An enzyme-linked immunoassay method, kit and application for detecting polycyclic aromatic hydrocarbons

Abstract

The application provides an enzyme-linked immunoassay method, a kit and application for detecting polycyclic aromatic hydrocarbons, and belongs to the technical field of environmental pollutant detection. The kit comprises a recombinant aromatic hydrocarbon receptor protein and an enzyme marker, the amino acid sequence of the recombinant aromatic hydrocarbon receptor protein is shown as SEQ ID NO: 2, and the enzyme marker is a coupling product of 1-pyrene butyric acid and horseradish peroxidase. The application further provides a detection method, which comprises the following steps: coating the recombinant aromatic hydrocarbon receptor protein on an enzyme-labeled plate, adding a sample to be detected or a standard sample and the enzyme marker for a competitive reaction, measuring the absorbance after color development, establishing a four-parameter fitting standard curve with benz[a]pyrene as a standard sample, and calculating the benz[a]pyrene equivalent concentration. The method can simultaneously detect 16 kinds of polycyclic aromatic hydrocarbons, and can be used for rapid screening of polycyclic aromatic hydrocarbons in environmental water samples for non-diagnostic purposes.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollutant detection technology, specifically relating to an enzyme-linked immunosorbent assay (ELISA) method, kit, and application for detecting polycyclic aromatic hydrocarbons (PAHs). Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants widely found in the environment, primarily originating from the incomplete combustion of organic matter, such as fossil fuel combustion, industrial emissions, and vehicle exhaust. These compounds exhibit significant carcinogenic, teratogenic, mutagenic, and bioaccumulative properties.

[0003] Currently, the detection of polycyclic aromatic hydrocarbons (PAHs) mainly relies on instrumental analysis methods such as gas chromatography-mass spectrometry (GC-MS). GC-MS combines the high separation efficiency of gas chromatography with the high resolution of mass spectrometry, enabling qualitative and quantitative analysis of individual PAHs. However, this method has significant limitations in practical applications. Conventional GC-MS methods have limited sensitivity and selectivity, especially for high molecular weight PAHs that are molecularly stable and difficult to break down, resulting in poor detection performance. Xie Danping et al. (2022) showed that for complex waste gas samples with significant matrix interference effects, conventional methods resulted in poor chromatographic peak responses for high-quality PAH monomers (such as dibenzo[a,h]anthracene and benzo[ghi]perylene), and the recovery rate of the extracted internal standard was less than 5% (Xie Danping et al. Formic acid acidification combined with gas chromatography-triple quadrupole tandem mass spectrometry for the detection of PAHs in complex waste gas [J]. Environmental Chemistry, 2022, 41(8): 2655-2661). Furthermore, GC-MS analysis typically requires complex sample pretreatment (such as extraction, purification, and concentration), involves expensive equipment, is time-consuming, and has low throughput, making it difficult to meet the needs of rapid screening of large-scale environmental samples. Antibody-based enzyme-linked immunosorbent assays (ELISA) are simple to operate and relatively inexpensive, and have seen some application in the detection of polycyclic aromatic hydrocarbons (PAHs) in recent years. However, traditional antibody preparation requires animal immunization and hybridoma screening, which is time-consuming and costly. Moreover, single antibodies usually only recognize a few specific PAH structures, failing to cover the dozens of structurally diverse compounds in the PAH family, resulting in a narrow detection range and an inability to comprehensively evaluate the overall toxicity of environmental samples.

[0004] In recent years, aromatic hydrocarbon receptors have attracted researchers' attention as a natural sensor for polycyclic aromatic hydrocarbons (PAHs). The PAS-B domain of the aromatic hydrocarbon receptor has a plastic hydrophobic binding pocket, which can recognize and bind a variety of PAHs and their analogues with different structures. Existing studies have used aromatic hydrocarbon receptors to construct cell-based bioassays, such as the chemically activated luciferase expression method. This type of method can detect the combined effect of all aromatic hydrocarbon receptor ligands in a sample and has a certain broad spectrum (Pieterse B, et al. (2013). PAH-CALUX, an optimized bioassay for AhR-mediated hazard identification of polycyclic aromatic hydrocarbons (PAHs) as individual compounds and in complex mixtures. Environmental Science & Technology, 47(20), 11651-11659). However, the cell culture process is complex, the culture cycle is long, and the requirements for the operating environment are high. Moreover, the instability of the cell state may affect the reproducibility of the detection results. There are few reports on cell-free detection methods based on recombinant aromatic hydrocarbon receptors, and they have not yet undergone systematic condition optimization. The detection sensitivity and stability need to be improved. Therefore, developing a cell-free, simple, broad-spectrum, and low-cost method for detecting polycyclic aromatic hydrocarbons based on recombinant aromatic hydrocarbon receptors has significant application value. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an enzyme-linked immunosorbent assay (ELISA) method, kit and application for detecting polycyclic aromatic hydrocarbons (PAHs), so as to achieve broad-spectrum, sensitive, simple and low-cost PAH detection.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting polycyclic aromatic hydrocarbons (PAHs), comprising: The recombinant aryl hydrocarbon receptor protein, the amino acid sequence of which is shown in SEQ ID NO: 2; and an enzyme label, wherein the enzyme label is a conjugate of 1-pyrenebutyric acid and horseradish peroxidase; the concentration of the recombinant aryl hydrocarbon receptor protein is 0.5-1.0 mg / mL.

[0007] Preferably, the coupling ratio of 1-pyrenebutyric acid to horseradish peroxidase is (5-7):1.

[0008] More preferably, it further comprises: a coating buffer, wherein the coating buffer is 25 mM Tris-HCl, 10 mM MgCl2, pH 8.0; a reaction buffer, wherein the reaction buffer is 40 mM Tris-HCl, 5 mM MgCl2, pH 8.0; and a standard, wherein the standard is benzo[a]pyrene.

[0009] Preferably, the polycyclic aromatic hydrocarbon is selected from at least one of the following: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, α, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indo[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene.

[0010] This invention also provides an enzyme-linked immunosorbent assay (ELISA) method for detecting polycyclic aromatic hydrocarbons (PAHs), comprising the following steps: The recombinant aromatic hydrocarbon receptor protein with the amino acid sequence shown in SEQ ID NO: 2 was diluted with coating buffer and adsorbed into the wells of an ELISA plate. The plate was incubated at 4°C for 15-20 h for coating. After blocking, the test sample or standard, along with a conjugate of 1-pyrene butyrate and horseradish peroxidase, were added simultaneously. The plate was then subjected to a competitive reaction in reaction buffer for 50-80 min. After washing, TMB substrate solution was added for color development in the dark. The reaction was terminated with stop solution, and the absorbance at 450 nm was measured using an ELISA reader. A four-parameter fitting standard curve was established with the logarithm of the benzo[a]pyrene standard concentration as the abscissa and the inhibition rate as the ordinate. The equivalent concentration of benzo[a]pyrene in the test sample was calculated. The coating buffer is 25 mM Tris-HCl, 10 mM MgCl2, pH 8.0; the reaction buffer is 40 mM Tris-HCl, 5 mM MgCl2, pH 8.0; the coupling ratio of 1-pyrene butyric acid to horseradish peroxidase is (5-7):1.

[0011] Preferably, the coating concentration of the recombinant aryl hydrocarbon receptor protein after dilution with coating buffer is 100-150 μg / mL.

[0012] Preferably, the working concentration of the 1-pyrene butyric acid-horseradish peroxidase conjugate is 0.5-2.0 mg / mL.

[0013] Preferably, the time for light-shielded color development is 8-12 minutes.

[0014] Preferably, the polycyclic aromatic hydrocarbon is selected from at least one of the following: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, α, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indo[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene.

[0015] The present invention also provides an application of the kit or the method described herein in the detection of polycyclic aromatic hydrocarbons in environmental water samples for non-diagnostic purposes.

[0016] Compared with the prior art, the present invention has the following advantages: This invention utilizes the recombinant aromatic hydrocarbon receptor (AhR) protein as a recognition element in an enzyme-linked immunosorbent assay (ELISA) system, successfully establishing a cell-free, broad-spectrum, and sensitive method for detecting polycyclic aromatic hydrocarbons (PAHs). Compared to traditional ELISA methods that rely on specific antibodies, this invention leverages the plastic hydrophobic binding pocket of the PAS-B domain of the AhR protein, enabling simultaneous recognition of 16 PAHs preferentially controlled by the US EPA, including naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, α, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indo[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene. This overcomes the limitation of single antibodies detecting only a few specific PAHs, achieving truly broad-spectrum detection. The half-maximal inhibitory concentration (IC50) for benzo[a]pyrene is [not specified in the original text]. 50 The concentration was as low as 3.90 ng / mL, with a detection limit of 1.97 ng / mL, and the IC50 of most PAHs was [missing value]. 50 The value is below 50 ng / mL, which meets the requirements for trace detection in environmental samples.

[0017] This invention utilizes recombinant AhR protein expression in *E. coli*, resulting in significantly lower preparation costs compared to monoclonal antibodies. It also exhibits good batch-to-batch consistency, requires no animal immunization, and is environmentally friendly. The entire detection process is based on a 96-well plate competitive ELISA platform, eliminating the need for cell culture, simplifying operation, and shortening the detection cycle, making it suitable for high-throughput screening of large-scale environmental samples. Detection results are expressed as benzo[a]pyrene equivalent concentrations (BaP-TEQ), comprehensively reflecting the total effect of all AhR ligands in the sample, thus overcoming the limitation of gas chromatography-mass spectrometry (GC-MS) which can only quantify pre-defined target analytes.

[0018] The method of this invention was used to detect actual environmental water samples, achieving spiked recoveries of 71.2%-108.8% and coefficients of variation of 2.1%-16.8%, showing a significant positive correlation with GC-MS results. Through the detection of actual samples such as Yellow River water samples, this method can rapidly classify samples according to their pollution levels into clean, light, moderate, and heavy grades, providing a targeted basis for subsequent precise analysis. This invention can serve as a high-throughput primary screening tool for polycyclic aromatic hydrocarbon (PAH) pollution, complementing GC-MS, and is particularly suitable for rapid screening of large-scale environmental samples in watershed surveys, emergency monitoring, and other similar scenarios. Attached Figure Description

[0019] Figure 1 The construction map of the recombinant expression plasmid pET-28b(+)-AhR.

[0020] Figure 2 SDS-PAGE analysis of the purified and refolded sample of recombinant aryl hydrocarbon receptor protein; M is the protein marker; 1 is the flow buffer; 2-7 are elution buffers with different imidazole concentrations, namely 20mM, 50mM, 250mM, 250mM, 250mM, and 500mM; 8-9 are purified samples after dialysis refolding with 250mM imidazole elution buffer.

[0021] Figure 3 This is the UV spectrum of the horseradish peroxidase-1-pyrenobutyric acid-horseradish peroxidase conjugate.

[0022] Figure 4 The infrared spectrum of the horseradish peroxidase-1-pyrenobutyric acid-horseradish peroxidase conjugate is shown.

[0023] Figure 5 This is the MALDI-TOF mass spectrum of the horseradish peroxidase-1-pyrenebutyrate-horseradish peroxidase conjugate.

[0024] Figure 6 This is a graph showing the effect of different combinations of Tris-HCl and MgCl2 concentrations in the coating buffer on the maximum absorbance value.

[0025] Figure 7 This is a graph showing the effect of different MgCl2 concentrations in the reaction buffer on the competitive inhibition effect.

[0026] Figure 8 This is a graph showing the effect of different Tris-HCl concentrations in the reaction buffer on the competitive inhibition effect.

[0027] Figure 9 The graph shows the effect of different competitive reaction times on the maximum absorbance and half-maximum inhibitory concentration.

[0028] Figure 10 The figure shows the standard curves of benzo[a]pyrene in different media; A in the figure is the standard curve in the reaction buffer solution, and B is the standard curve in the environmental sample blank matrix solution.

[0029] Figure 11 The figures show the correlation analysis between the method of this invention and gas chromatography-mass spectrometry (GC-MS). Figure A shows the correlation analysis between the total concentration of 16 PAHs measured by the method of this invention and GC-MS; Figure B shows the correlation analysis between the equivalent concentration of benzo[a]pyrene measured by the method of this invention and GC-MS. Detailed Implementation

[0030] This invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting polycyclic aromatic hydrocarbons (PAHs), comprising: The recombinant aryl hydrocarbon receptor protein, the amino acid sequence of which is shown in SEQ ID NO:2; and an enzyme marker, the enzyme marker being a conjugate of 1-pyrenebutyric acid and horseradish peroxidase.

[0031] In a specific embodiment of the present invention, the core components of the kit include a recombinant aryl hydrocarbon receptor protein and an enzyme marker. The recombinant aryl hydrocarbon receptor protein comprises an N-terminal His-tag and a thrombin cleavage site of pET-28b, and a human aryl hydrocarbon receptor (amino acids 22 to 474), with the following amino acid sequence: MGSSHHHHHHSSGLVPRGSHMVKPIPAEGIKSNPSKRHRDRLNTELDRLASLLPFPQDVINKLDKLSVLRLSVSYLRAKSFFDVALKSSPTERNGGQDNCRAANFREGLNLQEGEFLLQALNGFVLVVTTDALVFYASSTIQDYLGFQQSDVIHQSVYELIHTEDRAEFQRQLHWALNPSQCTESGQGIEEATGLPQTVVCYNPD QIPPENSPLMERCFICRLRCLLDNSSGFLAMNFQGKLKYLHGQKKKGKDGSILPPQLALFAIATPLQPPSILEIRTKNFIFRTKHKLDFTPIGCDAKGRIVLGYTEAELCTRGSGYQFIHAADMLYCAESHIRMIKTGESGMIVFRLLTKNNRWTWVQSNARLLYKNGRPDYIIVTQRPLTDEEGTEHLRKRNTKLPFMFTTGEAVLYEATNPFPAIMDPLPLRTKNGTSGKDSATTSTLSKDSLNPSSLLAAMMQQDESIYLYPASST (SEQ ID NO: 2). This recombinant protein was prepared using a prokaryotic expression system: the nucleotide sequence encoding the recombinant aryl hydrocarbon receptor protein is shown in SEQ ID NO: 1. This gene was synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into the pET-28b(+) vector. The complete nucleotide sequence of the resulting recombinant expression plasmid is shown in SEQ ID NO: 3. The above plasmid was transformed into Escherichia coli BL21(DE3), induced with 0.5 mM IPTG at 10 °C for 24 h, purified by nickel affinity chromatography, and renatured by dialysis with PBS. The final protein concentration was adjusted to 0.5-1.0 mg / mL, stored in PBS buffer containing 20% ​​glycerol, and kept at -20 °C for later use.The enzyme marker in the kit is a conjugate of 1-pyrenebutyric acid and horseradish peroxidase (PBA-HRP), which is prepared by an activated ester method: 1-pyrenebutyric acid is activated with dicyclohexylcarbodiimide and N-hydroxysuccinimide in DMF at room temperature for 2 h, and then reacted with horseradish peroxidase in carbonate buffer (pH 9.6) at room temperature for 4 h. After purification by dialysis, it is lyophilized and stored or prepared into a 1.0 mg / mL solution and stored at -20°C in the dark. The mass spectrometry identification shows a conjugate ratio of 5:1 to 7:1, preferably 6:1. For ease of detection, the kit preferably also includes coating buffer, reaction buffer, and standards. The coating buffer is 25 mM Tris-HCl, 10 mM MgCl2, pH 8.0; the reaction buffer is 40 mM Tris-HCl, 5 mM MgCl2, pH 8.0; the standard is benzo[a]pyrene, prepared in methanol or reaction buffer to a series of concentrations, such as 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL. In addition, the kit may also include blocking buffer (PBS with 2% w / v skim milk powder), washing buffer (PBS with 0.05% Tween-20), TMB chromogenic solution (10 mg TMB dissolved in 4 mL DMSO, mixed with substrate buffer and 30% H2O2 before use), and stop solution (2 MH2SO4). All the above components are aliquoted and stored according to the standard ELISA kit format.

[0032] This invention also provides an enzyme-linked immunosorbent assay (ELISA) method for detecting polycyclic aromatic hydrocarbons (PAHs), comprising the following steps: The recombinant aromatic hydrocarbon receptor protein with the amino acid sequence shown in SEQ ID NO:2 was diluted with coating buffer and adsorbed into the wells of an ELISA plate. The plate was incubated at 4°C for 15-20 h for coating. After blocking, the test sample or standard, along with a conjugate of 1-pyrene butyrate and horseradish peroxidase, were added and reacted competitively in reaction buffer for 50-80 min. After washing, TMB substrate solution was added for color development in the dark, and the reaction was terminated with stop solution. The absorbance at 450 nm was measured using an ELISA reader. A four-parameter fitting standard curve was established with the logarithm of the benzo[a]pyrene standard concentration as the abscissa and the inhibition rate as the ordinate. The equivalent concentration of benzo[a]pyrene in the test sample was calculated. The coating buffer is 25 mM Tris-HCl, 10 mM MgCl2, pH 8.0; the reaction buffer is 40 mM Tris-HCl, 5 mM MgCl2, pH 8.0; the coupling ratio of 1-pyrene butyric acid to horseradish peroxidase is (5-7):1.

[0033] In a specific embodiment of the present invention, the recombinant aromatic hydrocarbon receptor protein with the amino acid sequence shown in SEQ ID NO: 2 is first diluted to a suitable concentration with a coating buffer consisting of 25 mM Tris-HCl and 10 mM MgCl2 (pH 8.0). The preferred concentration of the diluted protein is 100-150 μg / mL, more preferably 150 μg / mL. The diluted protein solution is added to a 96-well microplate, 100 μL per well, and incubated at 4°C for 15-20 h, preferably 16 h, to allow the recombinant aromatic hydrocarbon receptor protein to be physically adsorbed and immobilized at the bottom of the wells. After incubation, the liquid in the wells is discarded, and each well is washed once with 250 μL of PBST washing buffer (PBS containing 0.05% Tween-20), and then blotted dry. Subsequently, blocking is performed: 200 μL of blocking buffer (PBS containing 2% w / v skim milk powder) is added to each well, and the plate is incubated at 37°C for 1 h to block the non-specific binding sites. After incubation, the plate is blotted dry. After sealing, proceed to the competitive reaction step: Add 50 μL of the sample to be tested (or a series of polycyclic aromatic hydrocarbon standards) and 50 μL of enzyme-labeled working solution to each well. The enzyme-labeled solution is a conjugate of 1-pyrene butyrate and horseradish peroxidase (PBA-HRP), with a working concentration preferably 0.5-2.0 mg / mL, more preferably 1.0 mg / mL, and a coupling ratio of 5:1 to 7:1, preferably 6:1. The above reaction is carried out in a reaction buffer, preferably 40 mM Tris-HCl and 5 mM MgCl2 (pH 8.0). Incubate the plate at 37°C for competitive reaction for 50-80 min, preferably 60 min. After the competitive reaction, discard the liquid in the wells, add 250 μL of PBST washing buffer to each well, wash three times, shaking for 30 s each time, and pat dry. Then proceed with color development: Add 100 μL of freshly prepared TMB substrate solution to each well (this substrate solution is prepared by dissolving 10 mg TMB in 4 mL DMSO, and should be mixed with substrate buffer and 30% H2O2 before use), and incubate at 37°C in the dark for 8-12 min, preferably 10 min. After color development, add 50 μL of stop solution (2 M H2SO4) to each well, gently vortex to mix, and immediately measure the absorbance (OD) at 450 nm using a microplate reader. 450 In the calculation, the concentration of benzo[a]pyrene standard (BaP) was used as the abscissa (logarithm), and the inhibition rate (B / B0) was used as the ordinate. A standard curve was established using a four-parameter fitting equation. Here, B represents the relative value of the absorbance of the sample or standard well minus the absorbance of the blank well, and B0 represents the relative value of the absorbance of the zero standard well minus the absorbance of the blank well. The four-parameter fitting equation is Y = (AD) / [1 + (x / C)]. BThe formula is: Y = A + D, where Y is the B / B0 value, x is the polycyclic aromatic hydrocarbon (PAH) concentration, A is the B / B0 value at the lowest PAH concentration, D is the B / B0 value at the highest PAH concentration, B is the slope factor of the curve, and C is the half-maximal inhibitory concentration (IC50). 50 The OD of the sample to be tested was measured. 450 Substituting the values ​​into the standard curve, the benzo[a]pyrene equivalent concentration (BaP-TEQ) of polycyclic aromatic hydrocarbons in the sample can be calculated.

[0034] In specific embodiments of the present invention, the polycyclic aromatic hydrocarbons that the method can detect include, but are not limited to, the following sixteen pollutants: naphthalene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, Chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene. Experimental results show that, except for phenanthrene and naphthalene which have lower sensitivity, the IC50 values ​​for the other polycyclic aromatic hydrocarbons are relatively high. 50 The values ​​were all below 50 ng / mL, with benzo[a]pyrene having the lowest IC50 value. 50The concentrations were as follows: 3.90 ng / mL for indo[1,2,3-cd]pyrene, 4.60 ng / mL for benzo[k]fluoranthene, 5.38 ng / mL for benzo[ghi]perylene, 4.83 ng / mL for benzo[a]anthracene, 5.66 ng / mL for benzo[b]fluoranthene, 6.97 ng / mL for fluoranthene, 7.33 ng / mL for dibenzo[a,h]anthracene, 13.86 ng / mL for fluorene, 15.86 ng / mL for anthracene, 15.77 ng / mL for acenaphthene, 26.18 ng / mL for acenaphthene, and 49.47 ng / mL for acenaphthene. The cross-reactivity of the above compounds with respect to benzo[a]pyrene ranges from 7.88% to 84.78%, with indo[1,2,3-cd]pyrene, benzo[ghi]perylene, and benzo[k]fluoranthene all exhibiting cross-reactivity exceeding 70%, demonstrating excellent recognition capabilities. Therefore, those skilled in the art will understand that the method described in this invention can broadly identify and quantitatively detect any one or more combinations of at least the above sixteen polycyclic aromatic hydrocarbons, and is suitable for rapid screening of total polycyclic aromatic hydrocarbons in environmental samples and assessment of benzo[a]pyrene equivalent concentrations.

[0035] The present invention also provides an application of the kit or the method described herein in the detection of polycyclic aromatic hydrocarbons in environmental water samples for non-diagnostic purposes.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0038] Example 1: Expression and purification of recombinant aryl hydrocarbon receptor protein The pET28b expression vector containing the human aryl hydrocarbon receptor gene was synthesized by GenScript. Its nucleotide sequence encoding the recombinant aryl hydrocarbon receptor protein is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. The complete nucleotide sequence of this expression vector is shown in SEQ ID NO: 3, and the plasmid map is shown below. Figure 1 The plasmid was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. The plates were then incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium, and cultured with shaking at 37°C until OD (out of control) was reached. 600The concentration was 0.6-0.8, and isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM. Induction was performed at 10°C for 24 h. Bacterial cells were collected, resuspended in lysis buffer (containing 8 M urea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), sonicated, and centrifuged to collect the supernatant. Purification was performed using a nickel ion affinity chromatography column, eluting sequentially with eluents containing 20 mM, 50 mM, 250 mM, and 500 mM imidazole, collecting the 250 mM imidazole eluent. The purified protein was dialyzed in PBS buffer to remove urea and imidazole, yielding the renatured recombinant aryl hydrocarbon receptor protein. SDS-PAGE analysis results are shown below. Figure 2 As shown, the target protein band appears at approximately 52 kDa, with a purity greater than 90%.

[0039] Example 2 Synthesis and characterization of 1-pyrene-butyrate-horseradish peroxidase enzyme markers An enzyme-labeled compound was prepared by coupling 1-pyrene-1-butyric acid (PBA) with horseradish peroxidase (HRP) using an activated ester method. The specific steps are as follows: 0.2 mmol of PBA was accurately weighed and dissolved in 100 mL of N,N-dimethylformamide (DMF). Then, 0.3 mmol of dicyclohexylcarbodiimide (DCC) and 0.6 mmol of N-hydroxysuccinimide (NHS) were added sequentially to achieve final concentrations of 0.3 mM and 0.6 mM, respectively. This mixture was magnetically stirred at room temperature for 2 h to obtain the activated ester solution (solution a). Separately, 100 mg of HRP was weighed and dissolved in 20 mL of water or carbonate buffer (CBS, pH=9.6), and stirred at room temperature until completely dissolved to obtain the HRP solution (solution b). Mix 10 mL of solution a with solution b and react magnetically at room temperature for 4 h. After the reaction, transfer the reaction solution to a dialysis bag and dialyze thoroughly against PBS buffer at 4 °C for 24 h, changing the dialysate 3-4 times during this period to remove uncoupled small molecules. The dialyzed enzyme-labeled products were aliquoted and stored at -20 °C for later use. Some samples were freeze-dried and stored as powder. The UV spectrum of the PBA-HRP conjugate is similar in shape to that of PBA, but a new absorption shoulder appears at ~340 nm, and there is a slight red shift compared to free PBA. Figure 3 Infrared spectroscopy shows the amide I band (~1650 cm⁻¹). -1 ) and amide II band (~1540cm) -1 Enhancement confirms successful coupling. Figure 4MALDI-TOF-MS showed a coupling ratio of approximately 6:1 (HRP molecular weight increased by approximately 2 kDa). Figure 5 ).

[0040] Example 3: Optimization of the working concentration of aromatic hydrocarbon acceptor and enzyme label This invention uses benzo[a]pyrene as a standard and employs a checkerboard titration method to optimize the coating concentration of recombinant aromatic hydrocarbon receptor protein and the optimal working concentration of PBA-HRP enzyme marker.

[0041] The recombinant aryl hydrocarbon receptor protein was diluted with coating buffer (20mM Tris-HCl, 30mM MgCl2, pH 8.0) to four concentration gradients of 50, 100, 150, and 200 μg / mL to coat the microplates. At the same time, the PBA-HRP enzyme marker was diluted to four concentration gradients of 0.05, 0.25, 0.5, and 1.0 mg / mL for a square matrix experiment, and the maximum absorbance value (A_max) at each concentration combination was measured.

[0042] The results are shown in Table 1. At the same aromatic receptor coating concentration, the A_max value increased with increasing enzyme label concentration; at the same enzyme label concentration, the A_max value generally increased with increasing aromatic receptor coating concentration. When the aromatic receptor concentration increased to 200 μg / mL, the A_max value exceeded the upper limit of the microplate reader (>4.0), and no valid reading could be obtained. Excessively high aromatic receptor coating concentrations combined with high enzyme label concentrations resulted in an overly strong colorimetric signal, exceeding the instrument's linear detection range and hindering accurate quantification. Considering both the appropriate intensity of the detection signal and the linear response range of the subsequent inhibition curve, the optimal working conditions were selected: an A_max value within the instrument's optimal detection range (typically 1.0-2.5) and a good concentration gradient response relationship, namely, an aromatic receptor coating concentration of 150 μg / mL and an enzyme label concentration of 1.0 mg / mL.

[0043] Table 1. Optimization of AhR and PBA-HRP working concentrations using the checkerboard method.

[0044] Note: A / A_max is the ratio of the absorbance value of benzo[a]pyrene at a concentration of 50 ng / mL to the absorbance value of the zero standard well.

[0045] Example 4: Optimization of optimal coating conditions Based on the optimal working concentration determined in Example 3, this invention optimizes the composition and coating conditions of the coating buffer.

[0046] (1) Optimization of coating buffer components The effects of different concentration combinations of Tris-HCl (0, 1, 10, 25, 50, 100 mM) and MgCl2 (0, 1, 10, 25, 50, 100 mM) on the A_max of the detection system were investigated. The results showed that the recombinant aryl hydrocarbon receptor protein was easily denatured and inactivated under strongly alkaline conditions (pH 9.6), rendering it unusable for subsequent detection. Therefore, a weakly alkaline Tris-HCl buffer (pH 8.0) was chosen as the coating buffer. At the same Tris-HCl concentration, the A_max value initially increased and then decreased with increasing MgCl2 concentration. Higher A_max values ​​were obtained for all Tris-HCl concentration groups at a MgCl2 concentration of 10 mM. At the same MgCl2 concentration, a higher A_max value was observed at a Tris-HCl concentration of 25 mM; excessively low or high Tris-HCl concentrations led to a decrease in signal strength. Therefore, the protein immobilization efficiency was highest under the conditions of 25mM Tris-HCl and 10mM MgCl2 (pH 8.0), which was determined to be the optimal ion concentration for the coating buffer.

[0047] (2) Optimization of coating temperature and time Two coating conditions were set up: incubation at 4℃ for 16 h and incubation at 37℃ for 4 h. The effects of different coating methods on the detection system's A_max, A_min (minimum absorbance value), and IC were compared. 50 The impact. Results as follows: Figure 6 The results show that the A_max value is 3.44, the A_min value is 1.63, and the IC value is [missing value] for coating at 4℃ for 16 hours. 50 The value is 3.20; while the A_max value for coating at 37℃ for 2 hours is 2.77, the A_min value is 1.87, and the IC value is... 50 The value was 2.71. The A_max value measured after coating at 4℃ for 16 hours was higher and the sensitivity was also higher (IC50). 50 (The value is lower), so the optimal coating condition was determined to be incubation at 4°C for 16 hours.

[0048] Example 5: Optimization of reaction buffer components This invention systematically optimizes the concentrations of MgCl2 and Tris-HCl based on fixed initial conditions of the reaction buffer.

[0049] (1) Optimization of MgCl2 concentration The Tris-HCl concentration in the reaction buffer was fixed at 20 mM. MgCl2 concentration gradients of 5, 10, 20, 30, 40, 50, and 100 mM were set, with 50 ng / mL benzo[a]pyrene as the inhibition concentration. The changes in A_max and A / A_max of the detection system under different MgCl2 concentrations were investigated. The results are as follows: Figure 7The results showed that when the MgCl2 concentration was 5 mM, the A / A_max value was the minimum at 0.5963 (range 0.5963-0.8716), indicating the optimal competitive inhibition effect at this concentration. As the MgCl2 concentration increased, the A / A_max value first increased, then decreased, and then increased again, resulting in a decrease in the overall inhibitory effect. Therefore, the optimal concentration of MgCl2 in the reaction buffer was determined to be 5 mM.

[0050] (2) Optimization of Tris-HCl concentration Based on the determination of the optimal MgCl2 concentration (5 mM), the Tris-HCl concentration was further optimized. The Tris-HCl concentration was set to 5, 10, 20, 30, 40, 50, and 100 mM, with 50 ng / mL benzo[a]pyrene as the inhibitory concentration, and the A / A_max value was measured under each condition. The results are as follows: Figure 8 The results showed that the minimum A / A_max value was 0.5091 (range 0.5091-0.7706) when the Tris-HCl concentration was 40 mM, indicating that the competitive inhibition effect was optimal at this concentration. Both excessively low and excessively high Tris-HCl concentrations led to an increase in the A / A_max value and a weakening of the inhibitory effect. Too low a Tris-HCl concentration might fail to stably maintain the pH of the reaction system, affecting the hydrogen bonds and π-π interactions between the aromatic hydrocarbon acceptor and polycyclic aromatic hydrocarbons; while too high a concentration might increase ionic strength and osmotic pressure, disrupting the protein surface hydration layer and specific binding interface, resulting in a decrease in competitive inhibition. Therefore, the optimal composition of the reaction buffer was determined to be 40 mM Tris-HCl, 5 mM MgCl2, and pH 8.0.

[0051] Example 6: Optimization of Competitive Response Time Based on the optimized conditions determined in Examples 3-5, this invention examines the impact of competitive response time on detection performance.

[0052] The competitive reaction times were set to 10, 20, 30, 60, and 90 min, and A_max and IC50 were measured at each time point. 50 Value. In terms of A_max / IC 50 The ratio serves as a comprehensive evaluation index; a larger ratio indicates that the system has higher sensitivity while ensuring signal quality.

[0053] The results are as follows Figure 9 The results show that as the competitive reaction time increases from 10 min to 90 min, the A_max value continues to increase, but the rate of increase gradually decreases; IC 50 The IC10 showed a trend of first increasing and then decreasing, with values ​​at 10 min and 20 min. 50The value was relatively high, decreased significantly at 30 minutes, reached its lowest value at 60 minutes, and thereafter, increasing the time had no significant effect on the A_max value. The A_max / IC ratio at each time point was calculated. 50 The ratios were: 10 min (0.034), 20 min (0.097), 30 min (0.293), 60 min (0.839), and 90 min (0.845). The ratios for 60 min and 90 min were similar, but the IC50 ratio for 60 min was higher. 50 Lower reaction times and shorter response times improve detection efficiency. Therefore, 60 minutes was determined to be the optimal competitive reaction time.

[0054] Example 7: Establishment of the Standard Curve Based on the recombinant aromatic hydrocarbon receptor protein prepared in Example 1, the PBA-HRP enzyme marker prepared in Example 2, and the optimized conditions determined in Examples 3-6 (aromatic hydrocarbon receptor coating concentration 150 μg / mL, PBA-HRP concentration 1.0 mg / mL, coating buffer 25 mM Tris-HCl + 10 mM MgCl2 pH 8.0, reaction buffer 40 mM Tris-HCl + 5 mM MgCl2 pH 8.0, and competitive reaction time 60 min), standard curves for 16 polycyclic aromatic hydrocarbons were established.

[0055] A series of standard solutions with concentrations of 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL were prepared using benzo[a]pyrene (BaP) as the standard. The absorbance values ​​at each concentration point were measured under optimized conditions. A standard curve was established using a four-parameter fitting equation, with the logarithm of the benzo[a]pyrene standard concentration (x) as the x-axis and the inhibition rate (B / B0) as the y-axis. Where Y is the B / B0 value (B represents the relative value of the absorbance of the sample well or standard well minus the absorbance of the blank well, and B0 represents the relative value of the absorbance of the zero standard well minus the absorbance of the blank well), x is the polycyclic aromatic hydrocarbon (PAH) concentration, A is the B / B0 value at the lowest PAH concentration, D represents the B / B0 value at the highest PAH concentration, B is the slope factor of the curve, and C is the half-maximum inhibition concentration (IC50). 50 ).

[0056] Standard curve such as Figure 10 The IC50 of benzo[a]pyrene in the reaction buffer was shown to be... 50 The concentration was 3.90 ng / mL, and the detection limit was 1.97 ng / mL; in environmental water sample matrix solution, the IC50 was 3.90 ng / mL. 50 The concentration was 4.95 ng / mL, and the ratio of the two was 1.27, indicating that the method has good tolerance to water sample matrix.

[0057] Standard curves for 16 polycyclic aromatic hydrocarbons were established using the same method, and the IC50 of each compound was calculated. 50 and cross-reactivity (CR). IC50 corresponding to benzo[a]pyrene. 50 The value is 100%, and the cross-reactivity rate is calculated using the following formula: The results are shown in Table 2. Except for phenanthrene (Phe) and naphthalene (Nap), the IC50 values ​​of the other polycyclic aromatic hydrocarbons... 50 The values ​​were all below 50 ng / mL. Among them, indo[1,2,3-cd]pyrene (InP), benzo[k]fluoranthene (BkF), and benzo[ghi]perylene (BghiP) had high cross-reactivity rates (>70%), indicating that the method has broad-spectrum detection capability.

[0058] Table 2 Standard curve parameters and cross-reactivity of 16 PAHs in reaction buffer.

[0059] Note: In the table, A, D, C, and B represent the four-parameter fitting equation Y = (AD) / [1 + (x / C)]. B The parameters in ]+D; CR is the cross-reactivity rate, expressed as the IC50 of benzo[a]pyrene. 50 Calculated at 100%; LOD is the detection limit.

[0060] The standard curve parameters for benzo[a]pyrene in environmental water samples are A=1.0362, D=0.002, C=4.9535, B=3, and the standard equation is: Y=(1.0362-0.002) / [1+(x / 4.9535) 3 +0.002.

[0061] Example 8: Enzyme-linked immunosorbent assay kit for detecting polycyclic aromatic hydrocarbons This invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting polycyclic aromatic hydrocarbons (PAHs), comprising the following components: (1) Recombinant aryl hydrocarbon receptor protein: The amino acid sequence is shown in SEQ ID NO: 2. The concentration is 0.5-1.0 mg / mL. It is stored in PBS buffer containing 20% ​​glycerol at -20℃.

[0062] (2) Enzyme label: 1-pyrene butyric acid conjugate with horseradish peroxidase (PBA-HRP), with a conjugation ratio of 6:1, is a lyophilized powder or liquid (1.0 mg / mL), and should be stored at -20°C protected from light.

[0063] (3) Coating buffer: 25mM Tris-HCl, 10mM MgCl2, pH 8.0, stored at 4℃.

[0064] (4) Reaction buffer: 40mM Tris-HCl, 5mM MgCl2, pH 8.0, stored at 4℃.

[0065] (5) Blocking solution: PBS solution containing 2% (w / v) skim milk powder, stored at 4°C.

[0066] (6) Washing solution: PBST (PBS containing 0.05% Tween-20), stored at room temperature.

[0067] (7) Colorimetric solution: TMB substrate solution (10 mg TMB dissolved in 4 mL DMSO), mixed with substrate buffer (Na2HPO4-citric acid buffer) and 30% H2O2 in an appropriate ratio before use, and prepared fresh.

[0068] (8) Termination solution: 2M H2SO4, stored at room temperature.

[0069] (9) Standards: Benzo[a]pyrene (BaP) series of concentration standard solutions, with concentrations of 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 ng / mL, in methanol or reaction buffer, stored at -20℃ protected from light.

[0070] Preparation of key components (1) Preparation of recombinant aryl hydrocarbon receptor protein The human aryl hydrocarbon receptor was expressed and purified according to the method described in Example 1. The purity was identified as >90% by SDS-PAGE. The concentration was adjusted to 0.5-1.0 mg / mL and then aliquoted and stored.

[0071] (2) Preparation of PBA-HRP enzyme markers Prepared according to the activated ester method described in Example 2. Specifically: 0.2 mmol of 1-pyrenebutyric acid was dissolved in DMF, and 0.3 mmol of DCC and 0.6 mmol of NHS were added, and the mixture was activated at room temperature for 2 h; the activated solution was mixed with 100 mg of HRP (dissolved in 20 mL of CBS, pH 9.6), and reacted at room temperature for 4 h; after dialysis purification, the mixture was lyophilized and stored. MALDI-TOF-MS confirmed that the coupling ratio was approximately 6:1.

[0072] Example 9: Enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons based on recombinant aromatic hydrocarbon receptors. This invention provides a complete operational procedure for detecting polycyclic aromatic hydrocarbons using the kit and optimized conditions of Example 8, including steps of coating, blocking, competitive loading, color development, detection, and result calculation.

[0073] (1) Wrapped The recombinant aromatic hydrocarbon receptor protein prepared in Example 1 was diluted to 150 μg / mL with coating buffer (25 mM Tris-HCl, 10 mM MgCl2, pH 8.0), and 100 μL was added to each well of a 96-well microplate and incubated at 4 °C for 16 h.

[0074] (2) Washing Discard the liquid in the wells, add 250 μL of PBST washing buffer (PBS containing 0.05% Tween-20) to each well, wash once, and pat dry.

[0075] (3) Closed Add 200 μL of blocking buffer (PBS containing 2% w / v skim milk powder) to each well, incubate at 37°C for 1 h, and pat dry.

[0076] (4) Competition for sample addition Add 50 μL of the sample to be tested (or a series of polycyclic aromatic hydrocarbon standards) and 50 μL of the PBA-HRP working solution (1.0 mg / mL) prepared in Example 2 to each well in reaction buffer (40 mM Tris-HCl, 5 mM MgCl2, pH 8.0) and react competitively at 37 °C for 60 min.

[0077] (5) Washing Discard the reaction solution, add 250 μL of PBST washing buffer to each well, wash 3 times, shaking for 30 seconds each time, and pat dry.

[0078] (6) Color development Add 100 μL of freshly prepared TMB substrate solution (10 mg TMB dissolved in 4 mL DMSO, mixed with substrate buffer and 30% H2O2 before use) to each well and develop color at 37 °C in the dark for 10 min.

[0079] (7) Termination Add 50 μL of stop solution (2M H2SO4) to each well and gently shake to mix.

[0080] (8) Detection The absorbance value (OD) at a wavelength of 450 nm was measured using an ELISA reader. 450 ).

[0081] (9) Calculation of results A standard curve was established using the four-parameter fitting equation from Example 7, with the logarithm of the benzo[a]pyrene standard concentration (x) as the x-axis and the inhibition rate (B / B0) as the y-axis. The OD of the sample to be tested was then... 450 Substitute the values ​​into the standard curve to calculate the corresponding benzo[a]pyrene equivalent concentration (BaP-TEQ).

[0082] Example 10: Actual Environmental Water Sample Testing Eight river water samples were collected from the Yellow River, filtered through a 0.45 μm filter membrane, and concentrated using a C18 solid-phase extraction column. The samples were then analyzed using the enzyme-linked immunosorbent assay (ELISA) method described in Example 9, with absorbance values ​​measured at 450 nm. The benzo[a]pyrene equivalent concentration was calculated by substituting the values ​​into the matrix-matched standard curve established in Example 7.

[0083] The spiked recovery experiments were conducted at three spiking levels (10, 50, and 80 ng / mL), with each concentration repeated six times. The results are shown in Table 3. The recoveries of each compound ranged from 71.2% to 108.8%, and the coefficients of variation (CV) ranged from 2.1% to 16.8%, indicating that the method has good accuracy and precision.

[0084] Table 3. Recovery rates and coefficients of variation of 16 PAHs in the enzyme-linked immunosorbent assay (ELISA) of this invention (n=6)

[0085] The test results of 8 actual water samples are shown in Table 4. The calculated equivalent concentration of benzo[a]pyrene ranges from 6.15 to 193.16 ng / mL.

[0086] Table 4. Determination of BaP equivalent concentration in environmental samples using the enzyme-linked immunosorbent assay (ELISA) method of the present invention.

[0087] Example 11: Comparison of results between this method and gas chromatography-mass spectrometry This invention compares the detection results of the same batch of water samples in Example 9 with those obtained by gas chromatography-mass spectrometry (GC-MS).

[0088] Qualitative and quantitative analyses of 16 polycyclic aromatic hydrocarbons (PAHs) were performed on eight water samples using GC-MS. The theoretical equivalent concentration of benzo[a]pyrene was obtained by multiplying the measured concentration of each PAH by its cross-reactivity and summing the results. The results are shown in Table 5. The total PAH concentration in the samples ranged from 13.62 to 55.51 ng / mL, with low-ring PAHs (2-3 rings) accounting for 18.6% to 78.9% and high-ring PAHs (4-6 rings) accounting for 21.1% to 81.4%.

[0089] Table 5. Total concentration, composition, and BaP equivalent concentration of PAHs in environmental samples determined by GC-MS.

[0090] The correlation between the measured benzo[a]pyrene equivalent (BaP-TEQ) by enzyme-linked immunosorbent assay (ELISA) in Example 9 and the total concentration of 16 PAHs measured by GC-MS and the theoretical BaP-TEQ was analyzed. Figure 11 A and Figure 11 (B) The results showed a significant positive correlation between the two (p<0.05, p<0.01). This indicates that the method is in good agreement with GC-MS in assessing PAH pollution levels in aquatic environmental samples. The BaP-TEQ value measured by this method is higher than the theoretical value of GC-MS. This phenomenon stems from the fundamental difference in their detection principles: this method is based on the broad-spectrum recognition characteristics of aromatic hydrocarbon receptors and can detect the combined effect of all ligands (including polycyclic aromatic hydrocarbons and their structural analogs) that can bind to aromatic hydrocarbon receptors in the sample, while GC-MS can only quantify 16 pre-defined target polycyclic aromatic hydrocarbons. Therefore, the detection results of this method more comprehensively reflect the comprehensive biotoxicity of environmental samples. The quantitative range of the standard curve of this method is 1-100 ng / mL (based on BaP equivalent), and the quantitative accuracy is high within this range; for samples outside this range, accurate results can be obtained by dilution and re-measurement. Since aromatic hydrocarbon acceptors have different binding affinities for each PAH (binding free energy ranges from -6.1 to -9.9 kcal / mol), the inhibition signal produced by high-affinity PAHs (such as BaP) is much stronger than that of low-affinity PAHs (such as naphthalene) at the same mass concentration. Therefore, the BaP-TEQ value output by this method reflects the total active equivalent concentration, rather than the total mass concentration of PAHs, and should not be directly compared with the total amount of GC-MS. However, the two methods are highly consistent in judging the pollution trend.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for detecting polycyclic aromatic hydrocarbons (PAHs), characterized in that, Include: A recombinant aryl hydrocarbon receptor protein, the amino acid sequence of which is shown in SEQ ID NO: 2; and an enzyme marker, the enzyme marker being a conjugate of 1-pyrenebutyric acid and horseradish peroxidase; The polycyclic aromatic hydrocarbon is selected from at least one of the following: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, α, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indo[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene.

2. The reagent kit according to claim 1, characterized in that, The coupling ratio of 1-pyrene butyric acid to horseradish peroxidase is (5-7):1; the concentration of the recombinant aryl hydrocarbon receptor protein is 0.5-1.0 mg / mL.

3. The kit according to claim 1 or 2, characterized in that, It also includes: a coating buffer consisting of 25 mM Tris-HCl, 10 mM MgCl2, pH 8.0; a reaction buffer consisting of 40 mM Tris-HCl, 5 mM MgCl2, pH 8.0; and a standard consisting of benzo[a]pyrene.

4. An enzyme-linked immunosorbent assay (ELISA) method for detecting polycyclic aromatic hydrocarbons, characterized in that, Includes the following steps: The recombinant aryl hydrocarbon receptor protein with the amino acid sequence shown in SEQ ID NO: 2 was diluted with coating buffer and adsorbed onto the wells of an ELISA plate for coating. After blocking, the test sample or standard and the conjugate of 1-pyrene butyric acid and horseradish peroxidase were added simultaneously and reacted competitively in the reaction buffer. After washing, TMB substrate solution was added for color development in the dark, and the reaction was terminated by adding stop solution. The absorbance was measured using an ELISA reader. A four-parameter fitting standard curve was established with the logarithm of the concentration of benzo[a]pyrene standard as the abscissa and the inhibition rate as the ordinate. The equivalent concentration of benzo[a]pyrene in the test sample was calculated. The coating buffer is 25 mM Tris-HCl, 10 mM MgCl2, pH 8.0; the reaction buffer is 40 mM Tris-HCl, 5 mM MgCl2, pH 8.0; the coupling ratio of 1-pyrenebutyric acid to horseradish peroxidase is (5-7):

1.

5. The enzyme-linked immunosorbent assay (ELISA) method according to claim 4, characterized in that, The recombinant aryl hydrocarbon receptor protein was diluted with coating buffer to a coating concentration of 100-150 μg / mL.

6. The enzyme-linked immunosorbent assay (ELISA) method according to claim 4, characterized in that, The working concentration of the 1-pyrene butyric acid-horseradish peroxidase conjugate is 0.5-2.0 mg / mL.

7. The enzyme-linked immunosorbent assay (ELISA) method according to claim 4, characterized in that, The time for developing color in the dark is 8-12 minutes.

8. The enzyme-linked immunosorbent assay (ELISA) method according to claim 4, characterized in that, The polycyclic aromatic hydrocarbon is selected from at least one of the following: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, α, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indo[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene.

9. The use of the kit according to any one of claims 1-3 or the method according to any one of claims 4-8 in the detection of polycyclic aromatic hydrocarbons in environmental water samples for non-diagnostic purposes.

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