Study On Immunoassay Of Butyl Benzyl Phthalate And Its Risk Assessment In Environmental Water Samples

Phthalate esters?PAEs?are important plasticizers,which are widely used in industrial and agricultural production and everyday life.PAEs has a strong bioaccumulation and anti-degradation,not only has a great impact on human reproduction and development,but also has a great harm to the nervous system,as well as carcinogenic risk and environmental ecological risk.The main detection methods of PAEs are instrumental methods and immunoassays.Because of its advantages of simplicity,high sensitivity,high throughput and high efficiency,immunoassays have significant advantages for the detection of PAEs in a large number of environmental samples,high throughput screening and environmental ecological risk.Benzyl butyl phthalate?BBP?is an important member of PAEs,because of its extensive use has caused a certain degree of environmental and ecological pollution.The research on simple and rapid analysis and detection technology and environmental risk assessment are of great significance for protecting the ecological environment safety and human health.In this paper,BBP was used to synthesize BBP hapten antigen and artificial antigen by chemical method,to prepare polyclonal antibody,to establish two methods of BBP immunoassays,and to evaluate the risk of BBP contamination in water environment.These include the following three areas of work:1.Preparation of antigens and antibodiesUsing 4-nitrophthalic anhydride as raw material,the hapten-antigen-BBP-A and hapten-antigen-BBP-B of two isomers containing-COOH were synthesized by multi-step chemical synthesis.To prepare artificial antigens,the hapten BBP-A and hapten BBP-B were separately conjugated to OVA to produce coating antigen BBP-A and coating antigen BBP-B by the mixed anhydride method,and separately conjugated to BSA to produce immunogen BBP-A and immunogen BBP-B by the carbodiimide method,and then immunized New Zealand white rabbits to prepare polyclonal antibodies PcAb BBP-A and PcAb BBP-B against BBP.2.Development of immunoassaysBased on the antigen and antibody,an enzyme-linked immunosorbent assay?ELISA?,was developed to evaluate the BBP polyclonal antibodies,and to optimize the concentration of antigen and antibody,ionic strength,pH value and content of methanol.Under the optimal conditions,the LOD of PcAb BBP-A was 7.8 ng/mL,IC₅₀0 was 119.9 ng/mL,and the detection range was 7.8-1854.1 ng/mL;the LOD of PcAb BBP-B was 2.5 ng/mL,IC₅₀0 was 79.4 ng/mL,the range of detection was2.5-2566.3 ng/mL.Both antibodies showed high specificity?CR<9.6%?.After the matrix effect of environmental samples was eliminated,the recoveries and coefficient of variations?CVs?were 76-116%and 4.7-13.7%,respectively.The real samples of environmental water samples and soil samples were tested.The BBP concentration in water samples was 2.7-22.9 ng/mL,the BBP concentration in soil samples was4.0-75.7 ng/mL.The results of HPLC have a good correlation to the developed ELISA.Based on high efficiency separation of magnetic beads and biotin-streptavidin?BAS?method,a magnetic bead immunoassay?MBI?method for BBP was developed.Compared with the ELISA,the detection limit was lower,the detection range was wider,the detection efficiency was higher,and the usage of antigen and antibody were reduced.Under the optimal conditions,the LOD was 0.57 ng/mL,IC₅₀0 was 119.61ng/mL,and the detection range was 0.57-24977.71 ng/mL.The cross reactivities was less than 4.5%toward the BBP structural analogues.The recoveries of water samples was 83.1-108.2%,the CVs were range from 3.1%to 12.4%,and the correlation coefficient with the results of HPLC analysis was 0.9786.3.Risk Assessment of BBP contamination in Water EnvironmentThirty-six environmental water samples were collected.A simple efficient and sensitive BAS-MBI method was used to detect the concentration of BBP,and the environmental risk,ecological risk and health risk of BBP were evaluated.The results showed that the health effects of BBP in the environment were within a controllable range,and the ASI was below 1.49.The highest value of ASII was 7.43,which should be paid attention to its harmful to the ecology.The entropy value of risk quotient was below 100,the highest was 59.47,which has a low risk to the environment and ecology,so it was necessary to strengthen the control.The non-carcinogenic risk of BBP exposure through drinking water was higher in females than that in males,which showed a slight risk of cancer,and the non-carcinogenic risk by the bath pathway was negligible.