Method and system for detecting estrogen endocrine disrupter in environmental water sample

Abstract

The invention belongs to the technical field of estrogen endocrine disrupter detection, and discloses a method for detecting estrogen endocrine disrupter in an environmental water sample, which detects the binding capacity of the environmental water sample with estrogen receptors ERalpha and ERbeta through fluorescence polarization competitive binding on the basis of the characteristic that a fluorescent probe E2-F is specifically bound with the estrogen receptors ERalpha and ERbeta. Compared with an existing method, the method does not need cell culture, and is short in period and high in repeatability; a detection system belongs to a homogeneous system and does not need physical separation; the detection is rapid and convenient, and the detection can be realized only by incubating the probe / receptor protein / environmental sample at room temperature for about 5 minutes; expensive commercial kits do not need to be purchased, so that the detection cost is relatively low; a detection instrument is relatively simple, detection can be performed through a fluorophotometer or a microplate reader, and the instrument is simple to operate and suitable for high-throughput popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of estrogen endocrine disruptor detection, and in particular relates to a method and system for detecting estrogen endocrine disruptors in environmental water samples. Background Art

[0002] Classical estrogen nuclear receptors ERs include two subtypes of receptors, namely ERα and ERβ. In the absence of estrogen ligands, the receptors are isolated in a multiprotein inhibitory complex in the cytoplasm or nucleus of the target cell. When the ligands bind to ERs, they induce activating conformational changes, promote receptor dimerization and high-affinity binding to specific estrogen response elements (EREs) in the regulatory region of the target gene, thereby regulating the expression of related target genes. ERα and ERβ play an important regulatory role in a variety of physiological and pathological processes in humans and animals, such as reproductive development, lipid metabolism, immune regulation, and cardiovascular system balance. More and more studies have shown that a series of environmental pollutants can interact with ERs to exert endocrine disrupting effects. Therefore, how to screen the binding ability of environmental samples to receptors is of great significance for evaluating the health impact of environmental samples. Although relevant methods have been reported for the detection of environmental estrogen receptor endocrine disruptors, there are still some limitations: 1) For example, the current screening of complex environmental samples is mainly based on the reporter gene method, which is based on in vitro cell (yeast recombinant cells or human / animal cells) culture, transient transfection or stable transfection of ERs-mediated reporter genes, and then the extracted environmental samples are exposed to the test. The entire experimental cycle takes at least 2-3 days, and then the reporter gene detection kit is used for detection. However, this method takes a long time to detect, and due to the influence of factors such as cell generation, state, and transfection efficiency, the experimental repeatability is poor, and the current commercial reporter gene detection kit is expensive, so the detection cost is high; 2) Receptor protein affinity chromatography column separation combined with mass spectrometry analysis detection, this method is similar to traditional chemical analysis. Although it can specifically identify and capture pollutants that can bind to ERs receptors, it still requires expensive mass spectrometry instruments, which is not conducive to high-throughput promotion and use; 3) Detection methods based on fluorescence polarization / fluorescence resonance energy transfer recruited by co-activators or co-repressors, and commercial detection kits are already available. However, the kit is relatively expensive and cumbersome to operate. It can only screen out ligands with coactivator or co-repressor recruitment enhancement or inhibition effects. It cannot detect ligands that have binding ability but no effect on coactivator recruitment. These ligands are also likely to compete with other activators or inhibitors for binding to receptors, thereby affecting the function of receptors; 4) Competitive binding detection method based on radioactive labeled probes. This method involves radioactive elements, requires physical separation of bound and free probes, and requires high requirements for radioactive element detection instruments, which is not convenient for large-scale promotion and use. Therefore, it is of great significance to establish a simple, easy-to-use and economically feasible method for the detection of high-throughput environmental samples.

[0003] Through the above analysis, the problems and defects of the prior art are as follows:

[0004] (1) Currently, the screening of complex environmental samples is mainly based on the reporter gene method, which takes a long time to detect. Due to the influence of factors such as cell generation, state, and transfection efficiency, the experimental repeatability is poor. In addition, the current commercial reporter gene detection kits are expensive, so the detection cost is high;

[0005] (2) Receptor protein affinity chromatography column separation combined with mass spectrometry analysis and detection. This method is similar to traditional chemical analysis. Although it can specifically identify and capture pollutants that can bind to ERs receptors, it still requires expensive mass spectrometry equipment, which is not conducive to high-throughput promotion and use;

[0006] (3) Fluorescence polarization / fluorescence resonance energy transfer detection methods based on the recruitment of coactivators or co-repressors, and commercial detection kits are available. However, the kits are relatively expensive and cumbersome to operate, and can only screen out ligands that have coactivator or co-repressor recruitment enhancing or inhibiting effects. This method cannot detect ligands that have binding ability but have no effect on coactivator recruitment, and these ligands are also likely to compete with other activators or inhibitors for binding to receptors, thereby affecting the function of receptors;

[0007] (4) Competitive binding detection method based on radioactive labeled probes. This method involves radioactive elements and requires physical separation of bound and free probes. In addition, the radioactive element detection instrument has high requirements and is not suitable for large-scale promotion and use. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a method for detecting estrogen endocrine disruptors in environmental water samples.

[0009] The present invention is achieved by a method for detecting estrogen endocrine disruptors in environmental water samples comprising:

[0010] Step 1: Based on the specific binding characteristics of the fluorescent probe E2-F to estrogen receptors ERα and ERβ, the binding ability of environmental water samples to ERα and ERβ was detected by fluorescence polarization competitive binding;

[0011] Step 2: When the environmental water sample to be tested can bind to ERα / ERβ, the probe can be competed off the receptor, making the fluorescent probe in a free small molecule state. Due to its small size, it rotates faster in the liquid phase, and the measured fluorescence polarization degree is also lower;

[0012] Step 3: On the contrary, if the environmental sample cannot bind to the receptor and cannot affect the binding of the fluorescent probe E2-F to the protein to form a macromolecular complex, the probe rotates slowly and the degree of fluorescence polarization detected is also higher.

[0013] Further, the E2-F synthesis method is as follows:

[0014] Step 1, synthesis of E2-002;

[0015] Step 2, synthesis of E2-003;

[0016] Step 3, synthesis of E2-F.

[0017] Further, the synthesis of E2-002:

[0018] 32 mg of lead acetate and 70 mg of triphenylphosphine were dissolved in 16 mL of diethylamine and stirred for 10 min in an argon atmosphere; then 54 mg of cuprous iodide and 766 mg of tert-butyl-4-bromobenzylcarbamate were added to the mixture and stirred for 5 min; then 800 mg of ethinyl estradiol was added, heated to 60 ° C and stirred for 6 h; finally, the solvent was removed by rotary evaporation, and the resulting solid mixture was separated by a silica gel column with an eluent of petroleum ether / ethyl acetate: 2:1 to obtain a yellow product E2-002.

[0019] Further, the synthesis of E2-003; 0.9 g of E2-002 was dissolved in 25 mL of ethanol, and 5 mL of HCl (6 M) was added dropwise; the resulting mixture was stirred at room temperature overnight, and the pH value was adjusted to 11 with NaHCO3; the reaction product was extracted with 20 mL of ethyl acetate, and the obtained organic phase was evaporated to remove the solvent, and then separated by silica gel column, and the eluent was dichloromethane / methanol: 10:1; finally, 0.36 g of white product E2-003 was obtained;

[0020] Further, the synthesis of E2-F; 0.36g E2-003 and 0.67g 5-carboxyfluorescein succinimide ester were dissolved in DMSO, and the mixture was stirred and reacted at room temperature in the dark for 16h; then 10mL H2O was added to the reaction solution, and 20mL ethyl acetate was used to extract three times; the organic phases obtained from the three extractions were combined, dried and evaporated to remove the solvent, and the crude product was separated and purified by preparative high performance liquid chromatography to obtain 0.15g of a yellow solid product E2-F.

[0021] Furthermore, the 5-carboxyfluorescein succinimidyl ester: CAS No.: 92557-80-7, Sigma-Aldrich.

[0022] Another object of the present invention is to provide a system for detecting estrogen endocrine disruptors in environmental water samples, comprising:

[0023] The detection module is used to detect the binding ability of environmental water samples with ERα and ERβ through fluorescence polarization competition binding based on the characteristics of the fluorescent probe E2-F specifically binding to estrogen receptors ERα and ERβ;

[0024] The judgment module is used to compete the probe from the receptor when the environmental water sample to be tested can bind to ERα / ERβ, so that the fluorescent probe is in a free small molecule state. Due to its small molecule, it rotates faster in the liquid phase, and the measured fluorescence polarization degree is also lower; on the contrary, if the environmental sample cannot bind to the receptor, it cannot affect the fluorescent probe E2-F binding to the protein to form a macromolecular complex, the probe rotates slower, and the detected fluorescence polarization degree is also higher.

[0025] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0026] First, compared with existing methods, this method does not require cell culture, has a short cycle and high repeatability; the detection system is a homogeneous system and does not require physical separation; the detection is fast and convenient, and this method only requires the probe / receptor protein / environmental sample to be incubated at room temperature for about 5 minutes for detection; there is no need to purchase expensive commercial kits, and the detection cost is low; the detection instrument is relatively simple, and can be detected by a fluorescence photometer or an enzyme reader. The instrument is easy to operate and suitable for high-throughput promotion and use.

[0027] Second, the expected benefits and commercial value after the transformation of the technical solution of the present invention are: the potential threats posed by the presence of new pollutants in the water environment to the human body and the ecological environment have gradually become prominent, and have received widespread attention at home and abroad in recent years. Endocrine disruptors are a new type of pollutants that are currently receiving much attention. The development of a method that can quickly and high-throughput screen endocrine disruptors in environmental water samples has important social and commercial value. Referring to the current imported commercial kit (ThermoScientific, item number A15885), the kit detects the binding of small molecules to ERα receptors based on coactivator fluorescence resonance energy transfer. The official price of the kit is 27,582 yuan, which is expensive. This patent can further develop related detection kits, and it is expected that the cost can be reduced by more than 50%. There are currently no related products of the same type in China.

[0028] The technical solution of the present invention fills the technical gap in the industry at home and abroad: the present invention is the first to use the fluorescence polarization method to detect estrogen endocrine disrupting mixtures in environmental water samples based on the ERα / ERβ receptor site-specific fluorescent probe E2-F.

[0029] Whether the technical solution of the present invention overcomes technical bias: the current commercial kit is based on the detection method of fluorescence polarization / fluorescence resonance energy transfer of coactivator or co-repressor recruitment, which can only screen out ligands with coactivator or co-repressor recruitment enhancement or inhibition effect, and cannot detect ligands that have binding ability but have no effect on coactivator / repressor recruitment, and these ligands are also likely to compete with other activators or inhibitors to bind to receptors and thus affect the function of receptors. The present invention can theoretically detect as long as the sample contains compounds that can bind to receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a flow chart of a method for detecting estrogen endocrine disruptors in environmental water samples provided by an embodiment of the present invention.

[0031] Figure 2 It is a block diagram of the system structure for detecting estrogen endocrine disruptors in environmental water samples provided by an embodiment of the present invention.

[0032] Figure 3 This is a synthetic route map of the fluorescent probe E2-F provided in an embodiment of the present invention.

[0033] Figure 4 It is a binding curve diagram of the fluorescent probe E2-F provided in the embodiment of the present invention and ERα / ERβ.

[0034] Figure 5 It is a competition binding curve diagram between the positive control E2 and ERα / ERβ provided in the examples of the present invention.

[0035] Figure 6 It is a competitive binding curve diagram between the actual environmental water sample and ERα / ERβ provided in the embodiment of the present invention.

[0036] Figure 7 It is a competitive binding curve diagram between the pure water sample and ERα / ERβ provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] like Figure 1 As shown, a method for detecting estrogen endocrine disruptors in environmental water samples provided by an embodiment of the present invention comprises the following steps:

[0039] S101, based on the fluorescent probe E2-F (structure Figure 3The specific binding characteristics of estrogen receptor ERα (estrogen receptor α subtype) and ERβ (estrogen receptor β subtype) were shown, and the binding ability of environmental water samples to ERα and ERβ was detected by fluorescence polarization competition binding;

[0040] S102, when the environmental water sample to be tested can bind to ERα / ERβ, it can compete with the probe from the receptor, making the fluorescent probe in a free small molecule state. Due to its small size, it rotates faster in the liquid phase, and the measured fluorescence polarization degree is also lower;

[0041] S103, on the contrary, if the environmental sample cannot bind to the receptor and cannot affect the binding of the fluorescent probe E2-F with the protein to form a macromolecular complex, the probe rotates slowly and the degree of fluorescence polarization detected is also higher.

[0042] The detection method of the embodiment of the present invention is based on the specific binding ability of the fluorescent probe E2-F to the estrogen receptors ERα and ERβ, and realizes the detection of estrogen endocrine disruptors in environmental water samples through fluorescence polarization competitive binding experiments. First, the fluorescent probe E2-F can form a stable macromolecular complex with the ERα or ERβ receptor. When there is no interference in the outside world, the binding of the probe to the receptor will not be disturbed. At this time, the complex molecules are large, the rotation speed is slow, and the fluorescence polarization signal is strong.

[0043] When the environmental water sample to be tested contains estrogen interfering substances, these substances can compete with ERα or ERβ receptors to displace the fluorescent probe from the receptor. The displaced fluorescent probe is in a small molecule free state, and its rotation speed in the liquid phase is significantly accelerated, resulting in a significant decrease in the fluorescence polarization signal. By measuring the change in the degree of fluorescence polarization, it can be determined whether the interfering substances in the environmental water sample are bound to the receptor.

[0044] The principle of fluorescence polarization competitive binding relies on the fact that the rotation speed of a molecule is related to its size. Large molecular complexes rotate slowly and have a high degree of polarization, while small molecules rotate quickly and have a low degree of polarization. Therefore, by comparing the degree of fluorescence polarization before and after adding water samples, it is possible to accurately detect whether there are endocrine disruptors in the sample that can bind to ERα or ERβ.

[0045] This fluorescence polarization competition-binding method can not only quickly and accurately detect estrogen endocrine disruptors in water samples, but also evaluate the potential biological activities of these compounds in the environment with high sensitivity and specificity.

[0046] Figure 3 As shown, the E2-F synthesis method provided by the embodiment of the present invention is as follows:

[0047] E2-002 (Structure as Figure 3Synthesis of

[0048] E2-003 (Structure as Figure 3 Synthesis of

[0049] Synthesis of E2-F.

[0050] Synthesis of E2-002 provided in the embodiment of the present invention:

[0051] 32 mg of lead acetate and 70 mg of triphenylphosphine were dissolved in 16 mL of diethylamine and stirred for 10 min in an argon atmosphere; then 54 mg of cuprous iodide and 766 mg of tert-butyl-4-bromobenzylcarbamate were added to the mixture and stirred for 5 min; then 800 mg of ethinyl estradiol was added, heated to 60 ° C and stirred for 6 h; finally, the solvent was removed by rotary evaporation, and the resulting solid mixture was separated by a silica gel column with an eluent of petroleum ether / ethyl acetate: 2:1 to obtain a yellow product E2-002.

[0052] Synthesis of E2-003 provided in an embodiment of the present invention; 0.9 g of E2-002 was dissolved in 25 mL of ethanol, and 5 mL of HCl (6 M) was added dropwise; the resulting mixed solution was stirred at room temperature overnight, and the pH value was adjusted to 11 with NaHCO3; the reaction product was extracted with 20 mL of ethyl acetate, and the obtained extracted organic phase was evaporated to remove the solvent, and then separated by column chromatography with a silica gel column, and the eluent was dichloromethane / methanol: 10:1; finally, 0.36 g of a white product E2-003 was obtained;

[0053] The synthesis of E2-F provided in an embodiment of the present invention is as follows: 0.36 g of E2-003 and 0.67 g of 5-carboxyfluorescein succinimide ester are dissolved in DMSO, and the mixture is stirred and reacted at room temperature in the dark for 16 hours; 10 mL of H2O is then added to the reaction solution, and then extracted three times with 20 mL of ethyl acetate; the organic phases obtained by the three extractions are combined, dried, and the solvent is removed by rotary evaporation, and the crude product is separated and purified by preparative high performance liquid chromatography to obtain 0.15 g of a yellow solid product E2-F.

[0054] The 5-carboxyfluorescein succinimidyl ester provided in the embodiment of the present invention has a CAS number of 92557-80-7, Sigma-Aldrich.

[0055] like Figure 2 As shown, a system for detecting estrogen endocrine disruptors in environmental water samples provided by an embodiment of the present invention includes:

[0056] The detection module is used to detect the binding ability of environmental water samples with ERα and ERβ through fluorescence polarization competition binding based on the characteristics of the fluorescent probe E2-F specifically binding to estrogen receptors ERα and ERβ;

[0057] The judgment module is used to compete the probe from the receptor when the environmental water sample to be tested can bind to ERα / ERβ, so that the fluorescent probe is in a free small molecule state. Due to its small molecule, it rotates faster in the liquid phase, and the measured fluorescence polarization degree is also lower; on the contrary, if the environmental sample cannot bind to the receptor, it cannot affect the fluorescent probe E2-F binding to the protein to form a macromolecular complex, the probe rotates slower, and the detected fluorescence polarization degree is also higher.

[0058] In order to further determine the working concentrations of ERα-LBD (ERα ligand binding threshold recombinant protein) and ERβ-LBD (ERβ ligand binding threshold recombinant protein) for competition experiments, 5 μL ERα-LBD (0-1000 nM) and 5 μL E2-F (10 nM) were mixed in 10 μL Tris-HCl (buffer) to a total volume of 20 μL. Three parallel experiments were set up. After incubation at room temperature for 5 min, the fluorescence polarization value was measured using a multifunctional microplate reader (excitation wavelength of 485 nm, emission wavelength of 530 nm). Finally, the fluorescence polarization value was plotted against the ERα-LBD concentration to obtain the receptor protein binding curve. Figure 4 As shown in a, as the concentration of ERα-LBD increases, more fluorescent probe E2-F binds to it, and the fluorescence polarization value gradually increases. Compared with the fluorescence polarization value detected when the reaction solution only contains E2-F (ERα-LBD concentration is 0 nM), 100 nM ERα-LBD can increase the fluorescence polarization value by 2.2 times, and as the concentration continues to increase, the fluorescence polarization value tends to a stable state. Therefore, the working concentration (saturation concentration) of ERα-LBD is 100 nM.

[0059] Similarly, ERβ-LBD at concentrations ranging from 0 to 1000 nM was reacted with 10 nM E2-F, and other conditions were the same as those for ERα-LBD. Figure 4 As shown in Figure b, with the increase of ERβ-LBD concentration, the fluorescence polarization value of the probe gradually increased. When the ERβ-LBD concentration reached 200 nM, the fluorescence polarization value reached 2.8 times. As the ERβ-LBD concentration continued to increase, the increasing trend of the fluorescence polarization value slowed down. Therefore, 200 nM was used as the working concentration of ERβ-LBD.

[0060] The above clarifies the ability of the probe to bind to ERα / ERβ, and obtains the working concentration of the receptor protein. In order to further verify the feasibility of the fluorescence competition binding method, 17β-estradiol (E2) was used as a positive control to verify the effectiveness of the ERα-LBD and ERβ-LBD competition method. The specific experimental steps are as follows: For ERα-LBD, different concentrations of E2 (0nM, 0.001nM, 0.01nM, 0.1nM, 0.6nM and 1nM), ERα-LBD (100nM) and E2-F (10nM) were mixed, incubated at room temperature for 5 minutes, and the fluorescence polarization value was detected using an enzyme reader. Figure 5 As shown in a, with the increase of E2 concentration, the binding of probe E2-F to ERα-LBD was effectively inhibited, resulting in a gradual decrease in the fluorescence polarization value, and its IC50 was 0.06 nM.

[0061] For ERβ-LBD, E2 (0 nM, 0.01 nM, 0.1 nM, 0.3 nM, 0.6 nM and 1 nM), ERβ-LBD (200 nM) and E2-F (10 nM) were mixed and incubated at room temperature for 5 min, and then the fluorescence polarization value was measured using an ELISA reader. Figure 5 As shown in b, with the increase of E2 concentration, the binding of probe E2-F to ERβ-LBD was effectively inhibited, resulting in a gradual decrease in the fluorescence polarization value. Its IC 50 IC 50 The smaller the value, the stronger the ligand affinity. Therefore, the experiment shows that E2 has high affinity for ERα-LBD / ERβ-LBD and high detection sensitivity. At the same time, E2 has stronger binding ability with ERα-LBD than ERβ-LBD, which is consistent with the results obtained by radiolabeling method in the literature, further illustrating the feasibility of the method.

[0062] In order to further verify the feasibility of the method for detecting environmental samples, a water sample from the Liuyang River Basin was taken as a representative for detection. The sample pretreatment method is as follows: 1000mL of water sample was taken, the water sample was filtered with a 1.2μm glass fiber filter, and extracted with a solid phase extraction column (6mL, 500mg). The extraction column was pre-activated with 5mL of dichloromethane, 5mL of dichloromethane / methanol (1:1), 5mL of methanol, and 5mL of water. Then the water sample was passed through the solid phase extraction column at a flow rate of 5mL / min, vacuum-dried, and then eluted step by step with 5mL of dichloromethane, 5mL of dichloromethane / methanol (1:1), and 5mL of methanol. The eluents were combined and slowly dried with nitrogen, and then dissolved in 100μl of DMSO to prepare a stock solution for testing (10,000 times concentrated relative to the original water sample).

[0063] The competitive binding detection method of ERα water samples is as follows: the pre-treated water sample stock solution is diluted 100 times (the final system concentration corresponds to 1 times the actual water sample) and 10 times (the final system concentration corresponds to 10 times the actual water sample) with DMSO. ERα-LBD (100nM) and E2-F (10nM) are mixed with water samples of different concentrations (stock solution, diluted 10 times, diluted 100 times) at room temperature for 5 minutes, and the fluorescence polarization value is detected. The results are shown in Figure 6 As shown in a, the actual water sample can compete the probe E2-F from ERα, resulting in a decrease in the fluorescence polarization value, and as the concentration gradient of the water sample increases, the competitive ability becomes stronger and stronger. When the actual water sample is concentrated 100 times, the competition rate is as high as 80%, indicating that the water sample contains chemical substances that can directly bind to ERα.

[0064] The competitive binding detection method of ERβ water samples is as follows: ERβ-LBD (100 nM), E2-F (10 nM) and water samples of different concentrations (stock solution, 10-fold dilution, 100-fold dilution) are mixed at room temperature for 5 minutes, and then the fluorescence polarization value is detected. Figure 6 As shown in b, as the concentration of the water sample increases, the competitive ability with ERβ becomes weaker, all below 20%, indicating that the water sample contains fewer chemical substances that can directly bind to ERβ.

[0065] In the method, ERα may be the protein described in a1), a2), or a3):

[0066] a1) a protein whose amino acid sequence is sequence 1 in the sequence listing;

[0067] a2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in Sequence 1 of the sequence listing;

[0068] a3) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, obtained by substituting and / or deleting and / or adding one or more amino acid residues.

[0069] The amino acid sequence 1 is as follows:

[0070] SAG DMRAANLWPS PLMIKRSKKN SLALSLTADQ MVSALLDAEP

[0071] PILYSEYDPT

[0072] RPFSEASMMG LLTNLADREL VHMINWAKRV PGFVDLTLHD

[0073] QVHLLECAWL EILMIGLVWR

[0074] SMEHPGKLLF APNLLLDRNQ GKCVEGMVEI FDMLLATSSR

[0075] FRMMNLQGEE FVCLKSIILL

[0076] NSGVYTFLSS TLKSLEEKDH IHRVLDKITD TLIHLMAKAG

[0077] LTLQQQHQRL AQLLLILSHI

[0078] RHMSNKGMEH LYSMKCKNVV PLYDLLLEML DAHRLHAPTS

[0079] RGGASVEETD QSHLATAGST

[0080] SSHSLQKYYI TGEAEGFPAT V

[0081] In the method, ERβ may be the protein of b1) or b2) or b3) as follows:

[0082] b1) a protein whose amino acid sequence is Sequence 2 of the sequence listing;

[0083] b2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in Sequence 2 of the sequence listing;

[0084] b3) A protein having the same function as that obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing.

[0085] The amino acid sequence 2 is as follows:

[0086] KAK RSGGHAPRVR ELLLDALSPE QLVLTLLEAE PPHVLISRPS

[0087] APFTEASMMM

[0088] SLTKLADKEL VHMISWAKKIPGFVELSLFD QVRLLESCWM

[0089] EVLMMGLMWRSIDHPGKLIF

[0090] APDLVLDRDE GKCVEGILEI FDMLLATTSR FRELKLQHKE

[0091] YLCVKAMILL NSSMYPLVTA

[0092] TQDADSSRKL AHLLNAVTDA LVWVIAKSGI SSQQQSMRLA

[0093] NLLMLLSHVR HASNKGMEHL

[0094] LNMKCKNVVP VYDLLLEMLN AHVLRGCKSS ITGSECSPAEDSKSKEGSQN PQSQ.

[0095] The invention belongs to the field of environmental analysis, and specifically relates to a method for detecting the binding ability of environmental water samples with estrogen receptors ERα and ERβ and a method for synthesizing a special fluorescent probe thereof.

[0096] The method of the present invention determines the level of estrogen endocrine disruptors in environmental water samples by specifically binding to the fluorescence polarization value of the fluorescent probe. Figure 1 As shown in the figure, when the probe binds to the estrogen receptor ERα / ERβ, the free rotation of the probe is restricted, the fluorescence polarization value increases, the maximum relative fluorescence polarization change value is set to 1, and the relative polarization value of the free state probe is set to 0. When the positive control estradiol (E2) is added to the probe + ERα / ERβ system, the probe can be competed off the receptor ERα / ERβ and converted from a bound state to a free state, resulting in a decrease in the fluorescence polarization value. When the E2 concentration is high enough, all probes can be converted into a free state, and the relative polarization value is 0 at this time. The results of the positive control can be used to verify the feasibility of the method on the one hand, and on the other hand, the E2 standard curve can be used to quantify the content of estrogen endocrine disruptors in environmental samples, expressed as E2 equivalent concentrations. As shown in the figure, Figure 5 As shown, we obtained a standard curve for the competitive binding of E2 with ERα / ERβ, indicating the feasibility of this method.

[0097] Similarly, when environmental water samples contain pollutants that can bind to ERα / ERβ receptors, the probes can be competed off ERα / ERβ, resulting in a decrease in the fluorescence polarization value. The higher the pollutant concentration, the lower the fluorescence polarization value (e.g. Figure 1 We collected a water sample from Liuyang River and tested it. The results are as follows: Figure 6 As shown in a, when the river water sample is not concentrated (concentration factor is 1), the probe polarization value can be reduced by 20%, and the sample concentration can cause the polarization value to decrease further. When the sample is concentrated 100 times, the probe polarization value decreases by 80%, indicating that the river water sample contains a high concentration of environmental pollutants that can bind to ERα. For ERβ, as the concentration factor of the water sample increases, the decrease in the probe fluorescence polarization value is weaker (such as Figure 6b), indicating that the concentration of environmental pollutants that can bind to ERβ in the river water sample is relatively low.

[0098] On the contrary, if there are no pollutants in the water sample that can bind to ERα / ERβ receptors, it will not affect the binding of the probe to ERα / ERβ receptors, and the fluorescence polarization value will remain unchanged. We used Wahaha pure water and environmental water samples for the same pretreatment and then tested them. The results are as follows Figure 7 As shown, even after the pure water is concentrated 100 times, it still cannot affect the binding of the probe to the ERα / ERβ receptor, indicating that the pollutants in the pure water that can bind to the ERα / ERβ receptor are almost negligible.

[0099] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for detecting estrogen endocrine disruptors in environmental water samples, characterized in that: The following steps are involved: The fluorescent probe E2-F was used to bind to the estrogen receptor ERα and / or ERβ to form a macromolecular complex; The treated environmental water sample is mixed with the fluorescent probe E2-F and ERα and / or ERβ receptors. If the water sample contains estrogen endocrine disruptors, the substances will compete with the receptors to displace the fluorescent probe E2-F from the receptors and convert it into a small molecule free state. The fluorescence polarization value of the mixture is measured, and the change in the fluorescence polarization degree is used to determine whether the water sample contains endocrine disruptors that can bind to ERα and / or ERβ.

2. The method for detecting estrogen endocrine disruptors in environmental water samples according to claim 1, characterized in that: The E2-F synthesis method is as follows: Synthesis of E2-002; Synthesis of E2-003; Synthesis of E2-F.

3. The method for detecting estrogen endocrine disruptors in environmental water samples as claimed in claim 2, characterized in that: Synthesis of E2-002: 32 mg of lead acetate and 70 mg of triphenylphosphine were dissolved in 16 mL of diethylamine and stirred for 10 min in an argon atmosphere; then 54 mg of cuprous iodide and 766 mg of tert-butyl-4-bromobenzylcarbamate were added to the mixture and stirred for 5 min; then 800 mg of ethinyl estradiol was added, heated to 60 ° C and stirred for 6 h; finally, the solvent was removed by rotary evaporation, and the resulting solid mixture was separated by a silica gel column with an eluent of petroleum ether / ethyl acetate: 2:1 to obtain a yellow product E2-002.

4. The method for detecting estrogen endocrine disruptors in environmental water samples as claimed in claim 2, characterized in that: The synthesis of the E2-003: 0.9 g of E2-002 was dissolved in 25 mL of ethanol, and 5 mL of HCl (6 M) was added dropwise; the resulting mixture was stirred at room temperature overnight, and the pH value was adjusted to 11 with NaHCO3; the reaction product was extracted with 20 mL of ethyl acetate, and the obtained extracted organic phase was evaporated to remove the solvent, and then separated by silica gel column, and the eluent was dichloromethane / methanol: 10:1; finally, 0.36 g of white product E2-003 was obtained.

5. The method for detecting estrogen endocrine disruptors in environmental water samples as claimed in claim 2, characterized in that: The synthesis of the E2-F: 0.36g E2-003 and 0.67g 5-carboxyfluorescein succinimide ester were dissolved in DMSO, and the mixture was stirred at room temperature in the dark for 16h; 10mL H2O was then added to the reaction solution, and 20mL ethyl acetate was used to extract three times; the organic phases obtained from the three extractions were combined, dried, and the solvent was removed by rotary evaporation, and the crude product was separated and purified by preparative high performance liquid chromatography to obtain 0.15g of a yellow solid product E2-F.

6. The method for detecting estrogen endocrine disruptors in environmental water samples according to claim 5, characterized in that: The 5-carboxyfluorescein succinimidyl ester: CAS number: 92557-80-7, Sigma-Aldrich.

7. The method for detecting estrogen endocrine disruptors in environmental water samples according to claim 1, characterized in that: The following steps are involved: S101, based on the specific binding characteristics of the fluorescent probe E2-F (concentration of 10 nM) with the estrogen receptor ERα-LBD (estrogen receptor α subtype ligand binding domain, concentration of 100 nM) and ERβ-LBD (estrogen receptor β subtype ligand binding domain, concentration of 200 nM), the binding ability of environmental water samples with ERα-LBD and ERβ-LBD was detected by fluorescence polarization competitive binding; S102, the environmental water sample was pre-treated and mixed with the probe E2-F and ERα-LBD or ERβ-LBD, incubated at room temperature for 5 minutes, and the fluorescence polarization value was measured using a fluorescence polarimeter; S103, judging whether there are chemical substances capable of binding to ERα-LBD or ERβ-LBD in the environmental water sample according to the change of fluorescence polarization value, a decrease in polarization value indicates that the chemical substances in the water sample compete with ERα-LBD or ERβ-LBD for binding.

8. The method for detecting estrogen endocrine disruptors in environmental water samples according to claim 7, characterized in that: ERα-LBD is the protein of a1) or a2) or a3) as follows: a1) The amino acid sequence is SAG DMRAANLWPS PLMIKRSKKN SLALSLTADQ MVSALLDAEP PILYSEYDPTRPFSEASMMG LLTNLADREL VHMINWAKRV PGFVDLTLHD QVHLLECAWL EILMIGLVWR SMEHPGKLLFAPNLLLDRNQ GKCVEGMVEI FDMLLATSSR FRMMNLQGEE FVCLKSIILL NSGVYTFLSS TLKSLEEKDHIHRVLDKITD TLIHLMAKAG LTLQQQHQRL AQLLLILSHI RHMSNKGMEH LYSMKCKNVV PLYDLLLEMLDAHRLHAPTS RGGASVEETD QSHLATAGST SSHSLQKYYI TGEAEGFPAT V; a2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in Sequence 1 of the sequence listing; a3) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing.

9. The method for detecting estrogen endocrine disruptors in environmental water samples according to claim 7, characterized in that: ERβ-LBD is the protein of b1) or b2) or b3) as follows: b1) The amino acid sequence is KAK RSGGHAPRVR ELLLDALSPE QLVLTLLEAE PPHVLISRPS APFTEASMMMSLTKLADKEL VHMISWAKKIPGFVELSLFD QVRLLESCWM EVLMMGLMWR SIDHPGKLIF APDLVLDRDEGKCVEGILEI FDMLLATTSR FRELKLQHKE YLCVKAMILL NSSMYPLVTA TQDADSSRKL AHLLNAVTDALVWVIAKSGI SSQQQSMRLA NLLMLLSHVR HASNKGMEHL LNMKCKNVVP VYDLLLEMLN AHVLRGCKSSITGSECSPAE DSKSKEGSQN PQSQ; b2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in Sequence 2 of the sequence listing; b3) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2 of the sequence listing.

10. A system for detecting estrogen endocrine disruptors in environmental water samples by implementing the method for detecting estrogen endocrine disruptors in environmental water samples as claimed in any one of claims 1 to 9, characterized in that: The system for detecting estrogen endocrine disruptors in environmental water samples comprises: The detection module is used to detect the binding ability of environmental water samples with ERα and ERβ through fluorescence polarization competition binding based on the characteristics of the fluorescent probe E2-F specifically binding to estrogen receptors ERα and ERβ; The judgment module is used to compete the probe from the receptor when the environmental water sample to be tested can bind to ERα / ERβ, so that the fluorescent probe is in a free small molecule state. Due to its small molecule, it rotates faster in the liquid phase, and the measured fluorescence polarization degree is also lower; on the contrary, if the environmental sample cannot bind to the receptor, it cannot affect the fluorescent probe E2-F binding to the protein to form a macromolecular complex, the probe rotates slower, and the detected fluorescence polarization degree is also higher.

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