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Abstract Phthalates (PAEs) compounds are a class of widely used environmental hormone plasticizers.Their harm to the environment and human health has caused a lot of attention. The detection of such substances, especially the simple and rapid detection, is of great significance for the protection of environmental food safety and consumer health. At present, a series of rapid immunoassay techniques have been applied to PAE detection with advantages of simple and fast operation, low cost, high sensitivity, high specificity and high throughput, achieving the goal of screening a large number of samples and making up for the shortcomings of chromatographic detection which uses expensive equipment with cumbersome operation, requires professional and technical personnel, and cannot achieve the target of screening a large number of samples. This paper introduced the research progress of PAE antibodies and their rapid immunoassay technology, and performed evaluation, in order to provide guidance and help for the development and application of PAE rapid detection technology.
Key words Phthalates; Plasticizers; Immunoassay; Research progress
Received: March 2, 2020Accepted: April 18, 2020
Supported by China Postdoctoral Fund (2016M601745); Advanced Talent Foundation of Jiangsu University of China (16JDG035).
Yin CUI (1994-), female, P. R. China, master, devoted to research about rapid analysis toxic and hazardous substances.
Corresponding author. E-mail: ddl@ujs.edu.cn.
Phthalic acid esters (PAEs, also known as phthalate esters) are a class of widely-used environmental hormone plasticizers, which are widely used as plasticizers, softeners, carriers and additives in plastics, automobiles, lubricants, cosmetics, clothing, pesticides and other industries［1-4］. PAEs have strong bioaccumulation and resistance to degradation, and not only have a great impact on human reproduction and development, but also have great harm to the nervous system, as well as carcinogenic risks and environmental ecological risks［5-8］. With long-term use and exposure, PAEs are released in environmental media and enter the food supply chain, resulting in different levels of detection in the atmosphere, water, soil and food, causing great risks to the environment and human body［9-11］.
The "Taiwan Plasticizer Storm" in 2011 and the "Inland Liquor Plasticizer Incident" in 2012 made PAEs well-known to consumers and caused some panic［12-13］. In recent years, the research on environmental food pollution and toxicology of PAEs has received extensive attention and has become a current research hotspot. There are many types of PAEs. More than 20 common varieties and their chemical structural formulas are shown in Table 1. The most common ones are DBP and DEHP, of which DEHP has been classified as a category 2B carcinogen by the US Environmental Protection Agency［14］.Many countries have identified multiple PAEs as environmental priority pollutants, and set limits or bans. The United States does not allow the addition of DBP, DIBP and DEHP to food contact materials. It is required that the total content of six PAEs (DEHP, DBP, BBP, DINP, DIDP, DnOP) in childrens toys or child care products should not exceed 0.1%［15］. The EU Directive 2008/105/EC stipulates that the limit of DEHP in surface water is 1.3 μg/L. The European Union does not allow DIBP to be added to food contact materials, and allow DBP and DEHP to be used in non-fat food contact materials with a migration limit of 0.3 mg/kg［16］. The Ministry of Health of China (Supervision Letter by the Office Bureau of Ministry of Health［2011］No.551) stipulates that the maximum residues of DBP, DEHP and DINP in food and food additives are 0.3, 1.5 and 9.0 mg/kg, respectively. The reference standards and limits the Chinese Sanitary Standards for Drinking Water Quality stipulate that the DEP and DBP limits are 0.3 and 0.003 mg/L, respectively, and the non-regular indices and limitations of water quality stipulate that the DEHP limit is 0.008 mg/L［17］. The research and reports of PAEs detection methods are mainly based on gas chromatography (GC), high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (HPLC-MS) and other chromatographic detection techniques and a series of rapid immunoassay techniques. Strengthening PAEs detection technology, especially high-sensitivity, simple and fast, high-throughput screening, and on-site detection methods, is particularly important for ensuring environmental ecological safety and food consumption safety. This paper focused on the research progress of PAE rapid immunoassay technology, which is widely concerned at home and abroad, with a view to providing ideas and directions for the development and application of rapid PAE immunoassay technology.
Development of PAE Antigens and Antibodies
The sensitivity and specificity of rapid immunoassay methods are closely related to the quality of antibodies and types of analytical methods. Haptens, artificial antigens, antibodies, and markers are the basic elements of immunoassays, of which antibodies are the core reagent in immunoassays. The synthesis of haptens and antigens is a key step in the preparation and analysis of small molecule compound antibodies［18］. Studies have shown that structures of haptens, lengths and structures of connecting arms and connection sites of active groups, and antibody acquisition pathways have varying degrees of influence on the specific recognition and sensitivity of antibodies.
Antigens
According to the chemical structural formula analysis of PAE plasticizers, this type of compounds possesses the same rigid planar aromatic hydrocarbon-phthalate group, and the difference lies in the two plastic non-linear fatty side chain groups. Therefore, the chemical synthesis and modification of PAE haptens also use similar approaches. According to current research reports, three strategies are mainly used to synthesize PAE haptens. The first strategy, for example, Ius et al. ［19］ used 4-hydroxyphthalic acid and methanol as raw materials to synthesize 4-hydroxy DMP, which chemically reacted with carboxymethoxylamine hemihydrochloride, obtaining the target DMP hapten with a -COOH spacer arm. The second strategy, for example, Yanaihara et al. ［20］ synthesized 4-nitro DMP by introducing -NO2 to the meta-position of the ester groups on the benzene ring of the DMP molecule using 4-nitrophthalic acid and methanol as raw materials and obtained 4-mmino DMP hapten after esterification and reduction. The third strategy, for example, Tang et al. ［21］ introduced -NH2 to the meta-position of the ester groups on the benzene ring of DMP, and then synthesized two kinds of DMP haptens whose -COOH spacer arms are of different lengths, through condensation reaction with carboxymethylhydroxylamine hemihydrochloride and succinic anhydride, respectively. In the immunoassays of PAEs, researchers mainly used the above-mentioned second strategy to chemically synthesize haptens by introducing a -NH2 active groups to the meta-position of the ester groups on the benzene ring of PAEs. In the subsequent study, the researchers also used the second strategy to synthesize 4-amino haptens of DCHP, DMP, DPrP, DIBP, DEP and DBP［22-31］. Based on the synthesis of PAE haptens, researchers usually use the diazo method to couple -NH2 haptens with bovine serum albumin (BSA) or chicken ovalbumin (OVA) to obtain immune antigens and coated antigens, respectively, and prepare artificial antigens by coupling the -COOH haptens with carrier proteins by the carbodiimide method.
Antibodies
Researchers immunized New Zealand white rabbits with the artificial immune antigens obtained, and purified from rabbit sera, DCHP polyclonal antibodies, DPrP polyclonal antibodies, DIBP polyclonal antibodies, DEP polyclonal antibodies, DBP polyclonal antibodies and DMP polyclonal antibodies［22,24-26,28,30,32-33］. Wei et al. and Zhou et al. ［29,31］ obtained DBP monoclonal antibodies through the procedures of mouse immunization, cell fusion, hybridoma cell screening and antibody production. Ius et al. and Tang et al. ［19,21］ prepared antigens from DMP hapten-conjugated proteins with long connecting arms and immunized New Zealand rabbits. The polyclonal antibodies obtained showed a high degree of affinity for DMP and other PAEs, which can be used as broad-spectrum antibodies for multi-component detection of various PAE plasticizers, indicating that a longer spacer arm is beneficial for the preparation of broad-spectrum PAE antibodies［19,21］.
Yin CUI et al. Research Advances on Immunoassays for Phthalic Acid Esters
PAE Rapid Immunoassay Detection
According to the different detection modes, carriers and markers, a variety of rapid immunoassay techniques have been developed for PAE analysis and detection. Researchers use various immunoassay models and labeling substances for rapid analysis and detection of PAEs, develop and obtain a series of immunoassay methods, and conduct detailed evaluation of the method performance, which greatly enriches the PAE analysis and detection technology and can reserve key techniques and important biochemical materials for PAE safety risk monitoring.
Micropore immunoassay detection
Enzyme immunoassay
As a classic method and basic method of immunoassay, enzyme-linked immunosorbent assay (ELISA) is widely used in the development and application of PAE analysis methods, in which the ELISA research for DMP and DBP is also the most in-depth. Zhang et al. ［23］ obtained enzyme-labeled antibodies by coupling DMP polyclonal antibodies with horse radish peroxidase (HRP), and established a direct competition ELISA (dc-ELISA) for the detection of DMP. The method showed a linear range of 0.1-2 000 ng/ml and a detection limit of 0.09 ng/ml, and had low cross-reactivity with other structural analogs (<8.8%). Sun et al. ［27］ established a biotin-streptavidin amplified ELISA method to detect DMP based on the biochemicalization of DMP antibodies. The detection range was 24-6 027 ng/ml, the IC 50 was 356 ng/ml, and the LOD was 8.2 ng/ml. The method had high specificity, and a cross-reaction rate with analogues was less than 10%. It showed high accuracy and precision in the detection of practical milk and milk samples, and the detection results were verified by GC-MS. Wei et al. ［29］ directly fixed the hapten 4-amino DBP by carboxylation of polyethylene microporous surface and EDC coupling reaction, establishing an indirect competition ELISA (ic-ELISA) method based on the monoclonal antibody DBP. Compared with fixing coated antigens on a microporous surface ( IC 50 : 106 ng/ml), the direct fixation of haptens greatly improved the sensitivity ( IC 50 : 14.6 ng/ml). In terms of specificity, except for the BBP cross-reaction rate of 21.3%, the rest were all below 7.98%. The method showed good accuracy and precision in food sample testing and was verified by GC-MS. Wan et al. ［34］ established an ELISA method for DBP detection in liquor and developed a successful kit for liquor sample detection. The method showed an IC 50 of 86.5 ng/ml and a detection range of 0-1 620 ng/ml. As to specificity, it had a 45% cross-reaction rate for DIBP, the rest were lower. Xu et al. ［30］ established an ic-ELISA method for the detection DBP in liquor with a detection limit of 64.5 ng/ml, a detection range of 64.5-1 606.2 ng/ml, and a correlation with GC-MS of 0.928. Sun and Zhuang［35］ biotinylated DBP polyclonal antibodies, establishing a biotin-avidin system amplification ELISA detection method, which had a detection limit reaching 5 pg/ml, an IC 50 of 0.36 ng/ml, a detection range of 0.45-7.06 ng/ml and high specificity<3.8%, and 0.45-7.06 ng/ml DBP was detected by the method in beverages and applied water. Zhou et al. ［31］ designed and synthesized DBP haptens and artificial antigens, prepared monoclonal antibodies, and established an ELISA method with a minimum detection limit of 0.06 ng/ml, an IC 50 of 7.34 ng/ml, and very high specificity (<1.25%). The method was 1 000 times more sensitive than polyclonal antibodies and 5 times higher than other reported monoclonal antibodies. It was applied to the analysis and detection of DBP content in human urine, and the results showed that among 1 246 urine samples, the detection rate was 72.87%, with a concentration of 0-42.98 ng/ml, and the concentration in young people was lower than that in the elderly. ELISA studies of several other PAEs mainly include DPrP, DEP and DEHP. Zhang et al. ［24］ coupled DPrP coated antigens with HRP to form a marker and established a direct competition chemiluminescence ELISA (CL-ELISA) method for the detection of DPrP in water and milk samples. The method showed an IC 50 of 0.19 ng/ml, an LOD of 0.03 ng/ml and a cross-reaction rate less than 9%. The samples without purification and concentration showed little matrix effect, indicating the simplicity of the immunoassay method. Zhang et al. ［26］ established an ic-ELISA detection method for DEP with a detection range of 0.005-18.6 ng/ml and a detection limit of 0.004 9 ng/ml. The cross-reactivity with other structural analogs was very low (<9%), and the recovery in juice, milk tea, pure milk and yogurt samples was in a range of 91.1-109.3%. Zhang et al. ［36］ prepared DEHP polyclonal antibodies and coupled them with HRP to form a detection probe and thereby established a dc-ELISA method. The method showed a minimum detection limit of 0.004 2 ng/ml, a detection range of 0.001-1 000 ng/ml, and cross-reactivity with structural analogs lower than 1%. It was successfully applied to DEHP detection in baby products.
Fluorescence immunoassay detection
Fluorescent immunoassay (FIA) uses fluorescent substances as markers, and the fluorescent signal gives the analysis method higher specificity and higher light quantum efficiency, which further improves the detection accuracy and sensitivity. Zhang et al. ［28］ established an indirect competition FIA method based on polyclonal antibodies. The method exhibited a detection limit of 0.02 ng/ml, an IC 50 of 10.53 ng/ml, a detection range of 0.1-300 ng/ml, and cross-reaction rates with other PAE plasticizers lower than 9.6%, and it was applied for added detection of paddy field water, river water, tap water and mineral water. Zhang et al. ［22］ coupled fluorescein isothiocyanate (FITC) and goat anti-rabbit secondary antibodies to prepare fluorescently labeled secondary antibodies, and established FIA combining with prepared DCHP rabbit-derived polyclonal antibodies. The method showed a DCHP detection range of 0.1-200 ng/ml, an LOD of 0.05 ng/ml and high specificity ( CR <8.7%), and it exhibited good reproducibility in various water samples. In the study of DMP by dc-ELISA conducted by Zhang et al. ［23］, DMP polyclonal antibodies were coupled with FITC to prepare fluorescently labeled antibodies to establish a DMP directly competition FIA (dc-FIA) method. The method showed a linear range of 0.05-30 ng/ml, a detection limit of 0.02 ng/ml, and sensitivity more than 4 times higher than that of dc-ELISA when using the same antigen and antibody, and it was applied to water sample detection［23］. Based on the preparation of specific DBP monoclonal antibodies, Zeng et al. ［37］ established an FIA analysis method to detect the distribution of DBP in mice, and established ic-ELISA to detect the accumulation of DBP in different organs of mice, thereby well applying immunoassay technology to the detection of living organisms. Cui et al. ［25］ prepared DIBP polyclonal antibodies and goat anti-rabbit-FITC and established an indirect competition FIA method for DIBP detection in edible oil samples. The method exhibited a detection limit reaching 5.82 ng/ml, an IC 50 of 61.2 ng/ml, a detection range of 10.47-357.06 ng/ml, and high specificity ( CR <1.5%). Homogeneous immunoassay
The change of immunoassay detection from heterogeneous mode to homogeneous mode will make the analysis and detection steps easier and the detection time significantly shortened. The application of polarization detection technology and magnetic separation technology can realize the detection in homogeneous mode, thereby simplifying the detection procedure and improving detection effectiveness. Tian et al. ［38］ labeled DEP antibodies with FITC and developed a DEP fluorescence polarization immunoassay (FPIA) with a detection limit of 6.0 ng/ml, an IC 50 value of 40.4 ng/ml, and a linear range of 10-200 ng/ml. The detection limits of bottled water samples, juice samples and drug capsule samples were 1.46 ng/ml, 340 ng/ml and 50 000 ng/g, respectively, the recovery was 85.9%-114.9%, and the relative standard deviation was 4.3%-17.0%. In our laboratory, Zhu et al. ［39］ directionally loaded DEP polyclonal antibodies on the surface of magnetic beads through goat anti-rabbit secondary antibodies, coupled coated antigens with a europium ion chelating agent to form a marker and established a magnetic bead homogeneous direct competition time-resolved fluorescence immunoassay (TRFIA) with a detection limit reaching 5.92 pg/ml. The method was applied to DEP distribution detection in the water environment of Zhenjiang City, and the DEP concentration was actually detected to be 2.98-306.19 ng/ml. These two PAE immunoassay techniques combine homogeneous mode, high efficiency of magnetic separation and high sensitivity of fluorescent signal, which significantly improves the detection ability, providing a reference for highly sensitive, homogeneous and efficient trace detection of other substances.
Ultra-sensitive PCR immunoassay
With the increase of human awareness of the environment and food safety, higher requirements have been put forward for the detection of toxic and hazardous substances. Under such circumstances, detection techniques that can accurately and sensitively detect ultra-low levels of toxic and hazardous substances in samples are greatly needed and welcomed. Sun et al. ［27］ reported that real-time quantitative immuno-PCR analysis technology based on gold nanoparticles was used for ultra-high sensitivity detection of DEP, with a detection range of 4-40 pg/ml, a detection limit of 1.06 fg/ml and high specificity CR (<5%), and then applied to DEP trace detection in food. The report combined immunoassay and ultra-sensitive PCR technology, which greatly improved the sensitivity of DEP detection (theoretically increased by more than 3 orders of magnitude). It is the most sensitive mark detection strategy at present, and has important guiding value for the development of ultra-trace detection of other toxic and hazardous substances. Multi-component immunoassay
With the long-term exposure of PAE plasticizers in the environment, the possibility of multiple PAEs present in environmental samples is greatly enhanced. The current methods mainly for single component detection are unable to meet the needs of simultaneous detection of multiple components. In order to expand the detection range of immunoassays, researchers have directed their research goals to the analysis of multiple analytes, also known as broad-spectrum specific analysis, multi-component analysis, or broad-selectivity analysis［40］. Different from single-component analysis, multi-component analysis can be used for total or quantitative detection of multiple analytes, which is a good method in primary screening or primary testing［41］. Ius et al. ［19］ biotinylated polyclonal antibodies and coupled streptavidin to a lanthanide complex using a two-way chelating agent (BCPDA) to form a marker, and established a broad-spectrum TRFIA method for PAE detection, which showed cross-reaction rates of 100%, 110%, 106%, 104% and 97% with DMP, DEP, DBP, BBP and DNOP, respectively, and values lower than 3.5% with other analogs. It can be used for the simultaneous analysis and detection of these five PAE plasticizers with similar cross-reaction rates, and the detection range was in the range of 0.5 pmol/ml-2 nmol/ml. Tang et al. ［21］ obtained polyclonal antibodies by synthesizing general haptens of PAEs, which can recognize a variety of PAEs (including DMP, DEP, DBP, DNOP, BBP, DEHP, DCHP), with cross-reaction rates between 63.9% and 103.6, IC 50 in the range of 17.12-102.57 ng/ml and minimum detection limits in the range of 0.012-0.042 ng/ml. The concentrations of PAEs in greenhouse soil samples were detected to be 1 260-3 580 ng/g, and the greenhouse soil used for 10 years was detected to have higher concentrations of PAEs than the soil used for 5 years. The development of PAE multi-component immunoassay technology provides a convenient screening method for the simultaneous presence of multiple PAEs in samples, which is of great value for simplifying initial screening procedure and improving detection efficiency.
Immunoaffinity chromatography analysis and detection
The immunoaffinity chromatography displays the immunoassay technology through chromatography technology, which is more intuitive and convenient for analyzing the substance to be tested, and can realize qualitative and semi-quantitative detection. The immunoassay of PAEs is mainly the methods reported in the preceding part of the text, and there have been no studies on PAEs using immunoaffinity chromatography technology. However, in practical applications, researchers use immunoaffinity chromatography technology to develop PAE detection cards. There are also many biochemical reagent companies producing and selling PAE plasticizer testing cards. PAE Immune Detection Devices and Their Application
The PAE immunoassay devices are mainly immunoassay kits and detection cards. These two kinds of devices respectively take ELISA and colloidal gold immunoaffinity chromatography as the core technology, and on this basis, products that can be used to detect PAE pollution in production practice can be obtained. At present, there are a number of biochemical reagent companies at home and abroad in the production and sale of PAE kits and test cards. For example, Beijing Kwinbon Biotechnology Co., Ltd. in China can provide ELISA kits, chemiluminescence kits and test cards for detecting DBP and DIBP in liquor samples, among which the ELISA kits have a sensitivity of 10 ng/ml and a sample detection limit of 100 ng/ml, stable detection recovery and coefficient of variation less than 10%. The DBP detection kits produced by Beijing Prime Bio Tek Co., Ltd. have a sensitivity of 30 ng/ml, a sample recovery of 95% ± 20%, and a coefficient of variation and cross-reaction rate less than 10%. The kits can be used for qualitative and quantitative detection of DBP in drinking water, beverages, wine and other samples. The detection sensitivity of the DBP kits of American REAGEN is 50 ng/ml, and the recovery is 70%-130%. The detection limit of DEHP kits of American GTX reaches 100 ng/ml, and they are suitable for DEHP detection in muscle and liver tissues, urine samples, serum and feeds. There are also reports on the application of the detection devices. Cao et al. ［42］ applied the plasticizer ELISA test kits produced by Beijing Prime Bio Tek Co., Ltd. to the rapid detection of DBP in red wine, and compared the test results by GC-MS. It showed that the kits could meet the initial screening goal of DBP, and have the advantages of simple sample preparation, rapid detection and high specificity. The production and supply of PAE test kits and test cards can provide convenient and fast guarantee for environmental and food safety testing.
The Development Trend of PAE Rapid Immune Detection Technology
As a simple and rapid technical means, PAE rapid immunoassay technology forms a technical reserve for the detection of common PAE plasticizers based on the preparation of early antigens and antibodies and the development of immunoassay methods, and for some main types, there have already been test kit and test card product reserve and corresponding commercialization. The development of this series of PAE immunoassay techniques can be used as alternative and supplementary methods for instrumental analysis and detection. Due to the differences in performance and unique advantages of these immunoassay techniques, they can meet different testing sites and testing needs, and consumers can choose flexibly in practical applications. However, there are still many PAEs on which no studies have been reported in the aspect of rapid immunoassay, and it is necessary to supplement and improve this deficiency as an important technical reserve. In addition, In view of the widespread presence of PAEs in the environment and food, their high exposure risk, and the difficulty of accurately detecting samples at ultra-trace levels, the quality requirements for immunoassay testing are also stricter. Whether immunoassay detection can achieve higher sensitivity and specificity, as well as simpler and faster operation, antibodies as a core reagent play a key role. However, the quality and traits of antibodies obtained from animals using traditional classic methods are difficult to change and improve. Therefore, strengthening the research of PAE antibodies and immunoassay methods, especially the application of new techniques and methods to prepare high-sensitivity, high-specificity and multi-functional antibodies, is of great significance to promote the application of rapid immunoassay technology to PAE analysis and detection. The current rapid immunoassay devices are mainly kits and test cards, and the fluorescent immunoassay and PCR immunoassay techniques with more significant sensitivity in the previous research, as well as the more convenient and efficient homogeneous immunoassay technique, should be better commercialized and promoted and applied, so as to further improve the rapid immune detection system, better protect the environment and food safety, and protect consumer health. References
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Editor: Yingzhi GUANGProofreader: Xinxiu ZHU
Key words Phthalates; Plasticizers; Immunoassay; Research progress
Received: March 2, 2020Accepted: April 18, 2020
Supported by China Postdoctoral Fund (2016M601745); Advanced Talent Foundation of Jiangsu University of China (16JDG035).
Yin CUI (1994-), female, P. R. China, master, devoted to research about rapid analysis toxic and hazardous substances.
Corresponding author. E-mail: ddl@ujs.edu.cn.
Phthalic acid esters (PAEs, also known as phthalate esters) are a class of widely-used environmental hormone plasticizers, which are widely used as plasticizers, softeners, carriers and additives in plastics, automobiles, lubricants, cosmetics, clothing, pesticides and other industries［1-4］. PAEs have strong bioaccumulation and resistance to degradation, and not only have a great impact on human reproduction and development, but also have great harm to the nervous system, as well as carcinogenic risks and environmental ecological risks［5-8］. With long-term use and exposure, PAEs are released in environmental media and enter the food supply chain, resulting in different levels of detection in the atmosphere, water, soil and food, causing great risks to the environment and human body［9-11］.
The "Taiwan Plasticizer Storm" in 2011 and the "Inland Liquor Plasticizer Incident" in 2012 made PAEs well-known to consumers and caused some panic［12-13］. In recent years, the research on environmental food pollution and toxicology of PAEs has received extensive attention and has become a current research hotspot. There are many types of PAEs. More than 20 common varieties and their chemical structural formulas are shown in Table 1. The most common ones are DBP and DEHP, of which DEHP has been classified as a category 2B carcinogen by the US Environmental Protection Agency［14］.Many countries have identified multiple PAEs as environmental priority pollutants, and set limits or bans. The United States does not allow the addition of DBP, DIBP and DEHP to food contact materials. It is required that the total content of six PAEs (DEHP, DBP, BBP, DINP, DIDP, DnOP) in childrens toys or child care products should not exceed 0.1%［15］. The EU Directive 2008/105/EC stipulates that the limit of DEHP in surface water is 1.3 μg/L. The European Union does not allow DIBP to be added to food contact materials, and allow DBP and DEHP to be used in non-fat food contact materials with a migration limit of 0.3 mg/kg［16］. The Ministry of Health of China (Supervision Letter by the Office Bureau of Ministry of Health［2011］No.551) stipulates that the maximum residues of DBP, DEHP and DINP in food and food additives are 0.3, 1.5 and 9.0 mg/kg, respectively. The reference standards and limits the Chinese Sanitary Standards for Drinking Water Quality stipulate that the DEP and DBP limits are 0.3 and 0.003 mg/L, respectively, and the non-regular indices and limitations of water quality stipulate that the DEHP limit is 0.008 mg/L［17］. The research and reports of PAEs detection methods are mainly based on gas chromatography (GC), high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (HPLC-MS) and other chromatographic detection techniques and a series of rapid immunoassay techniques. Strengthening PAEs detection technology, especially high-sensitivity, simple and fast, high-throughput screening, and on-site detection methods, is particularly important for ensuring environmental ecological safety and food consumption safety. This paper focused on the research progress of PAE rapid immunoassay technology, which is widely concerned at home and abroad, with a view to providing ideas and directions for the development and application of rapid PAE immunoassay technology.
Development of PAE Antigens and Antibodies
The sensitivity and specificity of rapid immunoassay methods are closely related to the quality of antibodies and types of analytical methods. Haptens, artificial antigens, antibodies, and markers are the basic elements of immunoassays, of which antibodies are the core reagent in immunoassays. The synthesis of haptens and antigens is a key step in the preparation and analysis of small molecule compound antibodies［18］. Studies have shown that structures of haptens, lengths and structures of connecting arms and connection sites of active groups, and antibody acquisition pathways have varying degrees of influence on the specific recognition and sensitivity of antibodies.
Antigens
According to the chemical structural formula analysis of PAE plasticizers, this type of compounds possesses the same rigid planar aromatic hydrocarbon-phthalate group, and the difference lies in the two plastic non-linear fatty side chain groups. Therefore, the chemical synthesis and modification of PAE haptens also use similar approaches. According to current research reports, three strategies are mainly used to synthesize PAE haptens. The first strategy, for example, Ius et al. ［19］ used 4-hydroxyphthalic acid and methanol as raw materials to synthesize 4-hydroxy DMP, which chemically reacted with carboxymethoxylamine hemihydrochloride, obtaining the target DMP hapten with a -COOH spacer arm. The second strategy, for example, Yanaihara et al. ［20］ synthesized 4-nitro DMP by introducing -NO2 to the meta-position of the ester groups on the benzene ring of the DMP molecule using 4-nitrophthalic acid and methanol as raw materials and obtained 4-mmino DMP hapten after esterification and reduction. The third strategy, for example, Tang et al. ［21］ introduced -NH2 to the meta-position of the ester groups on the benzene ring of DMP, and then synthesized two kinds of DMP haptens whose -COOH spacer arms are of different lengths, through condensation reaction with carboxymethylhydroxylamine hemihydrochloride and succinic anhydride, respectively. In the immunoassays of PAEs, researchers mainly used the above-mentioned second strategy to chemically synthesize haptens by introducing a -NH2 active groups to the meta-position of the ester groups on the benzene ring of PAEs. In the subsequent study, the researchers also used the second strategy to synthesize 4-amino haptens of DCHP, DMP, DPrP, DIBP, DEP and DBP［22-31］. Based on the synthesis of PAE haptens, researchers usually use the diazo method to couple -NH2 haptens with bovine serum albumin (BSA) or chicken ovalbumin (OVA) to obtain immune antigens and coated antigens, respectively, and prepare artificial antigens by coupling the -COOH haptens with carrier proteins by the carbodiimide method.
Antibodies
Researchers immunized New Zealand white rabbits with the artificial immune antigens obtained, and purified from rabbit sera, DCHP polyclonal antibodies, DPrP polyclonal antibodies, DIBP polyclonal antibodies, DEP polyclonal antibodies, DBP polyclonal antibodies and DMP polyclonal antibodies［22,24-26,28,30,32-33］. Wei et al. and Zhou et al. ［29,31］ obtained DBP monoclonal antibodies through the procedures of mouse immunization, cell fusion, hybridoma cell screening and antibody production. Ius et al. and Tang et al. ［19,21］ prepared antigens from DMP hapten-conjugated proteins with long connecting arms and immunized New Zealand rabbits. The polyclonal antibodies obtained showed a high degree of affinity for DMP and other PAEs, which can be used as broad-spectrum antibodies for multi-component detection of various PAE plasticizers, indicating that a longer spacer arm is beneficial for the preparation of broad-spectrum PAE antibodies［19,21］.
Yin CUI et al. Research Advances on Immunoassays for Phthalic Acid Esters
PAE Rapid Immunoassay Detection
According to the different detection modes, carriers and markers, a variety of rapid immunoassay techniques have been developed for PAE analysis and detection. Researchers use various immunoassay models and labeling substances for rapid analysis and detection of PAEs, develop and obtain a series of immunoassay methods, and conduct detailed evaluation of the method performance, which greatly enriches the PAE analysis and detection technology and can reserve key techniques and important biochemical materials for PAE safety risk monitoring.
Micropore immunoassay detection
Enzyme immunoassay
As a classic method and basic method of immunoassay, enzyme-linked immunosorbent assay (ELISA) is widely used in the development and application of PAE analysis methods, in which the ELISA research for DMP and DBP is also the most in-depth. Zhang et al. ［23］ obtained enzyme-labeled antibodies by coupling DMP polyclonal antibodies with horse radish peroxidase (HRP), and established a direct competition ELISA (dc-ELISA) for the detection of DMP. The method showed a linear range of 0.1-2 000 ng/ml and a detection limit of 0.09 ng/ml, and had low cross-reactivity with other structural analogs (<8.8%). Sun et al. ［27］ established a biotin-streptavidin amplified ELISA method to detect DMP based on the biochemicalization of DMP antibodies. The detection range was 24-6 027 ng/ml, the IC 50 was 356 ng/ml, and the LOD was 8.2 ng/ml. The method had high specificity, and a cross-reaction rate with analogues was less than 10%. It showed high accuracy and precision in the detection of practical milk and milk samples, and the detection results were verified by GC-MS. Wei et al. ［29］ directly fixed the hapten 4-amino DBP by carboxylation of polyethylene microporous surface and EDC coupling reaction, establishing an indirect competition ELISA (ic-ELISA) method based on the monoclonal antibody DBP. Compared with fixing coated antigens on a microporous surface ( IC 50 : 106 ng/ml), the direct fixation of haptens greatly improved the sensitivity ( IC 50 : 14.6 ng/ml). In terms of specificity, except for the BBP cross-reaction rate of 21.3%, the rest were all below 7.98%. The method showed good accuracy and precision in food sample testing and was verified by GC-MS. Wan et al. ［34］ established an ELISA method for DBP detection in liquor and developed a successful kit for liquor sample detection. The method showed an IC 50 of 86.5 ng/ml and a detection range of 0-1 620 ng/ml. As to specificity, it had a 45% cross-reaction rate for DIBP, the rest were lower. Xu et al. ［30］ established an ic-ELISA method for the detection DBP in liquor with a detection limit of 64.5 ng/ml, a detection range of 64.5-1 606.2 ng/ml, and a correlation with GC-MS of 0.928. Sun and Zhuang［35］ biotinylated DBP polyclonal antibodies, establishing a biotin-avidin system amplification ELISA detection method, which had a detection limit reaching 5 pg/ml, an IC 50 of 0.36 ng/ml, a detection range of 0.45-7.06 ng/ml and high specificity<3.8%, and 0.45-7.06 ng/ml DBP was detected by the method in beverages and applied water. Zhou et al. ［31］ designed and synthesized DBP haptens and artificial antigens, prepared monoclonal antibodies, and established an ELISA method with a minimum detection limit of 0.06 ng/ml, an IC 50 of 7.34 ng/ml, and very high specificity (<1.25%). The method was 1 000 times more sensitive than polyclonal antibodies and 5 times higher than other reported monoclonal antibodies. It was applied to the analysis and detection of DBP content in human urine, and the results showed that among 1 246 urine samples, the detection rate was 72.87%, with a concentration of 0-42.98 ng/ml, and the concentration in young people was lower than that in the elderly. ELISA studies of several other PAEs mainly include DPrP, DEP and DEHP. Zhang et al. ［24］ coupled DPrP coated antigens with HRP to form a marker and established a direct competition chemiluminescence ELISA (CL-ELISA) method for the detection of DPrP in water and milk samples. The method showed an IC 50 of 0.19 ng/ml, an LOD of 0.03 ng/ml and a cross-reaction rate less than 9%. The samples without purification and concentration showed little matrix effect, indicating the simplicity of the immunoassay method. Zhang et al. ［26］ established an ic-ELISA detection method for DEP with a detection range of 0.005-18.6 ng/ml and a detection limit of 0.004 9 ng/ml. The cross-reactivity with other structural analogs was very low (<9%), and the recovery in juice, milk tea, pure milk and yogurt samples was in a range of 91.1-109.3%. Zhang et al. ［36］ prepared DEHP polyclonal antibodies and coupled them with HRP to form a detection probe and thereby established a dc-ELISA method. The method showed a minimum detection limit of 0.004 2 ng/ml, a detection range of 0.001-1 000 ng/ml, and cross-reactivity with structural analogs lower than 1%. It was successfully applied to DEHP detection in baby products.
Fluorescence immunoassay detection
Fluorescent immunoassay (FIA) uses fluorescent substances as markers, and the fluorescent signal gives the analysis method higher specificity and higher light quantum efficiency, which further improves the detection accuracy and sensitivity. Zhang et al. ［28］ established an indirect competition FIA method based on polyclonal antibodies. The method exhibited a detection limit of 0.02 ng/ml, an IC 50 of 10.53 ng/ml, a detection range of 0.1-300 ng/ml, and cross-reaction rates with other PAE plasticizers lower than 9.6%, and it was applied for added detection of paddy field water, river water, tap water and mineral water. Zhang et al. ［22］ coupled fluorescein isothiocyanate (FITC) and goat anti-rabbit secondary antibodies to prepare fluorescently labeled secondary antibodies, and established FIA combining with prepared DCHP rabbit-derived polyclonal antibodies. The method showed a DCHP detection range of 0.1-200 ng/ml, an LOD of 0.05 ng/ml and high specificity ( CR <8.7%), and it exhibited good reproducibility in various water samples. In the study of DMP by dc-ELISA conducted by Zhang et al. ［23］, DMP polyclonal antibodies were coupled with FITC to prepare fluorescently labeled antibodies to establish a DMP directly competition FIA (dc-FIA) method. The method showed a linear range of 0.05-30 ng/ml, a detection limit of 0.02 ng/ml, and sensitivity more than 4 times higher than that of dc-ELISA when using the same antigen and antibody, and it was applied to water sample detection［23］. Based on the preparation of specific DBP monoclonal antibodies, Zeng et al. ［37］ established an FIA analysis method to detect the distribution of DBP in mice, and established ic-ELISA to detect the accumulation of DBP in different organs of mice, thereby well applying immunoassay technology to the detection of living organisms. Cui et al. ［25］ prepared DIBP polyclonal antibodies and goat anti-rabbit-FITC and established an indirect competition FIA method for DIBP detection in edible oil samples. The method exhibited a detection limit reaching 5.82 ng/ml, an IC 50 of 61.2 ng/ml, a detection range of 10.47-357.06 ng/ml, and high specificity ( CR <1.5%). Homogeneous immunoassay
The change of immunoassay detection from heterogeneous mode to homogeneous mode will make the analysis and detection steps easier and the detection time significantly shortened. The application of polarization detection technology and magnetic separation technology can realize the detection in homogeneous mode, thereby simplifying the detection procedure and improving detection effectiveness. Tian et al. ［38］ labeled DEP antibodies with FITC and developed a DEP fluorescence polarization immunoassay (FPIA) with a detection limit of 6.0 ng/ml, an IC 50 value of 40.4 ng/ml, and a linear range of 10-200 ng/ml. The detection limits of bottled water samples, juice samples and drug capsule samples were 1.46 ng/ml, 340 ng/ml and 50 000 ng/g, respectively, the recovery was 85.9%-114.9%, and the relative standard deviation was 4.3%-17.0%. In our laboratory, Zhu et al. ［39］ directionally loaded DEP polyclonal antibodies on the surface of magnetic beads through goat anti-rabbit secondary antibodies, coupled coated antigens with a europium ion chelating agent to form a marker and established a magnetic bead homogeneous direct competition time-resolved fluorescence immunoassay (TRFIA) with a detection limit reaching 5.92 pg/ml. The method was applied to DEP distribution detection in the water environment of Zhenjiang City, and the DEP concentration was actually detected to be 2.98-306.19 ng/ml. These two PAE immunoassay techniques combine homogeneous mode, high efficiency of magnetic separation and high sensitivity of fluorescent signal, which significantly improves the detection ability, providing a reference for highly sensitive, homogeneous and efficient trace detection of other substances.
Ultra-sensitive PCR immunoassay
With the increase of human awareness of the environment and food safety, higher requirements have been put forward for the detection of toxic and hazardous substances. Under such circumstances, detection techniques that can accurately and sensitively detect ultra-low levels of toxic and hazardous substances in samples are greatly needed and welcomed. Sun et al. ［27］ reported that real-time quantitative immuno-PCR analysis technology based on gold nanoparticles was used for ultra-high sensitivity detection of DEP, with a detection range of 4-40 pg/ml, a detection limit of 1.06 fg/ml and high specificity CR (<5%), and then applied to DEP trace detection in food. The report combined immunoassay and ultra-sensitive PCR technology, which greatly improved the sensitivity of DEP detection (theoretically increased by more than 3 orders of magnitude). It is the most sensitive mark detection strategy at present, and has important guiding value for the development of ultra-trace detection of other toxic and hazardous substances. Multi-component immunoassay
With the long-term exposure of PAE plasticizers in the environment, the possibility of multiple PAEs present in environmental samples is greatly enhanced. The current methods mainly for single component detection are unable to meet the needs of simultaneous detection of multiple components. In order to expand the detection range of immunoassays, researchers have directed their research goals to the analysis of multiple analytes, also known as broad-spectrum specific analysis, multi-component analysis, or broad-selectivity analysis［40］. Different from single-component analysis, multi-component analysis can be used for total or quantitative detection of multiple analytes, which is a good method in primary screening or primary testing［41］. Ius et al. ［19］ biotinylated polyclonal antibodies and coupled streptavidin to a lanthanide complex using a two-way chelating agent (BCPDA) to form a marker, and established a broad-spectrum TRFIA method for PAE detection, which showed cross-reaction rates of 100%, 110%, 106%, 104% and 97% with DMP, DEP, DBP, BBP and DNOP, respectively, and values lower than 3.5% with other analogs. It can be used for the simultaneous analysis and detection of these five PAE plasticizers with similar cross-reaction rates, and the detection range was in the range of 0.5 pmol/ml-2 nmol/ml. Tang et al. ［21］ obtained polyclonal antibodies by synthesizing general haptens of PAEs, which can recognize a variety of PAEs (including DMP, DEP, DBP, DNOP, BBP, DEHP, DCHP), with cross-reaction rates between 63.9% and 103.6, IC 50 in the range of 17.12-102.57 ng/ml and minimum detection limits in the range of 0.012-0.042 ng/ml. The concentrations of PAEs in greenhouse soil samples were detected to be 1 260-3 580 ng/g, and the greenhouse soil used for 10 years was detected to have higher concentrations of PAEs than the soil used for 5 years. The development of PAE multi-component immunoassay technology provides a convenient screening method for the simultaneous presence of multiple PAEs in samples, which is of great value for simplifying initial screening procedure and improving detection efficiency.
Immunoaffinity chromatography analysis and detection
The immunoaffinity chromatography displays the immunoassay technology through chromatography technology, which is more intuitive and convenient for analyzing the substance to be tested, and can realize qualitative and semi-quantitative detection. The immunoassay of PAEs is mainly the methods reported in the preceding part of the text, and there have been no studies on PAEs using immunoaffinity chromatography technology. However, in practical applications, researchers use immunoaffinity chromatography technology to develop PAE detection cards. There are also many biochemical reagent companies producing and selling PAE plasticizer testing cards. PAE Immune Detection Devices and Their Application
The PAE immunoassay devices are mainly immunoassay kits and detection cards. These two kinds of devices respectively take ELISA and colloidal gold immunoaffinity chromatography as the core technology, and on this basis, products that can be used to detect PAE pollution in production practice can be obtained. At present, there are a number of biochemical reagent companies at home and abroad in the production and sale of PAE kits and test cards. For example, Beijing Kwinbon Biotechnology Co., Ltd. in China can provide ELISA kits, chemiluminescence kits and test cards for detecting DBP and DIBP in liquor samples, among which the ELISA kits have a sensitivity of 10 ng/ml and a sample detection limit of 100 ng/ml, stable detection recovery and coefficient of variation less than 10%. The DBP detection kits produced by Beijing Prime Bio Tek Co., Ltd. have a sensitivity of 30 ng/ml, a sample recovery of 95% ± 20%, and a coefficient of variation and cross-reaction rate less than 10%. The kits can be used for qualitative and quantitative detection of DBP in drinking water, beverages, wine and other samples. The detection sensitivity of the DBP kits of American REAGEN is 50 ng/ml, and the recovery is 70%-130%. The detection limit of DEHP kits of American GTX reaches 100 ng/ml, and they are suitable for DEHP detection in muscle and liver tissues, urine samples, serum and feeds. There are also reports on the application of the detection devices. Cao et al. ［42］ applied the plasticizer ELISA test kits produced by Beijing Prime Bio Tek Co., Ltd. to the rapid detection of DBP in red wine, and compared the test results by GC-MS. It showed that the kits could meet the initial screening goal of DBP, and have the advantages of simple sample preparation, rapid detection and high specificity. The production and supply of PAE test kits and test cards can provide convenient and fast guarantee for environmental and food safety testing.
The Development Trend of PAE Rapid Immune Detection Technology
As a simple and rapid technical means, PAE rapid immunoassay technology forms a technical reserve for the detection of common PAE plasticizers based on the preparation of early antigens and antibodies and the development of immunoassay methods, and for some main types, there have already been test kit and test card product reserve and corresponding commercialization. The development of this series of PAE immunoassay techniques can be used as alternative and supplementary methods for instrumental analysis and detection. Due to the differences in performance and unique advantages of these immunoassay techniques, they can meet different testing sites and testing needs, and consumers can choose flexibly in practical applications. However, there are still many PAEs on which no studies have been reported in the aspect of rapid immunoassay, and it is necessary to supplement and improve this deficiency as an important technical reserve. In addition, In view of the widespread presence of PAEs in the environment and food, their high exposure risk, and the difficulty of accurately detecting samples at ultra-trace levels, the quality requirements for immunoassay testing are also stricter. Whether immunoassay detection can achieve higher sensitivity and specificity, as well as simpler and faster operation, antibodies as a core reagent play a key role. However, the quality and traits of antibodies obtained from animals using traditional classic methods are difficult to change and improve. Therefore, strengthening the research of PAE antibodies and immunoassay methods, especially the application of new techniques and methods to prepare high-sensitivity, high-specificity and multi-functional antibodies, is of great significance to promote the application of rapid immunoassay technology to PAE analysis and detection. The current rapid immunoassay devices are mainly kits and test cards, and the fluorescent immunoassay and PCR immunoassay techniques with more significant sensitivity in the previous research, as well as the more convenient and efficient homogeneous immunoassay technique, should be better commercialized and promoted and applied, so as to further improve the rapid immune detection system, better protect the environment and food safety, and protect consumer health. References
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