A method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials

Abstract

This invention discloses a method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials, belonging to the field of biochemical detection technology. The screening method includes: mixing recombinant nuclear receptor protein with phosphate buffer solution of two metal ions, allowing the mixture to react statically, collecting the precipitate, washing with water, and drying to obtain active and inactive metal ion-nuclear receptor protein nanomaterials, respectively. The active nanomaterial is mixed with the target environmental medium to bind biological effect substances in the medium. The active nanomaterial is then separated and subjected to liquid-solid extraction to obtain the screening sample. The inactive nanomaterial is used to obtain a control sample through the same procedure. Differential compounds between the two samples are screened and identified by searching chemical and mass spectrometry databases. This invention uses inactive nanomaterials as a negative control, significantly reducing false positives during the screening process and improving the screening efficiency and accuracy of endocrine disruptors in the environment.

Description

Technical Field

[0001] This invention belongs to the field of biochemical detection technology, specifically relating to a method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials. Background Technology

[0002] In recent years, with the rapid development of the global chemical industry, the number of synthetic chemical substances has exploded over the past few decades. According to the Chemical Abstracts Service (CAS), as of 2021, the number of registered chemical substances exceeded 250 million. While these synthetic chemicals have greatly improved social productivity and facilitated human life, they have also brought potential health risks. Among them, endocrine disruptors have attracted much attention because they can interfere with key processes such as the synthesis, secretion, transport, metabolism, binding, and clearance of signaling molecules in the body even at extremely low concentrations, thereby affecting the body's reproduction, development, and behavior, posing a serious threat to human health.

[0003] Screening for endocrine disruptors from complex environmental media remains a significant challenge due to the diverse range of chemicals present in the environment. In recent years, protein selective mass spectrometry (MS / MS) has emerged as a rapid, high-throughput, and cost-effective method, successfully used to screen for endocrine disruptors in complex environmental samples. MS / MS begins with a protein affinity assay, where a tagged recombinant nuclear receptor protein extract from an environmental sample is mixed to capture the chemical substance that binds to the protein. This is followed by ligand identification using non-targeted high-resolution mass spectrometry. To distinguish chemicals that bind nonspecifically to the protein, a negative control experiment is required, typically using an empty vector protein that does not express the recombinant nuclear receptor protein. While this method mitigates false positives from nonspecific binding to some extent, existing negative controls often fail to simultaneously capture the nonspecific binding characteristics of the target protein and avoid ligand capture functionality, potentially leading to false positives or false negatives.

[0004] Patent document CN117069858A discloses a metal ion-protein nanomaterial based on recombinant nuclear receptor protein and its preparation method. Metal ions are added to free recombinant nuclear receptor protein-phosphate buffer to obtain the metal ion-nuclear receptor protein nanomaterial, achieving enhanced stability while maintaining the receptor protein's activity. Since different metal ions have different effects in the protein immobilization process, it is possible to prepare metal ion-nuclear receptor protein nanomaterials that retain biological activity or to prepare materials that have lost their activity. Patent document CN109085361A discloses a high-throughput screening and identification method for biological effect substances based on recombinant nuclear receptor proteins. This method utilizes tagged, selected recombinant nuclear receptor proteins to selectively adsorb onto magnetic bead carriers. Based on the principle of affinity binding between nuclear receptors and small molecules, the recombinant nuclear receptor proteins specifically capture binding-active effect substances from complex environmental media. This is then combined with ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap high-resolution mass spectrometry for high-throughput, non-targeted analysis of effect substances, improving the specificity and efficiency of substance identification. This method can be applied to the rapid screening and structural identification of effect substances that bind to nuclear receptors in the environment, and can also be used for drug development. However, there are currently no reports on specific screening methods for biological effect substances based on metal ion-nuclear receptor protein nanomaterials. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies where negative controls cannot reflect the non-specific adsorption characteristics of target proteins, the present invention aims to provide a method for specific screening of bioeffect substances based on metal ion-nuclear receptor protein nanomaterials. This method utilizes active metal ion-nuclear receptor protein nanomaterials to bind with small molecules via affinity, and employs inactivated metal ion-nuclear receptor protein nanomaterials as a negative control. This enables specific screening of bioeffect substances in environmental samples. It is a high-throughput method for identifying non-targeted effect substances based on inactivated metal ion-nuclear receptor protein nanomaterials as a negative control, which can significantly reduce false positive results during screening and improve the efficiency and accuracy of screening for endocrine disruptors in the environment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials, comprising the following steps:

[0008] S1. The recombinant nuclear receptor protein was mixed with two metal ions in phosphate buffer solution, allowed to stand for reaction, the precipitate was collected, washed with water and dried to obtain metal ion-nuclear receptor protein active nanomaterials and metal ion-nuclear receptor protein inactivating nanomaterials, respectively.

[0009] S2. Mix the metal ion-nuclear receptor protein active nanomaterial with the environmental medium to be tested, so that it binds to the biological effect substances in the environmental medium;

[0010] S3. Separate the metal ion-nuclear receptor protein active nanomaterial and perform liquid-solid extraction on it to obtain a screening sample;

[0011] S4. Using the metal ion-nuclear receptor protein inactivation nanomaterial, a control sample is obtained based on the same operation process as steps S2 and S3.

[0012] S5. By comparing and screening the differential compounds between the screened samples and the control samples;

[0013] S6. Search and identify the above-mentioned differential compounds using chemical databases and mass spectrometry databases.

[0014] Further, in step S1, the recombinant nuclear receptor protein is selected from one or more of the following recombinant nuclear receptor proteins: AR, ERα, ERβ, ERγ, PR, GR, ERR, PPARα, PPARδ, PPARγ, RXRα, RXRβ, RXRγ, RARα, RARβ, RARγ, TRα, TRβ, VDR, PXR, CAR, LXRα, LXRβ, FXR, and AhR.

[0015] Further, in step S1, the recombinant nuclear receptor protein is a human nuclear receptor protein with a histidine (His) tag.

[0016] Furthermore, in step S1, the selection of the metal ion is related to the material activity and is selected from one or more of calcium ions, magnesium ions, iron ions, ferrous ions, copper ions, barium ions, divalent manganese ions, divalent nickel ions, divalent cobalt ions, and zinc ions.

[0017] Further, in step S1, the mass-volume concentration of the recombinant nuclear receptor protein in the phosphate buffer is 0.1–50 mg / mL; and / or the pH of the phosphate buffer is 5.0–9.0; and / or the concentrations of hydrogen phosphate and dihydrogen phosphate in the phosphate buffer are both 1–1000 mM; and / or the temperature of the static reaction is 0–40°C, and the static reaction time is 1 hour to one week; and / or the drying is carried out by one or more of the following methods: freeze drying, spray drying, vacuum drying at room temperature, hot air drying, and microwave drying.

[0018] Further, in step S2, the reaction conditions between the metal ion-nuclear receptor protein active nanomaterial and the environmental medium are as follows: after the metal ion-nuclear receptor protein active nanomaterial is mixed with the environmental medium, it is incubated at room temperature in the dark for 2 hours.

[0019] Furthermore, in step S3, the liquid-solid extraction uses ethyl acetate as the extractant and is performed three times.

[0020] Further, in step S5, the screened samples and control samples are subjected to high-resolution liquid chromatography-mass spectrometry detection, and the raw data are preprocessed using Compound Discoverer software, including feature extraction, alignment, and deconvolution. Differential compounds are screened based on the peak area fold change and the p-value of the significance test results between the screened samples and control samples. The screened samples are conjugates captured by metal ion-nuclear receptor protein active nanomaterials, containing specific ligands and non-specific substances, while the control samples are conjugates captured by metal ion-nuclear receptor protein inactivating nanomaterials, including non-specific conjugates.

[0021] Furthermore, in step S5, the screening criteria for the differential compounds are: fold change > 3, p < 0.05. To further eliminate the differences in experimental background and the materials themselves, two types of background controls are introduced in the data processing: First, by comparing the metal ion-nuclear receptor protein active nanomaterials with added environmental media and those without added environmental media, substances with no significant difference between the two groups (fold change > 3 and p < 0.05) are excluded; Second, by comparing the metal ion-nuclear receptor protein active nanomaterials without added environmental media and those without added environmental media, compounds caused by the differences in the materials themselves (fold change > 3 and p < 0.05) are removed.

[0022] Further, in step S6, the structure of the differentially expressed chemical is identified by searching the online chemical database EPA Toxcast in combination with mass spectrometry databases (mzCloud mass spectrometry database and local mass spectrometry database). The search and identification restrictions are: mass tolerance of 5 ppm, minimum element count of CH, maximum element count of C90H190Br10Cl10F10K2N10Na2O18P5S5, unsaturation of 0-40, and H / C ratio of 0.1-3.5.

[0023] The technical principle of this invention is to first utilize active metal ion-nuclear receptor protein nanomaterials to capture binding-active effector substances from complex environmental media, then use inactivated metal ion-nuclear receptor protein inactivated nanomaterials as a negative control, and finally combine ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-QE-MS / MS) for high-throughput, non-targeted biological effector substance analysis. Compared with the prior art, the beneficial effects of this invention are:

[0024] Unlike traditional protein selection mass spectrometry that uses empty carrier proteins as controls, this invention introduces metal ion-nuclear receptor protein inactivation nanomaterials as negative controls, providing the same non-specific binding sites as the target nuclear receptor protein. This not only effectively eliminates the non-specific adsorption effect of the target protein and significantly reduces false positive results, but also avoids false negatives caused by the lack of a control system, thus improving the accuracy of screening. It can be applied to the specific screening and structural identification of effector substances that bind to nuclear receptors in the environment, and can also be used for drug development. Attached Figure Description

[0025] Figure 1 The image shown is a scanning electron microscope (SEM) image of the Co-retinoic acid receptor protein-active nanomaterials in the examples.

[0026] Figure 2 This is a scanning electron microscope image of the Cu-retinoic acid receptor protein inactivated nanomaterial in the example.

[0027] Figure 3 The bar chart shows the recovery rates of the positive substance AM580 by the Co-retinoic acid receptor protein active nanomaterial and the Cu-retinoic acid receptor protein inactivating nanomaterial, respectively, in the examples.

[0028] Figure 4 The results show the screening results of empty carrier protein nanomaterials and Cu-retinoic acid receptor protein inactivating nanomaterials used as negative controls in the examples. Detailed Implementation

[0029] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0030] The following embodiments will use the identification of biologically effective substances in common environmental media as an example to illustrate the technical solution of the present invention in more detail.

[0031] The specific preparation conditions of the materials in the following examples, including buffer pH, protein concentration, metal ion concentration, reaction temperature and time, drying method, etc., can be found in patent document CN117069858A.

[0032] Example 1

[0033] This embodiment detects biological effectors in Yellow River water samples that specifically bind to the RARα recombinant nuclear receptor protein, as detailed below:

[0034] 1. Preparation of metal ion-nuclear receptor protein active nanomaterials and metal ion-nuclear receptor protein inactivating nanomaterials

[0035] 1) Prepare PBS buffer by weighing 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 and dissolving them in 500 mL of distilled water; prepare 40 mM cobalt chloride aqueous solution and 40 mM copper chloride aqueous solution.

[0036] 2) The recombinant retinoic acid receptor protein with histidine tag (commercially available or self-made) was replaced in the PBS solution of step 1 by desalting, dialysis and other methods, and its concentration was diluted to 0.5 mg / mL to obtain a protein solution.

[0037] 3) Preparation of metal ion-nuclear receptor protein active nanomaterials: Take 6 mL of the protein solution from step 2, add 0.12 mL of cobalt chloride solution, mix well, and let stand at 20 ℃ for 72 h. Collect the precipitate, wash with water, and dry under vacuum at 4 ℃ to obtain Co-retinoic acid receptor protein active nanomaterials. Its scanning electron microscope results are shown below. Figure 1 As shown.

[0038] 4) Preparation of metal ion-nuclear receptor protein inactivating nanomaterials: Using the same method, the protein solution from step 2 was mixed with an equal volume (0.12 mL) of copper chloride solution, allowed to stand for reaction, washed, and vacuum dried to obtain Cu-retinoic acid receptor protein inactivating nanomaterials. Their scanning electron microscopy results are shown below. Figure 2 As shown.

[0039] 5) Preparation of empty carrier protein materials: Metal ion-protein nanomaterials were prepared using empty carrier proteins, following the same steps as in step 3, to obtain empty carrier protein materials.

[0040] 2. Activity evaluation of the metal ion-recombinant nuclear acceptor nanomaterials prepared above

[0041] Take 0.5 mg of each of the above-mentioned materials, add 1 mL of water, and then add 10 μL of AM580, a positive control of retinoic acid receptor protein at a concentration of 100 ppb, to the system. Mix well and rotate at room temperature for 2 h. Centrifuge and discard the supernatant. Wash three times with 1 mL of ultrapure water, add 1 mL of ethyl acetate, shake and extract for 10 min, repeat three times, combine the extracts and blow dry with high-purity nitrogen, and make up to 100 μL with methanol. Determine the content of AM580 by liquid chromatography-tandem mass spectrometry and calculate the recovery rate.

[0042] Liquid chromatography-tandem mass spectrometry analysis was performed using an UltiMate 3000 ultra-high performance liquid chromatography system coupled with a TSQQuantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The detection conditions and parameter settings can be found in the method described in CN117069858A.

[0043] Evaluation results as follows Figure 3 As shown, the recovery rate of the positive substance AM580 by the Co-retinoic acid receptor protein active nanomaterial is higher than 90%, while the recovery rate of the positive substance AM580 by the Cu-retinoic acid receptor protein inactivating nanomaterial is only 8.5%.

[0044] 2. Detection of biological effect substances in Yellow River water samples that specifically bind to recombinant retinoic acid receptor protein, as detailed below:

[0045] 1) Yellow River water samples were filtered through a 0.7 μm glass fiber membrane (GF / C, Whatman) to remove suspended solids, and then enriched using an HLB solid-phase extraction column (Oasis, 500 mg / 6 mL, Waters, USA). The extraction column was activated sequentially with 6 mL of dichloromethane, 6 mL of methyl tert-butyl ether, 6 mL of methanol, and 6 mL of ultrapure water (Milli-Q). After loading the samples, they were eluted sequentially with 3 mL of methanol:methyl tert-butyl ether (1:1), 3 mL of methanol, and 3 mL of dichloromethane. The eluents were combined, dried under high-purity nitrogen, and dissolved in 1.5 mL of DMSO. 100 μL of each sample was used for subsequent protein affinity screening.

[0046] 2) Take 3 mg of the Co-retinoic acid receptor protein active nanomaterial prepared in Example 1, mix it with 60 μL of water sample extract and 6 mL of ultrapure water, and incubate it by rotation at room temperature for 2 h.

[0047] 3) After centrifugation, discard the supernatant, wash the material three times with 1 mL of ultrapure water, add 2 mL of ethyl acetate, shake and extract for 10 min, repeat the extraction three times, and combine the extracts.

[0048] 4) The combined extract was dried by blowing with high-purity nitrogen and diluted to 100 μL with methanol. High-resolution mass spectrometry analysis was performed to obtain the mass spectrometry data of the target biological effect substance for subsequent data analysis and structural identification.

[0049] 5) The operation process and conditions of the control experiment are the same as those above, except that the metal ion-nuclear receptor protein active nanomaterial is replaced with an equal amount of Cu-retinoic acid receptor protein inactivating nanomaterial or an equal amount of empty carrier protein material. A blank group experiment is set up. The experimental process is the same, but no water sample extract is added. Only an equal volume of DMSO is added as a solvent control.

[0050] 3. High-throughput material structure identification

[0051] 1) Samples were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-MS / MS). An ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters) was used for separation. Mobile phase A was ultrapure water, and mobile phase B was methanol. The gradient elution program was as follows: 0–3 min, linearly increasing from 5% B (95% A) to 60% B; 3–13 min, increasing from 60% B to 100% B and holding for 3 min; 16–16.1 min, gradient recovery to initial conditions and holding for 20 min. The flow rate was 0.3 mL / min, the column temperature was 40 ℃, the injection volume was 5 µL, and the sample chamber temperature was 10 ℃. Mass spectrometry was performed using Q Exactive UHPLC-MS / MS (Thermo Fisher), with positive and negative ion modes in Full MS-ddMS2 mode. The specific parameters are as follows: ionization method is H-ESI; spray voltage is 3500 V; carrier gas flow rate is 30 arb; auxiliary gas flow rate is 10 arb; ion transmission tube temperature is 320 ℃; auxiliary gas temperature is 350 ℃; scan range is 100-1200 m / z; primary mass spectrometry resolution is 70,000 FWHM; secondary mass spectrometry resolution is 17,500 FWHM; collision energy NCE is set to gradients of 10, 30, and 50 eV.

[0052] 2) After obtaining the raw data, Compound Discoverer 3.3 software was used for processing. The workflow included file input, spectrum selection, retention time comparison, unknown compound detection, gap filling, elemental composition prediction, Chemspider database search, and mzCloud database search. The parameters were set as follows: In the spectrum selection module, the retention time range was 0.5-18 min, and the mass range was 70-1000 Da; in the retention time comparison module, an adaptive curve model was used, with a maximum shift of 0.5 min and a mass tolerance of 5 ppm; in the unknown compound detection module, the mass tolerance was 5 ppm, the peak intensity tolerance was 30%, the signal-to-noise ratio was >3, and the minimum peak intensity was 1,000,000. In positive ion mode, the adducts considered include [M+H]+1, [M+Na]+1, [M+K]+1, [M+H-H2O]+1, [M+NH4]+1, and [M+H+MeOH]+1; in negative ion mode, the adducts considered include [MH]−1, [M–H-H2O]−1, [M–H+HAc]−1, and [M–H+FA]−1. In elemental composition prediction, the minimum element count is CH, and the maximum element count is C90H190Br10Cl10F10K2N10Na2O18P5S5; the unsaturation range is 0-40, and the H / C ratio is 0.1-3.5. In mzCloud matching, the ion activation energy tolerance is 30, and the matching factor threshold is 10. To reduce artifacts caused by peak extraction algorithms, the raw mass spectrometry data was converted to mzXML format using MSConvert software (ProteoWizard, Version 3.0.19064), and peak extraction was performed again using the R package XCMS to retain the ion features detected by both algorithms.

[0053] 3) Based on peak area data, differential ions meeting the following criteria were screened: the ratio of the average peak area of ​​the receptor protein group to the control group was >3, and the difference was statistically significant (t-test, p < 0.05). Furthermore, the ratio of the peak area of ​​this differential ion in the receptor protein group to the blank group was also >3, and the difference was significant (p < 0.05). These ions were considered potential receptor-active compounds. Experimental results showed that 973 differential ions were screened using Cu-retinoic acid receptor protein inactivating nanomaterials as a control; and 1476 differential ions were screened using empty carrier protein nanomaterials as a control.

[0054] 4) Compare the secondary mass spectra of the differentially expressed ions with the mzCloud library and the local mass spectrometry library. A successful match is considered to have at least two matching daughter ions (mass difference < 5 ppm). For compounds with successful matches, purchase standards and verify their consistency with the samples in retention time and secondary mass spectra to finally confirm the compound structure.

[0055] 5) After confirmation by standard samples, 14 chemical substances were identified using empty carrier protein nanomaterials as negative controls (Table 1), and 23 chemical substances were identified using Cu-retinoic acid receptor protein inactivation nanomaterials as negative controls (Table 2).

[0056] Table 1: Information on target substances identified using empty carrier protein nanomaterials as a negative control

[0057]

[0058]

[0059]

[0060] Table 2: Information on target substances identified using Cu-retinoic acid receptor protein inactivating nanomaterials as a negative control

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] 6) By reviewing relevant literature and the TOX21 program database, 12 compounds were found to have reported biological effects related to retinoic acid receptors. For the remaining compounds, whose biological effects were not yet reported, a yeast two-hybrid assay was used to verify their biological effects. The results are as follows: Figure 3 As shown, using Cu-retinoic acid receptor protein inactivating nanomaterials as a negative control, 23 bioactive substances were screened, while using empty carrier protein nanomaterials as a negative control only screened 12 bioactive substances, with 2 false positives. Comprehensive analysis indicates that using Cu-retinoic acid receptor protein inactivating nanomaterials as a negative control effectively eliminates non-specific adsorption, reduces false positives and false negatives, and thus improves the specificity of protein affinity mass spectrometry screening results.

[0067] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials, characterized in that, Includes the following steps: S1. The recombinant nuclear receptor protein was mixed with two metal ions in phosphate buffer solution, allowed to stand for reaction, the precipitate was collected, washed with water and dried to obtain metal ion-nuclear receptor protein active nanomaterials and metal ion-nuclear receptor protein inactivating nanomaterials, respectively. S2. Mix the metal ion-nuclear receptor protein active nanomaterial with the environmental medium to be tested, so that it binds to the biological effect substances in the environmental medium; S3. Separate the metal ion-nuclear receptor protein active nanomaterial and perform liquid-solid extraction on it to obtain a screening sample; S4. Using the metal ion-nuclear receptor protein inactivation nanomaterial, a control sample is obtained based on the same operation process as in steps S2 and S3. S5. By comparing and screening the differential compounds between the screened samples and the control samples; S6. Search and identify the above-mentioned differential compounds using chemical databases and mass spectrometry databases.

2. The method for specific screening of biological effect substances based on metal ion-nuclear acceptor protein nanomaterials according to claim 1, characterized in that, In step S1, the recombinant nuclear receptor protein is selected from one or more of the following recombinant nuclear receptor proteins: AR, ERα, ERβ, ERγ, PR, GR, ERR, PPARα, PPARδ, PPARγ, RXRα, RXRβ, RXRγ, RARα, RARβ, RARγ, TRα, TRβ, VDR, PXR, CAR, LXRα, LXRβ, FXR, and AhR.

3. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 1 or 2, characterized in that, In step S1, the recombinant nuclear receptor protein is a human nuclear receptor protein with a histidine tag.

4. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 1, characterized in that, In step S1, the metal ion is selected from one or more of calcium ions, magnesium ions, iron ions, ferrous ions, copper ions, barium ions, divalent manganese ions, divalent nickel ions, divalent cobalt ions, and zinc ions.

5. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 1, characterized in that, In step S1, the mass-volume concentration of the recombinant nuclear receptor protein in phosphate buffer is 0.1–50 mg / mL; And / or the pH of the phosphate buffer is 5.0 to 9.0; And / or the concentrations of hydrogen phosphate and dihydrogen phosphate in the phosphate buffer are both 1–1000 mM; And / or the temperature of the standing reaction is 0 to 40°C, and the standing reaction time is 1 hour to one week; And / or the drying process employs one or more of the following: freeze drying, spray drying, vacuum drying at room temperature, hot air drying, and microwave drying.

6. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 1, characterized in that, In step S2, the metal ion-nuclear receptor protein active nanomaterial is mixed with the test environment medium and then incubated at room temperature in the dark for 2 hours.

7. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 1, characterized in that, In step S3, the liquid-solid extraction uses ethyl acetate as the extractant and is performed three times.

8. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 1, characterized in that, In step S5, the screened samples and control samples are subjected to high-resolution liquid chromatography-mass spectrometry (HPLC-MS) detection, and the raw data are preprocessed using Compound Discoverer software, including feature extraction, alignment, and deconvolution. Differential compounds are screened based on the peak area fold change and the p-value of the significance test results between the screened samples and control samples. The screened samples are conjugates captured by metal ion-nuclear receptor protein active nanomaterials, including specific ligands and non-specific substances. The control samples are conjugates captured by metal ion-nuclear receptor protein inactivating nanomaterials, including non-specific conjugates.

9. The method for specific screening of biological effect substances based on metal ion-nuclear receptor protein nanomaterials according to claim 8, characterized in that, In step S5, the screening conditions for the differential compounds are: fold change > 3, p < 0.05; Two types of background controls were introduced in the data processing: First, by comparing metal ion-nuclear receptor protein active nanomaterials with added environmental media and those without added environmental media, substances with no significant difference between the two groups (fold change > 3 and p < 0.05) were excluded; Second, by comparing metal ion-nuclear receptor protein active nanomaterials without added environmental media and those without added environmental media, compounds caused by material-specific differences (fold change > 3 and p < 0.05) were removed.

10. The method for specific screening of biological effect substances based on metal ion-nuclear acceptor protein nanomaterials according to claim 1, characterized in that, In step S6, the structure of the differentially expressed chemical is identified by searching the online chemical database EPA Toxcast in conjunction with a mass spectrometry database. The search and identification conditions are as follows: mass tolerance is 5 ppm, minimum element count is CH, maximum element count is C90H190Br10Cl10F10K2N10Na2O18P5S5, unsaturation is set to 0-40, and H / C ratio is set to 0.1-3.5.

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