have been reported both at home and abroad, such as time-resolved fluoroimmunoassay(TR-FIA), direct ELISA method, competitive fluoroimmunoassay and ic-ELISA. However, these methods have disadvantages of narrow linear range, low sensitivity and the prepared antibody can only recognize a certain PAEs. It is difficult to meet the needs of simultaneous detection of multiple PAEs in real environment. For these reasons, we chose six PAEs concerned by USEPA and two commonly used plasticizers(DCHP, DNP) as detection objects, and designed and synthesized two haptens, and prepared immunogens, coating antigens and specific antibodies. We established ic-ELISA method and fiber optic immune sensing technique for PAEs detection, and the two methods were successfully applied to the rapid and sensitive detection of PAEs in facility vegetable soils. The main research achievements are summarized as follows:1. The two haptens(I and II) of PAEs had been successfully synthesized from4-nitroph phthalic acid via esterification, deoxidization and amidation. The characteristic absorption peak of the artificial antigen was the same as the carrierprotein and hapten. The preliminary results indicate that the coupling is successful. The concentrations of immunogens(hapten I-BSA and hapten II-BSA) were 4.28 and 5.35mg·m L-1, respectly. The concentrations of coating antigens(hapten I-OVA and hapten II-OVA) were 2.78 and 2.05 mg·m L-1, respectly. The coupling ratios of haptens(I and II) with BSA were obtained as 11:1 and 8.5:1, while those of haptens(I and II) with OVA were 6.5:1 and 4:1. The artificial antigen was synthized successfully via TLC,1HNMR and MS.2. The New Zealand white rabbits was immuned with artificial antigens, The antibody titer was 1:12800 and 1:25600 for A1 and A2(taking hapten I-BSA as immunogens), respectly. The antibody titer was 1:12800 and 1:12800 for B1 and B2(taking hapten II-BSA as immunogens), respectly. The higher antisera(1:25600) for A2 were used to conduct the immunoassay and the purified protein content was 2.959mg·m L-1.3. The detection conditions were optimized by ic-ELISA. The optimum detection conditions were obtained as follows: the coating antigen concentration and antisera dilution were 0.5 μg·m L-1and 1:3200, respectively. the p H of PBS is 7.4, and the salt concentration in PBS is 0.01 mol·L-1, the dilution of HRP-Ig G is 1:2000, the antigenprimary antibody reaction time is 60 min, and the methanol content in PAEs standard solution is 10%. Under these conditions, the standard calibration curves for the eight PAEs were obtained, taking the logarithm of PAEs concentration(log C) as horizontal coordinate and the binding inhibitory rate(B/B0) as longitudinal coordinate. The LODs were 0.013, 0.013, 0.012, 0.042, 0.035, 0.021, 0.017 and 0.016 μg·L-1for DMP, DEP,DBP, DEHP, Dn OP, BBP, DCHP and DNP, respectively. The cross reaction rates of antibody with the eight PAEs were 100%, 83.84%, 76.84%, 35.77%, 39.22%、44.01%,16.69% and 26.51%, which indicated that the antibody displayed a broad identification range. For DMP, the IC50 was 17.12 μg·L-1 and the linear range was 0.08~363.9 μg·L-1.The average recoveries of PAEs at the spiked levels of 5, 10 and 100 μg·kg-1 ranged from 63.9% to 103.6% with relative standard deviations(RSDs) below 11.32%. The average recoveries of PAEs by GC-MS ranged from 68.3% to 101.4% with RSDs below10.39%. The two methods obtained consistent results. The established ic-ELISA expanded linear range and reduced the detection limit of PAEs detection, simplified the sample pre-processing steps, the determination time was only 4.5 h. The developed ELISA method was successfully applied to determine PAEs in facility vegetable soils.4. The fiber optic immunosensor was constructed and antibody was labeled byCy5.5 NHS ester. The detection conditions were optimized by the fiber optic immunosensor, and the optimum detection conditions were as follows: the coating antigen concentration is 50 μg·m L-1, the p H value of PBS is 7.4, and the salt concentration in PBS is 0.01 mol·L-1, the PAEs-antibody pre-reaction time is 5 min.Under the optimum detection conditions, the standard calibration curves for the eight PAEs were obtained, taking the logarithm of PAEs concentration(log C) as horizontal coordinate and the binding inhibitory rate(B/B0) as longitudinal coordinate. The LODs were 0.147, 0.153, 0.148, 0.342, 0.337, 0.285, 0.317 and 0.156 μg·L-1 for DMP, DEP,DBP, DEHP, Dn OP, BBP, DCHP and DNP, respectively. The cross reaction rates of antibody with the eight PAEs were 100%, 71.94%, 62.66%, 31.14%, 51.8%, 42.85%,16.63% and 22.75%, which indicated that the antibody displayed a broad identification range. For DMP, the IC50 was 9.54 μg·L-1 and the linear range was 0.22~145 μg·L-1. The fiber optic immunosensor has good regeneration performance and stability. Using labeled antibodies with the same concentration, the probe was performed for 60 times,and the response signals were not significantly decreased. The average recoveries of PAEs at the spiked levels of 5, 10 and 100 μg·kg-1ranged from 61.5% to 106.7% with relative standard deviations(RSDs) below 13.41%. The average recoveries of PAEs by GC-MS ranged from 66.1% to 104.5% with RSDs below 11.63%. The two methods obtained consistent results. The established fiber optic immunosensor had a wide linear range and high sensitivity, the sample was simply extracted and the detection time was less than 15 min. the developed fiber optic immune sensing technique was successfully applied to determine PAEs in facility vegetable soils. |
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