New pollutant rapid screening method based on PPAR or ER protein affinity binding

Abstract

The invention belongs to the technical field of biochemical detection, and discloses a rapid new pollutant screening method based on PPAR or ER protein affinity binding, PPAR or ER protein with a His tag is selectively adsorbed to a Ni-magnetic bead carrier, and then a new pollutant is screened according to the principle of affinity binding of a nuclear receptor and ligand small molecules. Effect substances with binding activity are specifically captured from a complex environmental medium by using nuclear receptor protein, and the core is to convert environmental endocrine disruption effect activity into measurable signal output (fluorescence), so that rapid screening of new pollutants is realized. According to the method, the efficiency of screening new pollutants is improved, and the method can be applied to rapid screening of effector activity new pollutants combined with PPAR or ER protein in the environment.

Description

Technical Field

[0001] This application belongs to the field of biochemical detection technology, specifically relating to a novel rapid screening method for pollutants based on the affinity binding of PPAR or ER proteins. Background Technology

[0002] The rapid pace of industrialization and advancements in chemical technology have led to a surge in the types and quantities of synthetic chemicals. As the world's largest producer and consumer of chemicals, China sees a significant amount of chemicals intentionally or unintentionally released into the environment. Among these, endocrine disruptors (EDCs) have emerged as a new source of pollution and have garnered widespread attention. Numerous studies have demonstrated that EDCs in the environment bind to nuclear receptor proteins, interfering with metabolism and causing endocrine disorders, thereby impacting ecology and human health. Therefore, monitoring the biological endocrine disruption effects in the environmental media is crucial.

[0003] Because EDCs are generally characterized by wide sources, many types, low concentrations, and rapid increase, they have extremely high requirements for monitoring technologies and methods compared to other pollutants. Existing monitoring methods mainly use traditional gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry and other analytical methods, but these methods have problems such as low detection efficiency, long cycle time, high cost and lack of technical methods. Summary of the Invention

[0004] The purpose of this application is to address the problems of existing technologies by providing a novel rapid pollutant screening method based on the affinity binding of PPAR or ER proteins to rapidly screen and monitor the activity of biological endocrine disruption effects from environmental samples.

[0005] To address the technical problem, the technical solution of this application is: a novel rapid pollutant screening method based on PPAR or ER protein affinity binding, comprising the following steps: Step 1: Immobilize His-tagged PPAR or ER proteins onto pretreated magnetic bead suspensions at a ratio of 10-20 μg: 1 mL, incubate to bind, and form a recombinant nuclear receptor protein at a defined concentration. Step 2: The recombinant nuclear receptor protein at the concentration determined in Step 1 was bound to a known ligand labeled with CY-2 at the determined concentration, and incubated to obtain a control group; Step 3: Mix the recombinant nuclear receptor protein at the concentration determined in Step 1 with the environmental medium sample to be tested and incubate it to allow the recombinant nuclear receptor protein to bind to the biological effect substances in the environmental medium sample to be tested. Then mix and incubate it with a known ligand labeled with CY-2 fluorescence, wherein the concentration of the known ligand labeled with CY-2 fluorescence is the same as the concentration of the known ligand labeled with CY-2 fluorescence determined in Step 2, to obtain the sample group. Step 4: Place the sample group and control group on the magnetic rack, take the supernatant and put it into the analyzer to analyze the fluorescence concentration of the supernatant; Step 5: Compare the fluorescence concentration differences between the sample group and the control group, substitute them into the standard curve to determine the equivalent amount of biological effect substances that bind to recombinant nuclear receptor proteins in the environmental medium sample to be tested, and achieve rapid screening of new pollutants by detecting fluorescence concentration.

[0006] Preferably, the PPAR protein is human PPARγ protein, human PPARα protein, or human PPARβ protein, and the ER protein is human ERα protein or human ERβ protein.

[0007] Preferably, the magnetic beads are Ni magnetic beads with a high His-tag fusion protein loading capacity, including Ni-NTA agarose magnetic beads and Ni-IDA agarose magnetic beads.

[0008] Preferably, step 1 specifically involves: mixing the His-tagged PPAR or ER protein with the pretreated magnetic bead suspension at 4-6°C for 1-2 hours by rotation; the pretreatment process of the magnetic bead suspension is as follows: take Ni-NTA agarose magnetic beads or Ni-IDA agarose magnetic bead suspension into a centrifuge tube, place it on a magnetic separator, perform magnetic separation, and discard the supernatant; then add equilibration buffer, mix well, perform magnetic separation, discard the supernatant, and repeat several times to obtain the pretreated magnetic bead suspension; the equilibration buffer is composed of tris(hydroxymethyl)aminomethane and sodium chloride, with a weight ratio of tris(hydroxymethyl)aminomethane to sodium chloride of 1:10, and adjusting the pH to 7-7.8.

[0009] Preferably, step 3 specifically involves mixing the recombinant nuclear receptor protein with the pretreated environmental medium sample to be tested and then rotating and mixing at 4-6°C for 1-2 hours.

[0010] Preferably, the pretreatment of the environmental medium sample in step 3 is as follows: the water sample to be tested is filtered through a mixed fiber filter membrane and then enriched using a solid-phase extraction column. The solid-phase extraction column is activated with methanol and equilibrated with ultrapure water. The water sample to be tested is passed through the activated solid-phase extraction column at a flow rate of 1~2 mL / min. After rinsing with ultrapure water, the water sample to be tested is completely dried under vacuum to remove the water in the solid-phase extraction column. The target substance is then eluted with methanol and dried under a nitrogen flow. DMSO is dissolved and ultrapure water is added to make up to the target volume to obtain the environmental medium sample to be tested.

[0011] Preferably, the method also includes the separation of His-tagged PPAR or ER proteins from magnetic beads, specifically: adding an equal volume of elution buffer to the magnetic bead suspension after reaction, repeatedly pipetting to mix thoroughly, incubating at room temperature for 5-10 min on a mixer, magnetically separating for 1-5 min, and collecting the elution buffer; repeating this process multiple times and collecting the elution buffer each time, then adding 2 volumes of elution buffer, 2 volumes of deionized water, and 2 volumes of storage buffer to repeat the above steps, and finally adding 2 volumes of storage buffer and storing at 2-8°C.

[0012] Preferably, the elution buffer is imidazole or tromethamine, with a pH of 7-7.8; the storage buffer is 1×PBS buffer, with a pH of 7-7.8.

[0013] Preferably, the known ligand is rosiglitazone or estradiol.

[0014] Preferably, the absorbance (OD) formula for the biological effector substance binding to the recombinant nuclear receptor protein in the environmental medium sample to be tested is: OD = (Experimental group absorbance - Control group absorbance) × Dilution or enrichment coefficient; The formula for calculating the equivalent of biological effect substances is: Equivalent amount of biologically effective substance = a × OD + b; In the formula: a and b are determined based on the corresponding standard curves.

[0015] Compared with the prior art, the advantages of this application are: (1) In this application, selected nuclear receptor proteins with His tags are selectively adsorbed onto Ni-magnetic bead carriers. Then, based on the principle of affinity binding of nuclear receptor proteins with known ligand small molecules, the selected nuclear receptor proteins are selected by competing with known ligands with fluorescent labels for binding by biological effect substances with binding activity in complex environmental media samples. The activity of biological effect substances in environmental media samples is analyzed by comparing the differences in fluorescence concentration through instrument analysis, which has high detection efficiency. (2) This application has good environmental compatibility and can be applied to the rapid screening of almost all environmental media water samples. Existing chemical analysis generally requires cumbersome pretreatment to remove matrix interference. This application has stronger resistance to matrix interference and environmental media samples do not require complicated pretreatment, which greatly simplifies the operation process. (3) This application reduces the economic and time requirements in the analysis of environmental media samples, does not require high-end equipment, has a short experimental cycle, reduces operational complexity, and greatly improves efficiency; (4) Traditional chemical analysis methods tend to screen known substances, while the method of this application focuses more on identifying factors that have toxic effects on ecosystems and human health. The method of this application is not limited to detecting only chemical concentrations, but provides information based on toxicity and function. This model not only improves the comprehensiveness of pollution identification, but also provides data support for actual environmental risk assessment. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating a novel rapid pollutant screening method based on PPAR or ER protein affinity binding, as described in this application. Figure 2 This is the standard curve for CY-2 fluorescently labeled rosiglitazone in this application; Figure 3 This is the standard curve for E2 labeled with CY-2 in this application; Figure 4 This is a schematic diagram illustrating the accuracy evaluation of the method in this application, showing the difference in absorbance at different salinity concentrations; Figure 5 This diagram illustrates the accuracy evaluation of the method in this application, showing the difference in absorbance at different pH concentrations. Detailed Implementation

[0017] The present application is described in detail below with reference to the accompanying drawings and specific embodiments, but the present application is not limited to these embodiments. The present application covers any alternatives, modifications, equivalent methods, and solutions made within the spirit and scope of the present application. To provide the public with a thorough understanding of the present application, specific details are described in detail in the following embodiments, but those skilled in the art will fully understand the present application even without these detailed descriptions.

[0018] like Figure 1 As shown, this application proposes a novel rapid pollutant screening method based on PPAR or ER protein affinity binding, comprising the following steps: Step 1: Immobilize His-tagged PPAR or ER proteins onto pretreated magnetic bead suspensions. The ratio of His-tagged PPAR or ER proteins to pretreated magnetic bead suspensions is 10-20 μg: 1 mL. Incubate to bind and form a recombinant nuclear receptor protein at a defined concentration. Step 2: The recombinant nuclear receptor protein at the concentration determined in Step 1 was bound to a known ligand labeled with CY-2 at the determined concentration, and incubated to obtain a control group; Step 3: Mix the recombinant nuclear receptor protein at the concentration determined in Step 1 with the environmental medium sample to be tested and incubate it to allow the recombinant nuclear receptor protein to bind to the biological effect substances in the environmental medium sample to be tested. Then mix and incubate it with a known ligand labeled with CY-2 fluorescence, wherein the concentration of the known ligand labeled with CY-2 fluorescence is the same as the concentration of the known ligand labeled with CY-2 fluorescence determined in Step 2, to obtain the sample group. Step 4: Place the sample group and control group on the magnetic rack, take the supernatant and put it into the analyzer to analyze the fluorescence concentration of the supernatant; Step 5: Compare the fluorescence concentration differences between the sample group and the control group, substitute them into the standard curve to determine the equivalent amount of biological effect substances that bind to recombinant nuclear receptor proteins in the environmental medium sample to be tested, and achieve rapid screening of new pollutants by detecting fluorescence concentration.

[0019] Preferably, the PPAR protein is human PPARγ protein, human PPARα protein, or human PPARβ protein, and the ER protein is human ERα protein or human ERβ protein.

[0020] Preferably, the magnetic beads are Ni magnetic beads with a high His-tag fusion protein loading capacity, including Ni-NTA agarose magnetic beads and Ni-IDA agarose magnetic beads.

[0021] Preferably, step 1 specifically involves: mixing the His-tagged PPAR or ER protein with the pretreated magnetic bead suspension at 4-6°C for 1-2 hours by rotation; the pretreatment process of the magnetic bead suspension is as follows: take Ni-NTA agarose magnetic beads or Ni-IDA agarose magnetic bead suspension into a centrifuge tube, place it on a magnetic separator, perform magnetic separation, and discard the supernatant; then add equilibration buffer, mix well, perform magnetic separation, discard the supernatant, and repeat several times to obtain the pretreated magnetic bead suspension; the equilibration buffer is composed of tris(hydroxymethyl)aminomethane and sodium chloride, with a weight ratio of tris(hydroxymethyl)aminomethane to sodium chloride of 1:10, and adjusting the pH to 7-7.8.

[0022] Preferably, step 3 specifically involves mixing the recombinant nuclear receptor protein with the pretreated environmental medium sample to be tested and then rotating and mixing at 4-6°C for 1-2 hours.

[0023] Preferably, the pretreatment of the environmental medium sample in step 3 is as follows: the water sample to be tested is filtered through a mixed fiber filter membrane and then enriched using a solid-phase extraction column. The solid-phase extraction column is activated with methanol and equilibrated with ultrapure water. The water sample to be tested is passed through the activated solid-phase extraction column at a flow rate of 1~2 mL / min. After rinsing with ultrapure water, the water sample to be tested is completely dried under vacuum to remove the water in the solid-phase extraction column. The target substance is then eluted with methanol and dried under a nitrogen flow. DMSO is dissolved and ultrapure water is added to make up to the target volume to obtain the environmental medium sample to be tested.

[0024] Preferably, the method also includes the separation of His-tagged PPAR or ER proteins from magnetic beads, specifically: adding an equal volume of elution buffer to the magnetic bead suspension after reaction, repeatedly pipetting to mix thoroughly, incubating at room temperature for 5-10 min on a mixer, magnetically separating for 1-5 min, and collecting the elution buffer; repeating this process multiple times and collecting the elution buffer each time, then adding 2 volumes of elution buffer, 2 volumes of deionized water, and 2 volumes of storage buffer to repeat the above steps, and finally adding 2 volumes of storage buffer and storing at 2-8°C.

[0025] Preferably, the elution buffer is imidazole or tromethamine, with a pH of 7-7.8; the storage buffer is 1×PBS buffer, with a pH of 7-7.8.

[0026] Preferably, the known ligand is rosiglitazone or estradiol.

[0027] Preferably, the absorbance (OD) formula for the biological effector substance binding to the recombinant nuclear receptor protein in the environmental medium sample to be tested is: OD = (Experimental group absorbance - Control group absorbance) × Dilution or enrichment coefficient; The formula for calculating the equivalent of biological effect substances is: Equivalent amount of biologically effective substance = a × OD + b; In the formula: a and b are determined based on the corresponding standard curves.

[0028] Application Example 1 Detection of biological effectors that specifically bind to the PPARγ nuclear receptor protein in Qiantang River water samples: 1. A method for specifically capturing endocrine disruptors based on the principle of affinity binding: A. Take 100 ml of the water sample (the environmental medium sample to be tested), filter it through a mixed fiber membrane (0.22 μm pore size), and enrich it using an Oasis PRIME HLB solid-phase extraction column (30 mg, 1 cc, waters). Activate the solid-phase extraction column sequentially with 5 mL of methanol, equilibrate with 5 mL of ultrapure water, and control the flow rate of the water sample to pass through the activated HLB column at 1–2 mL / min. Elute with 5 mL of ultrapure water. After all the water sample has passed through the HLB column, vacuum dry it to remove all moisture from the column bed. Elute the target substance with 5 mL of methanol, dry it under a nitrogen stream, dissolve it in 100 μL of DMSO, and add ultrapure water to a final volume of 10 mL to obtain the water sample extract.

[0029] B. Transfer 1 ml of 10% Ni-NTA agarose magnetic bead suspension to a centrifuge tube, place it on a magnetic separator, and magnetically separate for 1 min. Discard the supernatant. Add 1 mL of equilibration buffer, and mix thoroughly by pipetting at least 5 times. Magnetically separate for 1 min, discard the supernatant, and repeat 2-3 times.

[0030] C. Take 50 μg of human PPARγ protein with histidine (His) tag (purchased commercially; for lyophilized recombinant proteins, reconstitution is required before aliquoting and storage or use), prepare a concentration of 20 μg / L with ultrapure water, mix with prepared Ni-NTA agarose beads, and incubate at 4°C with rotational mixing for 1 h to allow the PPARγ protein to fully bind to the Ni-NTA agarose beads.

[0031] D. Take 1 ml of the completed PPARγ protein and 1 ml of the water sample extract to be tested, incubate at 4℃ for 1 h, then add 2 mL of 20 μg / L CY-2 fluorescently labeled rosiglitazone (purchased from the market) to the reaction system, continue incubation for 1 h, repeat 2 times as experimental group.

[0032] E. Take 1 ml of the fully bound PPARγ protein, 1 ml of ultrapure water and 2 mL of 20 μg / L CY-2 fluorescently labeled rosiglitazone, and incubate at 4℃ for 1 h. Repeat the process in two replicates, with each replicate serving as a control group.

[0033] F. Place the reaction systems D and E on a magnetic rack and let stand for 1 min. Take 200 μL of the supernatant into each well of a 96-well plate and measure the absorbance at 506 nm using an ELISA reader.

[0034] G. Compare the absorbance differences between the control group and the experimental group to determine whether the water sample environment has endocrine-disrupting activity of PPARγ, as shown in Table 1: Table 1. Fluorescence values ​​of experimental and control groups of water samples from the Qiantang River 2. Calculation of activity equivalents for endocrine disrupting activity in water samples: A. Standard curve preparation for CY-2 fluorescently labeled rosiglitazone: CY-2 fluorescently labeled rosiglitazone at concentrations of 0.1, 1, 10, 50, and 100 μg / L were prepared in ultrapure water. After thorough mixing, 200 μL was added to each well of a 96-well plate, and the absorbance was measured at 506 nm using a microplate reader. The standard curve was then plotted. The experimental results are shown in the appendix. Figure 2 And Table 2: Table 2. Standard curve of CY-2 fluorescently labeled rosiglitazone B. Substitute the absorbance difference values ​​of the control group and the experimental group into the standard curve to calculate how much rosiglitazone activity equivalent (Rosi-BAE) the endocrine-disrupting activity in the environmental water sample is converted into.

[0035] The specific formula is: a × OD + b, where a and b are based on... Figure 2 The standard curve for rosiglitazone was determined by OD = (absorbance of experimental group - absorbance of control group) × dilution (enrichment) coefficient.

[0036] Application Example 2 Detection of biological effectors that specifically bind to the ERα nuclear receptor protein in water samples from rivers surrounding the factory: 1. A method for specifically capturing endocrine disruptors based on the principle of affinity binding: The specific chemical capture method based on the affinity binding principle is the same as the experimental procedure in Part 1 of Example 1, except that: A. Take 10 mL of the water sample to be tested, filter it through a mixed fiber membrane (0.22 μm pore size) to remove impurities.

[0037] C. Take 50 μg of human ERα protein with a histidine (His) tag (purchased commercially; for lyophilized recombinant proteins, reconstitution is required before aliquoting and storage or use), prepare a 10 μg / L solution, mix with the prepared Ni-magnetic beads, and incubate at 6°C with rotation for 2 h to allow the ERα protein to fully bind to the Ni-magnetic beads.

[0038] D. Take 1 ml of the completed ERα protein and 1 mL of the water sample to be tested, incubate at 6℃ for 2 h, then add 2 mL of 20 μg / L CY-2 fluorescently labeled estradiol (E2, purchased from the market) to the reaction system, and continue incubation for 1 h. Repeat 2 times as the experimental group.

[0039] E. Take 1 ml of the fully bound ERα protein, 1 ml of ultrapure water and 2 mL of 20 μg / L CY-2 fluorescently labeled E2, and incubate at 6℃ for 2 h. Repeat the process in two replicates, with each replicate serving as a control group.

[0040] G. Compare the absorbance differences between the control group and the experimental group to determine whether the water sample environment has endocrine-disrupting activity of ERα, as detailed in Table 3: Table 3. Fluorescence values ​​of water samples from the river surrounding the factory in the experimental and control groups. 2. Calculation of activity equivalents for endocrine disrupting activity in water samples: A. Standard curve preparation for CY-2 fluorescently labeled E2: CY-2 fluorescently labeled E2 at concentrations of 0.1, 1, 10, 50, and 100 μg / L were prepared in ultrapure water. After thorough mixing, 200 μL was added to each well of a 96-well plate, and the absorbance was measured at 506 nm using a microplate reader. A standard curve was then plotted. The experimental results are shown in the appendix. Figure 3 And Table 4: Table 4. Standard curve of E2 labeled with CY-2 fluorescence B. Substitute the absorbance difference values ​​of the control group and the experimental group into the standard curve to calculate how much E2 activity equivalent (E2-BAE) the endocrine interference activity in the environmental water sample is converted into.

[0041] The specific formula is: a × OD + b, where a and b are based on... Figure 3 The E2 standard curve was determined, and OD = (experimental group absorbance - control group absorbance) × dilution (enrichment) coefficient.

[0042] Application Example 3 Investigating the effects of environmental factors on the efficacy of biological effectors in water samples that specifically bind to nuclear receptor proteins: 1. In order to study the accuracy and versatility of the method and ensure its application in multiple scenarios, this study investigates the effect of salinity on the effectiveness of the method in this application.

[0043] A. Take 100 mL of the water sample to be tested, filter it through a mixed fiber membrane (0.22 μm pore size) to remove impurities. Based on the salinity of the aquatic environment, add NaCl granules to prepare a water sample with a salinity of 0–35‰.

[0044] B. Take 1 mL of 10% Ni-NTA agarose magnetic bead suspension into a centrifuge tube, place it on a magnetic separator, and magnetically separate for 1 min. Discard the supernatant. Add 1 mL of equilibration buffer, and mix thoroughly by pipetting at least 5 times. Magnetically separate for 1 min, discard the supernatant, and repeat 2-3 times.

[0045] C. Take 50 μg of human ERα protein with histidine (His) tag, prepare it to 10 μg / L, mix it with the prepared Ni-magnetic beads, and incubate it at 4℃ for 1 h by rotating to allow the ERα protein to fully bind to the Ni-magnetic beads.

[0046] D. Take 1 mL of the completed ERα protein and 1 mL of water sample, incubate at 4℃ for 1 h, then add 2 mL of 20 μg / L CY-2 fluorescently labeled E2 to the reaction system, and continue incubation for 1 h. Repeat 2 replicates as the experimental group.

[0047] E. Place the reaction system in D on a magnetic rack and let it stand for 1 min. Take 200 μL of the supernatant into a 96-well plate and measure the absorbance at 506 nm using an ELISA reader.

[0048] F. Compare the absorbance differences at various salinity concentrations; the experimental results are shown in the appendix. Figure 4 To determine the effect of salinity on the effectiveness of the method in this application.

[0049] like Figure 4 As shown in the figure, the absorbance differences at various salinity concentrations are small, indicating that salinity, as an environmental factor, does not significantly affect the experimental results. The salinity of environmental water bodies is generally 0.5% (freshwater) to 3.5% (seawater). This study verifies that this application is applicable to the rapid screening of endocrine-disrupting activities of novel pollutants in almost all environmental water bodies, ensuring that this method can be applied to water bodies with varying salinity levels.

[0050] 2. In order to study the accuracy of the method and ensure that the method can be applied in multiple scenarios, the effect of pH on the effectiveness of the method in this application was investigated.

[0051] The specific operating method is the same as the experimental steps in Part 1 of Example 3, except that: A. Take 100 mL of the water sample to be tested, filter it through a mixed fiber membrane (0.22 μm pore size) to remove impurities. Based on the salinity of the aquatic environment, add HCl and NaOH solutions to prepare a water sample with a pH range of 2-13.

[0052] C. Take 50 μg of human PPARγ protein with histidine (His) tag, prepare it to 10 μg / L, mix it with the prepared Ni-magnetic beads, and incubate it at 4℃ for 1 h by rotating to allow the PPARγ protein to fully bind to the Ni-magnetic beads.

[0053] D. Take 1 mL of the completed PPARγ protein and 1 mL of water sample, incubate at 4℃ for 1 h, then add 2 mL of 20 μg / L CY-2 fluorescently labeled rosiglitazone to the reaction system, and continue incubation for 1 h. Repeat the process in two replicates as the experimental group.

[0054] F. Compare the absorbance differences at different pH concentrations; the experimental results are shown in the appendix. Figure 5 To determine the effect of pH on the effectiveness of the method in this application.

[0055] like Figure 5 As shown, the absorbance varies at different pH concentrations. The same sample exhibits different absorbance under acidic, neutral, or alkaline conditions, indicating that pH, as an environmental factor, can significantly affect the experimental results. This study demonstrates that pH interferes with the rapid screening of endocrine-disrupting activities of novel pollutants in environmental water bodies using the method described in this application. When using this method, it is crucial to maintain the pH of the environmental water sample and the control group at the same level to avoid significant errors in the results.

[0056] This application first utilizes the selective adsorption of selected nuclear receptor proteins with His tags onto Ni-magnetic bead carriers. Then, based on the principle of affinity binding between nuclear receptor proteins and known ligand small molecules, it utilizes the competitive binding of bioactive substances in complex environmental media samples with known fluorescently labeled ligands to the selected nuclear receptor proteins. By comparing the differences in fluorescence concentration through instrumental analysis, the activity of bioactive substances in environmental media samples can be analyzed, resulting in high detection efficiency.

[0057] This application has good environmental compatibility and can be applied to the rapid screening of almost all environmental media water samples. Existing chemical analysis generally requires cumbersome pretreatment to remove matrix interference. This application has stronger resistance to matrix interference, and environmental media samples do not require complicated pretreatment, which greatly simplifies the operation process.

[0058] This application reduces economic and time requirements in environmental media sample analysis, eliminates the need for high-end equipment, shortens the experimental cycle, reduces operational complexity, and greatly improves efficiency.

[0059] Traditional chemical analysis methods tend to screen known substances, while the method proposed in this application focuses more on identifying factors that have toxic effects on ecosystems and human health. This method is not limited to detecting only chemical concentrations, but provides information based on toxicity and function. This approach not only improves the comprehensiveness of pollution identification, but also provides data support for actual environmental risk assessment.

[0060] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application.

[0061] Many other changes and modifications can be made without departing from the concept and scope of this application. It should be understood that this application is not limited to the specific embodiments, and the scope of this application is defined by the appended claims.

Claims

1. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding, characterized in that, Includes the following steps: Step 1: Immobilize His-tagged PPAR or ER proteins onto pretreated magnetic bead suspensions at a ratio of 10-20 μg to 1 mL, incubate to bind, and form a recombinant nuclear receptor protein at a defined concentration. Step 2: The recombinant nuclear receptor protein at the concentration determined in Step 1 was bound to a known ligand labeled with CY-2 at the determined concentration, and incubated to obtain a control group; Step 3: Mix the recombinant nuclear receptor protein at the concentration determined in Step 1 with the environmental medium sample to be tested and incubate it to allow the recombinant nuclear receptor protein to bind to the biological effect substances in the environmental medium sample to be tested. Then mix and incubate it with a known ligand labeled with CY-2 fluorescence, wherein the concentration of the known ligand labeled with CY-2 fluorescence is the same as the concentration of the known ligand labeled with CY-2 fluorescence determined in Step 2, to obtain the sample group. Step 4: Place the sample group and control group on the magnetic rack, take the supernatant and put it into the analyzer to analyze the fluorescence concentration of the supernatant; Step 5: Compare the fluorescence concentration differences between the sample group and the control group, substitute them into the standard curve to determine the equivalent amount of biological effect substances that bind to recombinant nuclear receptor proteins in the environmental medium sample to be tested, and achieve rapid screening of new pollutants by detecting fluorescence concentration.

2. The novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 1, characterized in that, The PPAR protein is human PPARγ protein, human PPARα protein, or human PPARβ protein, and the ER protein is human ERα protein or human ERβ protein.

3. The novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 1, characterized in that, The magnetic beads are Ni magnetic beads with high His-tag fusion protein loading capacity, including Ni-NTA agarose magnetic beads and Ni-IDA agarose magnetic beads.

4. The novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 3, characterized in that: Step 1 specifically involves: mixing His-tagged PPAR or ER proteins with a pretreated magnetic bead suspension at 4-6°C for 1-2 hours by rotation; the pretreatment process of the magnetic bead suspension is as follows: take Ni-NTA agarose magnetic beads or Ni-IDA agarose magnetic bead suspension into a centrifuge tube, place it on a magnetic separator, perform magnetic separation, and discard the supernatant; then add equilibration buffer, mix well, perform magnetic separation, discard the supernatant, and repeat several times to obtain the pretreated magnetic bead suspension; the equilibration buffer is composed of tris(hydroxymethyl)aminomethane and sodium chloride, with a weight ratio of tris(hydroxymethyl)aminomethane to sodium chloride of 1:10, and adjusting the pH to 7-7.

8.

5. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 1, characterized in that, Step 3 specifically involves mixing the recombinant nuclear receptor protein with the pretreated environmental medium sample and then rotating and mixing it at 4-6°C for 1-2 hours.

6. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 5, characterized in that, The pretreatment of the environmental medium sample in step 3 is as follows: the water sample to be tested is filtered through a mixed fiber filter membrane and then enriched using a solid phase extraction column. The solid phase extraction column is activated with methanol and equilibrated with ultrapure water. The water sample to be tested is passed through the activated solid phase extraction column at a flow rate of 1~2 mL / min and then rinsed with ultrapure water. After all the water sample to be tested has passed through the solid phase extraction column, it is vacuum dried to remove all the water in the solid phase extraction column. The target substance is then eluted with methanol and dried under a nitrogen flow. DMSO is dissolved and ultrapure water is added to make up to the target volume to obtain the environmental medium sample to be tested.

7. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 1, characterized in that, It also includes the separation of His-tagged PPAR or ER proteins from magnetic beads. Specifically, the magnetic bead suspension after reaction is added to an equal volume of elution buffer, and the mixture is repeatedly pipetted to ensure thorough mixing. The mixture is then incubated at room temperature for 5-10 minutes, followed by magnetic separation for 1-5 minutes. The elution buffer is collected. This process is repeated several times, and the elution buffer is collected each time. Subsequently, 2 volumes of elution buffer, 2 volumes of deionized water, and 2 volumes of storage buffer are added to repeat the above steps. Finally, 2 volumes of storage buffer are added, and the mixture is stored at 2-8°C.

8. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 7, characterized in that, The elution buffer is imidazole or tromethamine, with a pH of 7-7.8; the storage buffer is 1×PBS buffer, with a pH of 7-7.

8.

9. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 1, characterized in that, The known ligands are rosiglitazone or estradiol.

10. A novel rapid pollutant screening method based on PPAR or ER protein affinity binding as described in claim 1, characterized in that, The absorbance (OD) formula for the biological effector substances that bind to the recombinant nuclear receptor protein in the environmental medium sample to be tested is: OD = (Experimental group absorbance - Control group absorbance) × Dilution or enrichment coefficient; The formula for calculating the equivalent of biological effect substances is: Equivalent amount of biologically effective substance = a × OD + b; In the formula: a and b are determined according to the corresponding standard curve.

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