Hindawi Journal of Chemistry Volume 2018, Article ID 8673476, 12 pages https://doi.org/10.1155/2018/8673476 Research Article A Chemical Synthesis of Benzo[a]pyrene Hapten and Its Application in a Streptavidin-Horseradish Peroxidase-Based Enzyme-Linked Immunological Analysis Zhen Ma and Huisheng Zhuang School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China Correspondence should be addressed to Huisheng Zhuang; [email protected] Received 15 August 2017; Revised 30 November 2017; Accepted 5 December 2017; Published 4 February 2018 Academic Editor: Jorge F. Fernandez-Sanchez Copyright © 2018 Zhen Ma and Huisheng Zhuang. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A specifc BaP hapten was synthesized and BaP antigens (immunogen, coating antigen) were prepared using diverse methods. Based on these works, a streptavidin-horseradish peroxidase-based enzyme-linked immunological analysis was developed and frstly used to detect BaP.Several physiochemical factors that may infuence the assay performance were optimized in the assay. Under optimal −1 −1 conditions, a detection limit of 0.0094 ng mL was obtained and good linearity was achieved within a range of 0.03–35.40 ng mL . Satisfactory recovery of spiked samples (91.12–109.23%) was obtained and the coefcient of variation was acceptable. Finally, the detection results of BaP in environmental and food samples were consistent with those obtained using high-performance liquid chromatography. Te proposed immunoassay is reliable and has great potential for detecting trace amounts of BaP. 1. Introduction BaPisagreatpotentialhazardtohumanhealth.Itis a type of carcinogenic compound, which can cause lung, Polycyclic aromatic hydrocarbons (PAHs) are persistent gastric, bladder, and gastrointestinal cancers [12, 13]. Recent organic pollutants that contain 2–8 aromatic rings in linear, studies of BaP have also demonstrated its involvement in the angular, or cluster arrangements [1]. Due to their toxicity development of cardiovascular diseases [14]. BaP is muta- andcarcinogenicity,PAHshaveattractedtheattentionof genic and teratogenic; it can even pass through the placenta environmental chemists and regulatory agencies for several to endanger the embryogenesis [15]. Dietary intake of BaP decades [2, 3]. Among the PAHs, benzo[a]pyrene (BaP) has is a major source of human exposure [16]. Related studies been widely studied. In recent years, much attention has been have shown that people in the general population without devoted to the existence of BaP in the environment. BaP is substantial environmental and occupational exposure had considered the most dangerous of PAHs because of its chem- a higher exposure to BaP by food ingestion [17]. Probable ical and physical characteristics and ease of accumulation sources of BaP in food are environmental contamination in the food chain [4, 5]. BaP is usually found in exhaust from soil, water, and the thermal treatment used for the gas produced by the incomplete combustion of fuels, in all preparation of food, such as roasting. smokeresultingfromthecombustionoforganicmaterial Up to now, many diferent methods have been described and in charbroiled food [6–8]. BaP can enter into water for the determination of BaP residues. Te main meth- bodies mainly via atmospheric fallout, municipal/industrial ods are gas chromatography and mass spectrometry (GC- efuents, urban runof, and oil spillage [9, 10]; it is emitted to MS) and high-performance liquid chromatography (HPLC); the atmosphere from diferent sources, including industrial however, these chromatographic methods usually require processes, combustion of fossils and fuels, and irrational long periods of detection processes that are time-consuming application of pesticide [11]. and the follow-up maintenance is expensive. Compared 2 Journal of Chemistry Washing Yellow Substrate Blue BaP Streptavidin Horseradish peroxidase Coating antigen Bio-pAb-BaP Figure 1: Principle of the SA-ELISA method. BaP coating antigens which are covered on the surface of the tubes competed with BaP analytes to combine with the bio-pAb-BaP. Te horseradish peroxidase-conjugated streptavidin (SA-HRP) is fxed on the wall through the reaction with bio-pAb-BaP. As the increase of BaP concentration, less bio-pAb-BaP can be attached to the solid carrier. In this case, the amount of SA-HRP is less, and then quantitative analysis could be performed afer sufcient colour development. −1 −1 with chromatographic techniques, immunoassays are cost- [10 mmol L ] and KCl [2.7 mmol L ], pH 7.40), PBST (PBS efective, with adequate sensitivity and high selectivity with 0.05% Tween 20, pH 7.40), and carbonate bufer (CBS: −1 −1 [18]. Terefore, immunochemical techniques have been Na2CO3 [15 mmol L ]andNaHCO3 [34.9 mmol L ], pH increasingly considered as complementary methods for 9.60)wereusedintheassay. residue analysis. Among these immunoassays, enzyme- linked immunosorbent assay (ELISA) is well suited for 2.2. Materials and Chemicals. Te BaP standard, other detecting trace amounts of pollutants. Some improved types of PAHs, chemicals for BaP hapten and antigen immunoassays were researched based on the conventional synthesis, 25% glutaraldehyde solution, polyethylene gly- ELISA, such as fuorescence-enzyme immunoassay (FL- col 20,000 (PEG 20,000), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were purchased from J&K ELISA) and chemiluminescence immunoassay (CL-ELISA) � � [19, 20]. Accompanying the improved immunoassays above, Chemical (Beijing, China). 3,3 ,5,5 -Tetramethyl benzidine the streptavidin-enzyme based ELISA can reduce nonspecifc (TMB), hydrogen peroxide (H2O2), keyhole limpet hemo- reactions with reagents due to the higher specifcity and cyanin (KLH), poly-L-Lysine (PLL), polyvinyl alcohol (PVA), afnity between streptavidin and biotin, and it has great skimmed milk powder, H2SO4,HCl,gelatin,Tween20, potential for detecting trace amounts of BaP. However, as Coomassie Brilliant Blue G250, and inorganic salts were we know, no information on detecting BaP by streptavidin- purchased from Sinopharm Chemical Reagent Co. Ltd. enzyme based ELISA has been reported. (Shanghai, China). Bovine serum albumin (BSA), ovalbumin In this research, a specifc BaP hapten was synthesized. (OVA), dialysis bags, biotinylated N-hydroxysuccinimide Diverse BaP antigens and biotinylated polyclonal BaP anti- ester (BNHS), and SA-HRP were all purchased from Sangon body (bio-pAb-BaP) were prepared. Based on these works, Co. Ltd. (Shanghai, China). BaP hapten was purifed through a streptavidin-horseradish peroxidase-based enzyme-linked column chromatography using silica gel (40 �maverage immunoassay was developed (SA-ELISA) and frstly used particle size) obtained from Shanghai Sanpont Co. Ltd. for the sensitive detection of BaP. Some environmental and (Shanghai, China). food samples were collected in Shanghai, China. Several physiochemical factors that may infuence the assay perfor- 2.3. Synthesis of BaP Hapten. BaP molecule does not contain mance were optimized, and the accuracy and reliability of the functional groups that can connect with proteins. Terefore, immunoassay were validated. Figure 1 shows the detection a specifc BaP hapten was synthesized (Figure 2). Ten, the process of SA-ELISA method. BaP antigens and bio-pAb-BaP were prepared. For the prepa- ration of BaP hapten, antigens, and pAb-BaP, we followed the 2. Materials and Methods methods of Ma and Zhuang 2017 [21]. BaP (200 mg), n-iodosuccinimid (NIS, 210 mg), and 2.1. Bufers and Solutions. Phosphate-bufer saline (PBS: acidic alumina (5 g) were dissolved in toluene (15 mL); −1 −1 NaCl [137 mmol L ],KH2PO4 [2 mmol L ], Na2HPO4 the mixture was allowed to react with stirring for 4 days Journal of Chemistry 3 NIS Al2O3 4-Pentene-2-OL LiCl Toluene Pa(OAr)2 C6H15N DMF I O Figure 2: Synthesis of BaP hapten. DCC NHS BSA O NaBH4 HN O BSA C CMO NH O (C4H9) N O HN O 3 OVA O C OH C5H9ClO2 O O HN O O NH OVA Figure 3: Synthesis of BaP immunogen and coating antigen. ∘ at 20 C. Te residual acidic alumina was washed with was allowed to react in absence of light. Afer a completed dichloromethane (25 mL × 4). Aferwards, the organic phase reaction, sodium borohydride (36 mg) dissolved in ddH2O was collected and extracted by saturated sodium sulphite, (300 �L) was added to the reaction mixture and allowed to saturated salt water, and double-distilled water (ddH2O) reactfor5hatambienttemperature.Afertheorganicphase successively. Afer being dried with MgSO4,themixture was evaporated to dryness, the crude product was washed was concentrated down to near dryness; then, the yellow with HCl solution (pH 2.0) and ddH2O successively. Afer powder (6-I-BaP, 225 mg, yield: 75.3%) was obtained by being dried, the yellow product BaP-COOH was obtained. recrystallization with dichloromethane and petroleum ether. BaP-COOH (0.155 g) was dissolved in DMF (1 mL). � Next, 6-I-BaP (135 mg), palladium acetate (5 mg), and N-Hydroxysuccinimide (NHS) and N, N -dicyclohexyl- LiCl (10 mg) were dissolved in DMF (10 mL). Ten, 4- carbodiimide (DCC) were dissolved in DMF (1 mL), respec- pentene-2-alcohol (35 �L) and triethylamine (100 �L) were tivelyandaddedtothesolutionabovesuccessively.Te ∘ added. Te mixture was allowed to react for 15 h at 120 C. mixturewasallowedtoreactwithstirringfor8hatroomtem- Afer a completed reaction, toluene