Method and device for extracting and analyzing endocrine disrupter

Abstract

The invention discloses an endocrine disrupter extraction and analysis method and device, relates to the technical field of endocrine disrupter analysis, and aims to extract and analyze residual endocrine disrupter in a human body and collect a urine sample. A solid phase extraction column is used to enrich and purify endocrine disrupters in a sample. Removing high-molecular-weight substances by adopting gel permeation chromatography after solid-phase extraction; and selectively adsorbing the target endocrine disrupter by adopting the molecularly imprinted polymer. Rapid screening of specific endocrine disruptors is realized by adopting an immunoassay technology, so that an analysis result is quicker and more accurate. And performing data analysis and compound identification by using software. Extraction and analysis of the endocrine disrupter remaining in the body are achieved, the concentration of the endocrine disrupter remaining in the body can be directly reflected, and therefore a more accurate inducement diagnosis result of the endocrine dyscrasia symptom is given.

Description

Technical Field

[0001] The present invention relates to the technical field of endocrine disruptor analysis, and in particular to a method and device for extracting and analyzing endocrine disruptors. Background Art

[0002] Some artificially synthesized chemicals can interfere with the human endocrine system in various ways, such as blocking the action of hormones or accelerating the decomposition of hormones, affecting the body's normal hormone levels and causing endocrine abnormalities. Therefore, these endocrine disrupting substances from the external environment are collectively referred to as environmental endocrine disruptors (EDCs). After these exogenous compounds enter the body, they activate or inhibit the function of the endocrine system, interfere with the synthesis, release, transport, metabolism and binding of normal endocrine substances in the body, thereby destroying the role of the endocrine system in maintaining and regulating the balance of the body's internal environment. Therefore, these substances are also called environmental hormones.

[0003] EDCs can be generally divided into two categories: pesticides and industrial compounds, most of which are organic compounds. Pesticides account for about 60%, including herbicides, insecticides, fungicides, fumigants, etc.; industrial compounds include resin raw materials and plasticizers, surfactant degradation products, insulating oils, preservatives, flame retardants, industrial by-products and other heavy metals.

[0004] Endocrine disrupting chemicals (EDCs) are a class of chemicals present in the environment that can interfere with the normal function of the endocrine system in humans and animals. Currently, the extraction and analysis techniques for this type of compounds mainly include the following:

[0005] Chemical and biological analysis methods: These methods are used to detect endocrine disruptors in water. They cover sample pretreatment techniques as well as subsequent chemical and biological analysis.

[0006] Biological method and mass spectrometry: Biological method is a method to determine whether a compound is an endocrine disruptor and its estrogenic activity, including whole organ method, cell method and non-cell method. Mass spectrometry has high sensitivity and accuracy in the quantitative analysis of endocrine disruptors, but the sample pretreatment process has a great influence on the accuracy of the entire test result.

[0007] The above extraction techniques have some drawbacks:

[0008] Complex pretreatment process: The sample pretreatment process usually consumes a lot of time and resources and may affect the accuracy of the results.

[0009] Use of organic solvents: Traditional extraction methods often require the use of large amounts of organic solvents, which not only pollute the environment but may also pose health risks to experimenters.

[0010] Detection limit and accuracy issues: Some methods may not have low enough detection limits or insufficient accuracy when detecting trace levels of endocrine disruptors.

[0011] Moreover, the above methods cannot directly detect the endocrine disruptors remaining in the human body. Summary of the invention

[0012] In order to solve the technical problem of detecting endocrine disruptors remaining in the human body, the present invention provides a method and device for extracting and analyzing endocrine disruptors. The following technical solution is adopted:

[0013] A method for extracting and analyzing endocrine disruptors comprises the following steps:

[0014] Step 1, collecting urine samples;

[0015] Step 2, pre-treating the urine sample at low temperature;

[0016] Step 3, extracting using solid phase extraction technology;

[0017] Step 4, using gel permeation chromatography to purify and remove high molecular weight substances;

[0018] Step 5, using molecular imprinting polymers to selectively adsorb target endocrine disruptors;

[0019] Step 6, using immunoassay technology to detect target endocrine disruptors;

[0020] Step 7, the test results are input into the endocrine disruptor analysis module, and the endocrine disruptor analysis module establishes a standard curve according to the OD value of the standard, substitutes the OD value of the sample into the standard curve, and calculates the concentration of the target endocrine disruptor.

[0021] By adopting the above technical solution, the extraction and analysis of endocrine disruptors remaining in the human body is a complicated process. Urine samples are used for analysis here, and 24-hour urine samples are collected. Ensure that the sample is uniform for subsequent processing. If the sample is not tested within 24 hours, the urine sample needs to be frozen to prevent the degradation of endocrine disruptors. If the test is performed within 24 hours, low-temperature pretreatment is required to try to avoid the degradation interference of endocrine disruptors in the sample;

[0022] Solid Phase Extraction (SPE): Use solid phase extraction columns to enrich and clean up endocrine disruptors in samples.

[0023] After solid phase extraction, gel permeation chromatography (GPC) is used to remove high molecular weight substances; molecular imprinting polymers (MIPs) can selectively adsorb target endocrine disruptors.

[0024] Immunoassay technology, such as enzyme-linked immunosorbent assay (ELISA), can be used to quickly screen specific endocrine disruptors, making the analysis results faster and more accurate. Software is used for data analysis and compound identification. Through these steps, the extraction and analysis of residual endocrine disruptors in the body can be achieved, which can directly reflect the concentration of residual endocrine disruptors in the body, thereby providing more accurate diagnostic results of the causes of endocrine disorders.

[0025] Optionally, in step 2, the urine sample is placed in a sample container of a low-temperature pretreatment device, the temperature is adjusted to 2°C-5°C, and the stirring blade speed of the sample container is set to 10 revolutions per minute to wait for extraction.

[0026] By adopting the above technical solution, the urine sample is kept at a low temperature and the homogenization stirring is maintained at a low speed, which can maintain the uniform distribution of sample components and avoid precipitation or stratification. For some samples that are easy to precipitate, stirring can help maintain their dissolved state, providing a more stable sample for subsequent solid phase extraction.

[0027] Optionally, step 3 includes the following sub-steps:

[0028] Step 31, selecting a suitable SPE column according to the properties of the target endocrine disruptors;

[0029] Step 32, column activation, using 5-10% v / v methanol or acetonitrile for activation;

[0030] Step 33, loading the sample, passing the extract through the SPE column, and controlling the flow rate at 1-2 mL / min;

[0031] Step 34, washing, using an aqueous solution containing 5-10% of an organic solvent to wash and remove impurities;

[0032] Step 35, drying, turning off the vacuum pump, allowing the SPE column to dry naturally or blow dry with nitrogen;

[0033] Step 36, elution, uses 15-20% v / v methanol or acetonitrile for elution.

[0034] By adopting the above technical solution, select the appropriate SPE column: select a suitable SPE column (such as C18, HLB, silica gel, etc.) according to the properties of the target endocrine disruptors. Activation: Methanol or acetonitrile is usually used for activation. Loading: Pass the extract through the SPE column with a flow rate controlled at 1-2mL / min. Washing: Use an aqueous solution containing 5-10% organic solvent to wash and remove impurities. Drying: Turn off the vacuum pump and let the SPE column dry naturally or blow it dry gently with nitrogen. Elution: Use a solution containing a higher proportion of organic solvent (such as methanol or acetonitrile) for elution.

[0035] Optionally, step 4 includes the following sub-steps:

[0036] Step 41, diluting the extract to a recommended GPC concentration;

[0037] Step 42, loading the sample into the GPC system;

[0038] Step 43, through the GPC column, the high molecular weight substances are eluted first, and the low molecular weight target substances are collected later;

[0039] Step 44, collecting the target endocrine disruptor fractions using the GPC fraction collector.

[0040] By adopting the above technical scheme, Gel Permeation Chromatography (GPC) is a technique used to determine the molecular weight and molecular weight distribution of polymers. Determining the sample concentration suitable for GPC requires consideration of multiple factors, and different specifications of chromatographic columns have different tolerances to sample concentration. Generally, the finer the filler of the chromatographic column, the lower the sample concentration should be. The chemical properties, molecular weight range, polarity of the sample, and the solubility of the sample in the solvent will affect the choice of concentration. The solvent must be able to fully dissolve the sample without damaging the chromatographic column.

[0041] Consult the operating manual provided by the column manufacturer, which usually gives the recommended sample concentration range. You can also use different sample concentrations to observe the response of the column by performing a series of preliminary experiments. Determine the concentration range: Low concentration: You can start with a lower concentration, such as 0.1-1.0% (w / v). Medium concentration: If the detector signal is too weak at low concentrations, you can gradually increase the concentration, for example to 2-5% (w / v). High concentration: For very sensitive detectors or non-polymer samples, the concentration may be higher, but it is generally not recommended to exceed 10% (w / v).

[0042] Optionally, step 5 includes the following sub-steps:

[0043] Step 51, preparing MIPs, preparing specific MIPs for target endocrine disruptors;

[0044] Step 52, mixing the target endocrine disruptor fraction obtained in step 4 with the MIPs;

[0045] Step 53, washing the MIPs with a washing solvent to remove non-specifically adsorbed substances;

[0046] Step 54, transfer the MIPs bound to the target to a buffer solution with a set pH value of 25°C-30°C, stir at a constant speed of 100-300 rpm, and elute the target bound to the MIPs.

[0047] By adopting the above technical scheme, the washing step of molecularly imprinted polymers (MIPs) is a key process to ensure that unreacted monomers, crosslinkers, initiators and other impurities are removed from the polymer. An appropriate solvent is a solvent that can effectively remove these impurities without destroying the polymer structure or affecting its specificity. The following are factors that should be considered when defining an appropriate solvent: Choose a polar solvent that is compatible with the impurities, usually those that can dissolve monomers and crosslinkers. If the impurities are non-polar, a non-polar solvent should be selected. The washing solvent should have appropriate volatility so that it can be easily evaporated from the polymer after the washing process. The washing solvent should not react with the functional groups in the polymer to avoid destroying the selectivity of the imprinted site. The selected solvent should be as environmentally friendly as possible, harmless to the human body, and easy to handle.

[0048] Commonly used cleaning solvents include methanol, ethanol, acetone, ethyl acetate, dichloromethane, ether, etc.

[0049] Directly transfer the MIPs bound to the target to a buffer solution with a specific pH value, stir or shake at an appropriate temperature for a period of time, so that the change in pH affects the binding of the target to the MIPs. The elution time depends on the binding strength of the target to the MIPs and the effect of the pH change. The change in pH can affect the non-covalent interactions between the target and the polymer, such as hydrogen bonding, ion exchange, and hydrophobic interactions.

[0050] Optionally, step 6 includes the following sub-steps:

[0051] Step 61, adding 100 μL of sample to each microplate;

[0052] Step 62, sealing the microplate with a sealing film;

[0053] Step 63, incubating at room temperature or set temperature for 1-2 hours;

[0054] Step 64, removing the sealing film and pouring out the liquid in the well;

[0055] Step 65, fill each microwell with a washing solution, let it stand for 10 minutes and then pour it out, repeating 3-5 times;

[0056] Step 66, adding enzyme-labeled antibody, adding 100 μL of enzyme-labeled antibody to each microwell, sealing the plate and incubating for 60 minutes to 80 minutes, and repeating step 65 to remove unbound enzyme-labeled antibody;

[0057] Step 67, adding 100 μL of substrate solution to each microwell, and incubating at room temperature for a certain time of 15-30 minutes to allow the enzyme to catalyze the substrate to produce a color reaction;

[0058] Step 68, adding 50 μL of stop solution to each microwell to terminate the enzyme reaction;

[0059] Step 69, using an ELISA reader to read the absorbance OD value at a set wavelength.

[0060] By adopting the above technical scheme, sample preparation: if necessary, the sample is extracted, purified and diluted to meet the requirements of the kit. Sample addition: add the standard or diluted sample to the microplate, usually duplicate wells for each standard and sample. Add to the antibody-coated microplate, usually 100μL to each microwell. Seal the plate: seal the microplate with a sealing film to prevent water evaporation and cross contamination. Incubate at room temperature or a specific temperature for a certain time (such as 1-2 hours) to allow the antigen to bind to the solid phase antibody. Wash: remove the sealing film and pour out the liquid in the well. Fill each microwell with washing solution, let it stand for a period of time and then pour it out. Repeat the washing step 3-5 times to remove unbound substances. Add enzyme-labeled antibody: add enzyme-labeled antibody to each microwell, usually 100μL. Seal the plate and incubate for a certain time (such as 1 hour) to allow the enzyme-labeled antibody to bind to the antigen-antibody complex on the solid phase. Wash again: repeat the washing step to remove unbound enzyme-labeled antibody. Add substrate: Add substrate solution to each microwell, usually 100μL. Incubate at room temperature or a specific temperature for a certain period of time (such as 15-30 minutes) to allow the enzyme to catalyze the substrate to produce a color reaction. Stop reaction: Add stop solution to each microwell, usually 50μL, to stop the enzyme reaction. Read the plate: Use a microplate reader to read the absorbance (OD value) at an appropriate wavelength (usually 450nm).

[0061] Optionally, in step 7, use Excel, GraphPadPrism or Python software to draw a standard curve, and enter the following data in the software: first column: concentration of the standard; second column: average OD value corresponding to each concentration; set the standard concentration as the X-axis and the OD value as the Y-axis; draw a scatter plot, where each point represents the concentration of a standard and the corresponding OD value;

[0062] The four-parameter logistic regression curve fitting method was selected to fit the curve. After the curve was fitted, the software provided an equation for calculating the sample OD value.

[0063] Optionally, the software provides the following equation:

[0064]

[0065] Where Y is the OD value, X represents the sample concentration, A is the maximum OD value, D is the minimum OD value, C is the inflection point of the OD value, and C is the sample concentration when C is equal to (A+D) / 2.

[0066] By adopting the above technical scheme, a large number of standards with known concentrations and measured OD values ​​are required. The concentrations of these standards will cover the concentration range you expect to detect in the samples. Use Excel, GraphPadPrism, R, Python or other graphing software to draw the standard curve. Enter the following data into the software: First column: concentration of the standard (usually logarithmic or linear dilution). Second column: average OD value corresponding to each concentration (if there are duplicate wells, calculate the average). Set the standard concentration as the X-axis and the OD value as the Y-axis. Draw a scatter plot with each point representing the concentration of a standard and the corresponding OD value. Choose an appropriate curve fitting method. For ELISA, the following methods are commonly used: Four-parameter logistic regression (4-PL): Provides the best fit and can handle a wide range of concentrations. Three-parameter logistic regression (3-PL): Use when the lower limit of the standard curve is close to zero. Straight line (if the data is linear in the detection range). Use the software's curve fitting function to fit the selected model.

[0067] After the curve is fitted, the software provides an equation to calculate the concentration of unknown samples using the standard curve equation.

[0068] Substitute the OD value of the sample into the equation and solve for X, the sample concentration.

[0069] An endocrine disruptor analysis device comprises a data entry module, a memory and an endocrine disruptor input analysis module, wherein the data entry module communicates and interacts with the memory to input standard concentration, corresponding OD value data and OD value of a sample, the endocrine disruptor input analysis module comprises a computer and a display, the computer is communicatively connected with the memory, the computer is pre-installed with Excel, GraphPadPrism or Python software, and is pre-installed with an analysis program designed in step 7 of an endocrine disruptor extraction and analysis method, the data interactively inputted from the memory is entered into the analysis program to establish a standard curve, and the OD value of the sample is substituted into the standard curve to calculate the concentration of the target endocrine disruptor.

[0070] A computer is pre-installed with an analysis program designed according to step 7 of an endocrine disruptor extraction and analysis method, and the concentration of endocrine disruptors is analyzed based on the analysis program.

[0071] In summary, the present invention includes at least one of the following beneficial technical effects:

[0072] The present invention can provide a method and device for extracting and analyzing endocrine disruptors, extract and analyze endocrine disruptors remaining in the human body, and collect 24-hour urine samples. A solid phase extraction column is used to enrich and purify endocrine disruptors in the sample. After solid phase extraction, gel permeation chromatography is used to remove high molecular weight substances; molecular imprinting polymers are used to selectively adsorb target endocrine disruptors. Immunoassay technology is used to achieve rapid screening of specific endocrine disruptors, making the analysis results faster and more accurate. Software is used for data analysis and compound identification. The extraction and analysis of residual endocrine disruptors in the body can directly reflect the concentration of residual endocrine disruptors in the body, thereby providing a more accurate diagnosis result of the cause of endocrine disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is a schematic flow chart of a method for extracting and analyzing endocrine disruptors of the present invention;

[0074] Figure 2 It is a schematic diagram of the component connection principle of an endocrine disruptor analysis device of the present invention.

[0075] Explanation of the accompanying drawings: 1. Data entry module; 2. Memory; 31. Computer; 32. Display. DETAILED DESCRIPTION

[0076] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0077] The embodiment of the invention discloses a method and a device for extracting and analyzing endocrine disruptors.

[0078] Reference Figure 1 and Figure 2 , Example 1, a method for extracting and analyzing endocrine disruptors, comprising the following steps:

[0079] Step 1, collecting urine samples;

[0080] Step 2, pre-treating the urine sample at low temperature;

[0081] Step 3, extracting using solid phase extraction technology;

[0082] Step 4, using gel permeation chromatography to purify and remove high molecular weight substances;

[0083] Step 5, using molecular imprinting polymers to selectively adsorb target endocrine disruptors;

[0084] Step 6, using immunoassay technology to detect target endocrine disruptors;

[0085] Step 7, the test results are input into the endocrine disruptor analysis module, and the endocrine disruptor analysis module establishes a standard curve according to the OD value of the standard, substitutes the OD value of the sample into the standard curve, and calculates the concentration of the target endocrine disruptor.

[0086] Extracting and analyzing endocrine disruptors remaining in the human body is a complex process. Urine samples are used for analysis here, and urine samples are collected for 24 hours. Ensure that the sample is uniform for subsequent processing. If the sample is not tested within 24 hours, the urine sample needs to be frozen to prevent the degradation of endocrine disruptors. If the test is performed within 24 hours, low-temperature pretreatment is required to try to avoid degradation interference of endocrine disruptors in the sample;

[0087] Solid Phase Extraction (SPE): Use solid phase extraction columns to enrich and clean up endocrine disruptors in samples.

[0088] After solid phase extraction, gel permeation chromatography (GPC) is used to remove high molecular weight substances; molecular imprinting polymers (MIPs) can selectively adsorb target endocrine disruptors.

[0089] Immunoassay technology, such as enzyme-linked immunosorbent assay (ELISA), can be used to quickly screen specific endocrine disruptors, making the analysis results faster and more accurate. Software is used for data analysis and compound identification. Through these steps, the extraction and analysis of residual endocrine disruptors in the body can be achieved, which can directly reflect the concentration of residual endocrine disruptors in the body, thereby providing more accurate diagnostic results of the causes of endocrine disorders.

[0090] In Example 2, in step 2, the urine sample is placed in a sample container of a low-temperature pretreatment device, the temperature is adjusted to 2°C-5°C, and the stirring blade speed of the sample container is set to 10 revolutions per minute to wait for extraction.

[0091] Keeping urine samples at low temperatures and maintaining a low-speed homogenization stirring can maintain uniform distribution of sample components and avoid precipitation or stratification. For some samples that are prone to precipitation, stirring can help maintain their dissolved state, providing a more stable sample for subsequent solid phase extraction.

[0092] Embodiment 3, step 3 comprises the following sub-steps:

[0093] Step 31, selecting a suitable SPE column according to the properties of the target endocrine disruptors;

[0094] Step 32, column activation, using 5-10% v / v methanol or acetonitrile for activation;

[0095] Step 33, loading the sample, passing the extract through the SPE column, and controlling the flow rate at 1-2 mL / min;

[0096] Step 34, washing, using an aqueous solution containing 5-10% of an organic solvent to wash and remove impurities;

[0097] Step 35, drying, turning off the vacuum pump, allowing the SPE column to dry naturally or blow dry with nitrogen;

[0098] Step 36, elution, uses 15-20% v / v methanol or acetonitrile for elution.

[0099] Select the appropriate SPE column: Select a suitable SPE column (such as C18, HLB, silica gel, etc.) according to the properties of the target endocrine disruptors. Activation: Methanol or acetonitrile is usually used for activation. Loading: Pass the extract through the SPE column with a flow rate of 1-2mL / min. Washing: Use an aqueous solution containing 5-10% organic solvent to wash and remove impurities. Drying: Turn off the vacuum pump and let the SPE column dry naturally or blow it dry gently with nitrogen. Elution: Use a solution containing a higher proportion of organic solvent (such as methanol or acetonitrile) for elution.

[0100] Embodiment 4, step 4 comprises the following sub-steps:

[0101] Step 41, diluting the extract to a recommended GPC concentration;

[0102] Step 42, loading the sample into the GPC system;

[0103] Step 43, through the GPC column, the high molecular weight substances are eluted first, and the low molecular weight target substances are collected later;

[0104] Step 44, collecting the target endocrine disruptor fractions using the GPC fraction collector.

[0105] Gel Permeation Chromatography (GPC) is a technique used to determine the molecular weight and molecular weight distribution of polymers. There are many factors to consider when determining the sample concentration suitable for GPC. Columns of different specifications have different tolerances to sample concentration. Generally, the finer the column filler, the lower the sample concentration should be. The chemical properties of the sample, the molecular weight range, the polarity, and the solubility of the sample in the solvent will affect the choice of concentration. The solvent must be able to fully dissolve the sample without damaging the column.

[0106] Consult the operating manual provided by the column manufacturer, which usually gives the recommended sample concentration range. You can also use different sample concentrations to observe the response of the column by performing a series of preliminary experiments. Determine the concentration range: Low concentration: You can start with a lower concentration, such as 0.1-1.0% (w / v). Medium concentration: If the detector signal is too weak at low concentrations, you can gradually increase the concentration, for example to 2-5% (w / v). High concentration: For very sensitive detectors or non-polymer samples, the concentration may be higher, but it is generally not recommended to exceed 10% (w / v).

[0107] Embodiment 5, step 5 comprises the following sub-steps:

[0108] Step 51, preparing MIPs, preparing specific MIPs for target endocrine disruptors;

[0109] Step 52, mixing the target endocrine disruptor fraction obtained in step 4 with the MIPs;

[0110] Step 53, washing the MIPs with a washing solvent to remove non-specifically adsorbed substances;

[0111] Step 54, transfer the MIPs bound to the target to a buffer solution with a set pH value of 25°C-30°C, stir at a constant speed of 100-300 rpm, and elute the target bound to the MIPs.

[0112] The washing step of molecularly imprinted polymers (MIPs) is a critical process to ensure that unreacted monomers, crosslinkers, initiators, and other impurities are removed from the polymer. An appropriate solvent is one that can effectively remove these impurities without destroying the polymer structure or affecting its specificity. The following are factors that should be considered when defining an appropriate solvent: Choose polar solvents that are compatible with the impurities, usually those that can dissolve monomers and crosslinkers. If the impurities are non-polar, a non-polar solvent should be selected. The washing solvent should have appropriate volatility so that it can be easily evaporated from the polymer after the washing process. The washing solvent should not react with the functional groups in the polymer to avoid destroying the selectivity of the imprinted site. The selected solvent should be as environmentally friendly, harmless to the human body, and easy to handle as possible.

[0113] Commonly used cleaning solvents include methanol, ethanol, acetone, ethyl acetate, dichloromethane, ether, etc.

[0114] Directly transfer the MIPs bound to the target to a buffer solution with a specific pH value, stir or shake at an appropriate temperature for a period of time, so that the change in pH affects the binding of the target to the MIPs. The elution time depends on the binding strength of the target to the MIPs and the effect of the pH change. The change in pH can affect the non-covalent interactions between the target and the polymer, such as hydrogen bonding, ion exchange, and hydrophobic interactions.

[0115] Embodiment 6, step 6 comprises the following sub-steps:

[0116] Step 61, adding 100 μL of sample to each microplate;

[0117] Step 62, sealing the microplate with a sealing film;

[0118] Step 63, incubating at room temperature or set temperature for 1-2 hours;

[0119] Step 64, removing the sealing film and pouring out the liquid in the well;

[0120] Step 65, fill each microwell with a washing solution, let it stand for 10 minutes and then pour it out, repeating 3-5 times;

[0121] Step 66, adding enzyme-labeled antibody, adding 100 μL of enzyme-labeled antibody to each microwell, sealing the plate and incubating for 60 minutes to 80 minutes, and repeating step 65 to remove unbound enzyme-labeled antibody;

[0122] Step 67, adding 100 μL of substrate solution to each microwell, and incubating at room temperature for a certain time of 15-30 minutes to allow the enzyme to catalyze the substrate to produce a color reaction;

[0123] Step 68, adding 50 μL of stop solution to each microwell to terminate the enzyme reaction;

[0124] Step 69, using an ELISA reader to read the absorbance OD value at a set wavelength.

[0125] Sample preparation: If necessary, extract, purify and dilute the sample to meet the requirements of the kit. Sample addition: Add the standard or diluted sample to the microplate, usually duplicate each standard and sample. Add to the antibody-coated microplate, usually 100μL to each microwell. Seal: Seal the microplate with a sealing film to prevent water evaporation and cross-contamination. Incubate at room temperature or a specific temperature for a certain period of time (such as 1-2 hours) to allow the antigen to bind to the solid phase antibody. Wash: Remove the sealing film and pour out the liquid in the well. Fill each microwell with washing solution, let it stand for a period of time, then pour it out. Repeat the washing step 3-5 times to remove unbound substances. Add enzyme-labeled antibody: Add enzyme-labeled antibody to each microwell, usually 100μL. Seal the plate and incubate for a certain period of time (such as 1 hour) to allow the enzyme-labeled antibody to bind to the antigen-antibody complex on the solid phase. Wash again: Repeat the washing step to remove unbound enzyme-labeled antibody. Add substrate: Add substrate solution to each microwell, usually 100μL. Incubate at room temperature or a specific temperature for a certain period of time (such as 15-30 minutes) to allow the enzyme to catalyze the substrate to produce a color reaction. Stop reaction: Add stop solution to each microwell, usually 50μL, to stop the enzyme reaction. Read the plate: Use a microplate reader to read the absorbance (OD value) at an appropriate wavelength (usually 450nm).

[0126] In Example 7, in step 7, Excel, GraphPadPrism or Python software is used to draw a standard curve, and the following data are input into the software: first column: concentration of the standard; second column: average OD value corresponding to each concentration; set the concentration of the standard as the X-axis and the OD value as the Y-axis; draw a scatter plot, where each point represents the concentration of a standard and the corresponding OD value;

[0127] The four-parameter logistic regression curve fitting method was selected to fit the curve. After the curve was fitted, the software provided an equation for calculating the sample OD value.

[0128] The software provides the following equation:

[0129]

[0130] Where Y is the OD value, X represents the sample concentration, A is the maximum OD value, D is the minimum OD value, C is the inflection point of the OD value, and C is the sample concentration when C is equal to (A+D) / 2.

[0131] You will need a large number of standards of known concentrations and have measured OD values. The concentrations of these standards will cover the range of concentrations you expect to detect in your samples. Use Excel, GraphPadPrism, R, Python, or other graphing software to plot the standard curve. Enter the following data into the software: First column: concentration of the standard (usually a logarithmic or linear dilution). Second column: average OD value corresponding to each concentration (calculate the average if there are duplicate wells). Set the standard concentration as the X-axis and the OD value as the Y-axis. Plot a scatter plot with each dot representing the concentration of a standard and the corresponding OD value. Choose an appropriate curve fitting method. For ELISA, the following methods are commonly used: Four-parameter logistic regression (4-PL): Provides the best fit and can handle a wide range of concentrations. Three-parameter logistic regression (3-PL): Use when the lower limit of the standard curve is close to zero. Straight line (if the data is linear over the detection range). Use the software's curve fitting function to fit the selected model.

[0132] After the curve is fitted, the software provides an equation to calculate the concentration of unknown samples using the standard curve equation.

[0133] Substitute the OD value of the sample into the equation and solve for X, the sample concentration.

[0134] Embodiment 8, an endocrine disruptor analysis device, comprising a data entry module 1, a memory 2 and an endocrine disruptor input analysis module, the data entry module 1 communicates and interacts with the memory 2 to input the standard concentration, the corresponding OD value data and the OD value of the sample, the endocrine disruptor input analysis module comprises a computer 31 and a display 32, the computer 31 is communicatively connected to the memory 2, the computer 31 is pre-installed with Excel, GraphPadPrism or Python software, and is pre-installed with an analysis program designed in step 7 of an endocrine disruptor extraction and analysis method, the data interactively inputted from the memory 2 is entered into the analysis program to establish a standard curve, and the OD value of the sample is substituted into the standard curve to calculate the concentration of the target endocrine disruptor.

[0135] Embodiment 9, a computer is pre-installed with an analysis program designed according to step 7 of a method for extracting and analyzing endocrine disruptors, and the concentration of endocrine disruptors is analyzed based on the analysis program.

[0136] The above are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting and analyzing endocrine disruptors, characterized in that: The following steps are involved: Step 1, collecting urine samples; Step 2, pre-treating the urine sample at low temperature; Step 3, extracting using solid phase extraction technology; Step 4, using gel permeation chromatography to purify and remove high molecular weight substances; Step 5, using molecular imprinting polymers to selectively adsorb target endocrine disruptors; Step 6, using immunoassay technology to detect target endocrine disruptors; Step 7, the test results are input into the endocrine disruptor analysis module, and the endocrine disruptor analysis module establishes a standard curve according to the OD value of the standard, substitutes the OD value of the sample into the standard curve, and calculates the concentration of the target endocrine disruptor.

2. The method for extracting and analyzing endocrine disruptors according to claim 1, characterized in that: In step 2, the urine sample is placed in a sample container of a low-temperature pretreatment device, the temperature is adjusted to 2°C-5°C, and the stirring blade speed of the sample container is set to 10 revolutions per minute to wait for extraction.

3. The method for extracting and analyzing endocrine disruptors according to claim 2, characterized in that: Step 3 includes the following sub-steps: Step 31, selecting a suitable SPE column according to the properties of the target endocrine disruptors; Step 32, column activation, using 5-10% v / v methanol or acetonitrile for activation; Step 33, loading the sample, passing the extract through the SPE column, and controlling the flow rate at 1-2 mL / min; Step 34, washing, using an aqueous solution containing 5-10% of an organic solvent to wash and remove impurities; Step 35, drying, turning off the vacuum pump, allowing the SPE column to dry naturally or blow dry with nitrogen; Step 36, elution, uses 15-20% v / v methanol or acetonitrile for elution.

4. The method for extracting and analyzing endocrine disruptors according to claim 3, characterized in that: Step 4 includes the following sub-steps: Step 41, diluting the extract to a recommended GPC concentration; Step 42, loading the sample into the GPC system; Step 43, through the GPC column, the high molecular weight substances are eluted first, and the low molecular weight target substances are collected later; Step 44, collecting the target endocrine disruptor fractions using the GPC fraction collector.

5. The method for extracting and analyzing endocrine disruptors according to claim 4, characterized in that: Step 5 includes the following sub-steps: Step 51, preparing MIPs, preparing specific MIPs for target endocrine disruptors; Step 52, mixing the target endocrine disruptor fraction obtained in step 4 with the MIPs; Step 53, washing the MIPs with a washing solvent to remove non-specifically adsorbed substances; Step 54, transfer the MIPs bound to the target to a buffer solution with a set pH value of 25°C-30°C, stir at a constant speed of 100-300 rpm, and elute the target bound to the MIPs.

6. The method for extracting and analyzing endocrine disruptors according to claim 5, characterized in that: Step 6 includes the following sub-steps: Step 61, adding 100 μL of sample to each microplate; Step 62, sealing the microplate with a sealing film; Step 63, incubating at room temperature or set temperature for 1-2 hours; Step 64, removing the sealing film and pouring out the liquid in the well; Step 65, fill each microwell with a washing solution, let it stand for 10 minutes and then pour it out, repeating 3-5 times; Step 66, adding enzyme-labeled antibody, adding 100 μL of enzyme-labeled antibody to each microwell, sealing the plate and incubating for 60 minutes to 80 minutes, and repeating step 65 to remove unbound enzyme-labeled antibody; Step 67, adding 100 μL of substrate solution to each microwell, and incubating at room temperature for a certain time of 15-30 minutes to allow the enzyme to catalyze the substrate to produce a color reaction; Step 68, adding 50 μL of stop solution to each microwell to terminate the enzyme reaction; Step 69, using an ELISA reader to read the absorbance OD value at a set wavelength.

7. The method for extracting and analyzing endocrine disruptors according to claim 6, characterized in that: In step 7, use Excel, GraphPadPrism or Python software to draw a standard curve. Enter the following data in the software: First column: concentration of the standard; Second column: average OD value corresponding to each concentration; Set the standard concentration as the X-axis and the OD value as the Y-axis; Draw a scatter plot, where each point represents the concentration of a standard and the corresponding OD value; The four-parameter logistic regression curve fitting method was selected to fit the curve. After the curve was fitted, the software provided an equation for calculating the sample OD value.

8. The method for extracting and analyzing endocrine disruptors according to claim 7, characterized in that: The software provides the following equation: Where Y is the OD value, X represents the sample concentration, A is the maximum OD value, D is the minimum OD value, C is the inflection point of the OD value, and C is the sample concentration when C is equal to (A+D) / 2.

9. An endocrine disruptor analysis device, characterized in that: The invention comprises a data entry module (1), a memory (2) and an endocrine disruptor analysis module, wherein the data entry module (1) communicates and interacts with the memory (2) to enter the concentration of a standard substance, corresponding OD value data and the OD value of a sample, and the endocrine disruptor analysis module comprises a computer (31) and a display (32), wherein the computer (31) is communicatively connected with the memory (2), the computer (31) is pre-installed with Excel, GraphPadPrism or Python software, and is pre-installed with an analysis program designed in step 7 of the endocrine disruptor extraction and analysis method according to claim 8, the data interactively entered into the memory (2) is entered into the analysis program to establish a standard curve, and the OD value of the sample is substituted into the standard curve to calculate the concentration of the target endocrine disruptor.

10. A computer, characterized in that: An analysis program designed in step 7 of the method for extracting and analyzing endocrine disruptors according to claim 8 is preinstalled, and the concentration of endocrine disruptors is analyzed based on the analysis program.

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