| Di(2-ethylhexyl)phthalate(DEHP),is a kind of phthalate esters(PAEs)and is commonly known as environmental hormone.DEHP is a type of plasticer with the largest amount of production and usage in PAEs.It is used to enhance flexibility in polyethylene plastic products and has great commercial utility value.It is generally used in hundreds of cosmetics,food packaging,and pesticides.The harm of long-term exposure to high levels of DEHP are as follows:the toxicity of its monoester metabolites was significantly stronger than itself and the cumulative effect of toxicity is caused by a decrease in blood cells,which leads to liver cancer and renal insufficiency and can cause endocrine disorders,fetal malformations,young children’s gender disorders,and loss of reproductive system function.At present,the national standard detection method for DEHP detection method mainly includes instrument detection method.In recent years,immunological testing has gradually been applied to large-scale detection of small and medium-sized chemical substances in commodities,such as common ELISA kits and colloidal gold test strips.The ELISA method has the advantages of simple principle,simplicity,low cost,sensitivity,and suitability for large-scale sample detection.Therefore,the ELISA method can be used to detect PAEs.According to the basic principle of hapten design,a hapten structure(DBCP)that meets the requirements of adding two carbon arms to the structure of DEHP was designed.Anti-DEHP serum was prepared by immunizing mice with DEHP-BSA.DEHP-OVA was used as a coating antigen to develop an IC-ELISA method for detecting DEHP.At the same time,DEHP-OVA was coupled with a carboxyl magnetic bead to prepare a magnetic bead probe.An indirect competition ELISA(IC-ELISA)method and DEHP indirect competition ELISA(IMB-IC-ELISA)basedon magnetic beads were developed,were used to detect DEHP in liquid food samples.A solid phase detection antigen(DEHP-OVA)was prepared by coupling a small molecule of DEHP with ovalbumin(OVA)using a mixed anhydride method.The indirect detection principle was that the target analyte and the solid phase antigen in the microtiter plate competed with the specific polyclonal antibody.Competitive ELISA(IC-ELISA).After the optimization of the main conditions of IC-ELISA detection,the main steps after optimization are as follows: concentration of detection antigen is 2 μg/m L,coating condition is 37℃,4 h or 4℃,12 h incubation;blocked condition is 1% skim milk powder,37℃,0.5 h;The optimal dilution of antibody and working conditions is 1: 16000,37℃,1 h;The optimal dilution of HRP Goat anti-mice antibody and working conditions is 1: 10000,37℃,1 h;TMB substrate coloring solution 37℃,15 min;H2SO4 solution terminated.The linear regression equation was: y=69.873x-79.811,R2=0.9945,IC50=63.19 ng/m L,the method detection range was 16.91-236.11 ng/m L;Adding DEHP 200.00 ng/m L,62.50ng/m L,and 31.25 ng/m L respectively to negative water and beverage samples,negative oil samples,and negative wine samples to get the corresponding recoveries for water and beverage samples were respectively 116.51%,112.99%,and 112.06%;the corresponding recoveries for the oil samples were 110.70%,102.84%,94.20%;the corresponding recovery rates of oil samples were 91.84%,92.26%,and 95.12%respectively;105 pieces of samples were purchased from the supermarket.According to the standard curve,the content of DEHP in the actual samples was measured.The detection antigen DEHP-OVA is coupled with a carboxyl magnetic bead to prepare a magnetic bead probe to used as a detection antigen,under the action of a magnetic field,it is equivalent to a coating step.The principle of the IMB-IC-ELISA is the same as that of an ordinary IC-ELISA.Methods The main conditions were optimized.The best procedure for IMB-IC-ELISA were as follows: the optimal amount of magnetic bead probe was 60 μg,the optimal dilution of antibody and the working condition was 1:4000,incubated for 1 hour at 37℃;The HRP Goatanti-mice antibody dilution and working conditions were 1:6000,incubated at 37℃ for 40 min;TMB substrate solution was incubated at 37℃ for 10 min,H2SO4 solution terminated.The linear regression equation of DEHP is: y=68.482x-112.61,R2=0.9924.The detection range of DEHP is 58.16-824.15 ng/m L;IC50=218.94ng/m L.The actual samples were spiked with DEHP 500 ng/m L,250 ng/m L,and 100ng/m L.DEHP content was calculated according to the standard curve established.The results showed that the all samples met the national testing standards.The results of the IC-ELISA and IMB-IC-ELISA developed in this experiment were basically consistent with the results of the GC-MS method.Compared with IC-ELISA,IMB-IC-ELISA method has a higher cost,and the result is closer to the national standard method result. |
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