Method for detecting estradiol by using organic metal framework enriched combined enzyme-linked immunoassay

Abstract

This invention belongs to the field of analytical detection technology and relates to a method for detecting estradiol using a combination of organometallic framework enrichment and enzyme-linked immunosorbent assay (ELISA). A magnetic organometallic framework solid-phase extractant is used to enrich estradiol in the sample, followed by detection of the enriched estradiol using ELISA. The magnetic organometallic framework solid-phase extractant is prepared by subjecting mercaptoacetic acid-modified Fe3O4 particles, zirconium salt, and 2-amino-terephthalic acid to a solvothermal reaction at 110–130°C for 22–26 hours. The detection method provided by this invention has advantages such as short detection time, high sensitivity, good specificity, and high accuracy, thereby enabling the detection of trace amounts of estradiol in environmental water samples.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology and relates to a method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA). Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Estradiol (17β-estradiol, E2) is a natural estrogen secreted by mature ovarian follicles and is the most potent estrogen. In animal husbandry, adding estradiol can affect dairy cow growth, improve feed conversion rate, and promote estrus. However, some pharmaceutical and chemical plants discharge wastewater containing estradiol into rivers and groundwater systems, potentially impacting drinking water sources and the environment. According to the inventors' research, the concentration of E2 in environmental water samples is currently extremely low, making it difficult to detect using existing methods. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA). The detection method provided by the present invention has advantages such as short detection time, high sensitivity, good specificity, and high accuracy, thereby enabling the detection of trace amounts of estradiol in environmental water samples.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, a magnetic organometallic framework solid-phase extractant is used to enrich estradiol by subjecting mercaptoacetic acid-modified Fe3O4 particles, zirconium salt, and 2-amino-terephthalic acid to a solvothermal reaction at 110–130 °C for 22–26 h.

[0007] Organometallic frameworks exhibit a regular porous structure and a large specific surface area, making them excellent materials for solid-phase extraction. Magnetic solid-phase extraction (SPE) introduces magnetic groups into solid-phase extraction technology, allowing analytes to be easily separated from the liquid phase using a magnet. Magnetic organometallic framework materials, based on organometallic frameworks, simplify experimental operations and are more suitable for rapid detection conditions.

[0008] Enzyme-linked immunosorbent assay (ELISA) uses the principle of antigen-antibody reaction to detect analytes. It has the advantages of simple operation, high sensitivity, and good specificity, and is widely used in the field of rapid food safety testing. However, it is easily affected by factors such as organic solvents and structural analogs, which can easily lead to false positive and false negative test results.

[0009] This invention has found that enriching estradiol using the magnetic organometallic framework solid-phase extractant provided by this invention, followed by detection using enzyme-linked immunosorbent assay (ELISA), not only improves detection sensitivity but also has the advantages of high specificity and accuracy, thereby overcoming the defects of false positive and false negative results in ELISA detection.

[0010] On the other hand, a method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) involves enriching estradiol in the sample using the aforementioned magnetic organometallic framework solid-phase extractant, and then detecting the enriched estradiol using ELISA.

[0011] Thirdly, the application of the above-mentioned detection method in monitoring the concentration of estradiol in surface water.

[0012] The beneficial effects of this invention are as follows:

[0013] 1. This invention utilizes a magnetic organometallic framework solid-phase extractant to enrich estradiol in environmental water samples, and then uses ELISA to detect it, which has the advantages of short detection time, high sensitivity, good specificity, and high accuracy.

[0014] 2. The detection limit of the detection method provided by this invention is 50 times higher than that of the ELISA kit. It can effectively serve as an early warning for the detection of trace amounts of estradiol in environmental water samples. By tracing the source from low to high concentration, it helps to find the source of estradiol pollution, effectively identify pollution sources, and ensure the safety of drinking water for the public. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is a curve showing the effect of different pH values ​​on the recovery rate in an embodiment of the present invention;

[0017] Figure 2 This is a graph showing the comparative results of the determination of several estradiol structural and functional analogs at different concentrations by magnetic MOF enrichment-ELISA in an embodiment of the present invention.

[0018] Figure 3 This is a statistical chart showing the estradiol recovery rate of the magnetic MOFs materials reused in this invention embodiment;

[0019] Figure 4 This is a graph showing the results of five consecutive measurements using the magnetic MOFs-ELISA detection method in an embodiment of the present invention. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] Given that existing methods are difficult to detect trace amounts of estradiol in environmental water samples, this invention proposes a method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA).

[0023] In a typical embodiment of the present invention, a magnetic organometallic framework solid-phase extractant is provided for enriching estradiol. The extractant is obtained by solvothermal reaction of mercaptoacetic acid-modified Fe3O4 particles, zirconium salt, and 2-amino-terephthalic acid at 110–130 °C for 22–26 h.

[0024] The zirconium salts described in this invention are compounds whose cation is zirconium ion, such as zirconium sulfate, zirconium nitrate, zirconium chloride, etc.

[0025] In some embodiments, the mass ratio of mercaptoacetic acid-modified Fe3O4 particles, zirconium salt, and 2-amino-terephthalic acid is 1:7 to 9:5 to 7, preferably 1:7.5 to 8.5:5.5 to 6.5.

[0026] The solvothermal reaction described in this invention refers to a reaction in which the solvent of the reaction system is an organic compound, and the reaction is carried out under closed conditions and heated to form a high-pressure system. In some embodiments, the solvent for the solvothermal reaction is N,N-dimethylformamide.

[0027] In some embodiments, the preparation process of thioglycolic acid-modified Fe3O4 particles is as follows: Fe3O4 particles are added to a solution of thioglycolic acid and dispersed evenly, followed by room temperature modification treatment. Specifically, the even dispersion is achieved through ultrasonic treatment. The room temperature mentioned in this invention refers to the indoor ambient temperature, generally 15–30°C. Specifically, the modification treatment time is 10–14 hours. Specifically, the ratio of Fe3O4 particles to thioglycolic acid is 15–25:1.

[0028] Fe3O4 particles can be obtained commercially or synthesized in-house. In some embodiments, the Fe3O4 particles are prepared by adding ferrous and ferric salts to deoxygenated water, adding ammonia, heating and stirring the mixture under an inert atmosphere, followed by magnetic separation. Specifically, the reaction temperature is 70–90°C.

[0029] The ferrous salts described in this invention are compounds whose cation is ferrous ion, such as ferrous chloride, ferrous sulfate, and ferrous nitrate.

[0030] The iron salts described in this invention are compounds whose cations are ferric ions, such as ferric chloride, ferric sulfate, and ferric nitrate.

[0031] Specifically, the molar ratio of ferrous salt to ferric salt is 1:1.8 to 2.2.

[0032] Another embodiment of the present invention provides a method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA). The method uses the above-mentioned magnetic organometallic framework solid-phase extractant to enrich estradiol in the sample to be tested, and then uses ELISA to detect the enriched estradiol.

[0033] In some embodiments, the enrichment process is as follows: the magnetic organometallic framework solid-phase extractant is added to the sample to be tested and shaken for extraction, a first magnetic separation is performed, estradiol in the solid obtained from the first magnetic separation is eluted with an eluent, a second magnetic separation is performed, and the eluent from the second magnetic separation is collected.

[0034] In one or more embodiments, acetone was used as the eluent. Studies have shown that acetone can better elute estradiol from magnetic organometallic framework solid-phase extractants.

[0035] In one or more embodiments, agitation is performed during the elution process to accelerate the elution rate.

[0036] In one or more embodiments, impurities are washed with an acetonitrile aqueous solution during a single magnetic separation process. Specifically, the mass concentration of the acetonitrile aqueous solution is 1-3%.

[0037] In some embodiments, the magnetic organometallic framework solid-phase extractant is added to the sample to be tested, and the pH is adjusted to 4–7. Studies have shown that this condition is more conducive to improving the recovery rate of estradiol.

[0038] In some embodiments, the concentration of estradiol in the sample to be tested is between 0.02 and 5 pmol / L. Studies have shown that detection within this range yields better accuracy.

[0039] A third embodiment of the present invention provides an application of the above-described detection method in monitoring the concentration of estradiol in surface water.

[0040] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, all reagents used in the following embodiments are commercially available.

[0041] Example

[0042] Synthesis of Fe3O4 nanoparticles:

[0043] Ultrapure water was deoxygenated with high-purity nitrogen. 9g of FeCl₂·4H₂O and 24g of FeCl₃·6H₂O were dissolved in 500mL of deoxygenated ultrapure water. 25mL of ammonia (28 w / w%) was added, and high-purity nitrogen was continuously bubbled through the solution. The mixture was heated to 80°C and stirred. After 40 minutes, it was allowed to cool naturally to room temperature. Stirring was stopped, and the precipitate formed by the reaction was separated using an external magnetic field. The product was washed several times with ethanol and ultrapure water. The product was then stored in a container filled with nitrogen-removed ultrapure water, ensuring no air was trapped at the top, and the container was tightly capped and refrigerated.

[0044] Modification of functional groups in thioglycolic acid:

[0045] Take 0.1g of the synthesized Fe3O4 particles, add 200mL of 0.3mM mercaptoacetic acid in ethanol solution, sonicate for 1h, and oscillate at room temperature for 12h for modification. The modified product is recovered by an external magnetic field to obtain mercaptoacetic acid modified Fe3O4 particles. The product is stored in ultrapure water filled with nitrogen-free water, with no air left in the upper part, and the bottle cap is tightened and stored in the refrigerator.

[0046] Preparation of organometallic framework magnetic nanoparticles:

[0047] Take 30 mL of N,N-dimethylformamide (DMF), and add 0.1 g of mercaptoacetic acid-modified Fe3O4 particles, 0.8 g of anhydrous ZrCl4 and 0.6 g of 2-amino-terephthalic acid sequentially into N,N-dimethylformamide (DMF). Disperse by sonication for 10 min. Transfer the mixture to a reaction vessel containing polytetrafluoroethylene and react at 120 °C for 24 h. After cooling, magnetically separate the reaction solution, discard the supernatant, wash three times with N,N-dimethylformamide (DMF), wash five times with ethanol, and vacuum dry at 60 °C to obtain magnetic organometallic framework (MOF) particles.

[0048] Sample processing:

[0049] Take 1L of environmental water sample into a small-bottomed glass bottle, add 0.2g of magnetic MOF particles, shake and extract for 1min, then let stand for 10min. Use a magnet to attract the magnetic MOF particles near point A at the bottom of the small-bottomed glass bottle, pour out the solution, remove the magnet, and add 50mL of 2% acetonitrile aqueous solution twice to wash away impurities. Use the magnet to attract the magnetic MOF particles near point A at the bottom of the container again, and pour out the solution. Add 3mL of acetone, remove the magnet, shake for 1min, and use a pipette to remove the solution. Dry the solution with nitrogen gas, add 0.5mL of PBS buffer (pH=7) to obtain the test solution. Add an estradiol (E2) ELISA kit (Meizheng) and perform the test according to the instructions.

[0050] result:

[0051] The effect of water sample pH on measurement results:

[0052] In addition to extremely low estradiol levels, the environmental water samples also exhibited significant pH fluctuations. Water samples were spiked to 0.5 pmol / L, divided into several portions, and the pH was adjusted to 4, 5, 6, 7, 8, and 9 respectively. The samples were then analyzed using a magnetic MOFs enrichment-ELISA kit. The results are shown in [Figure number missing]. Figure 1 The recovery rate was greater than 90% when the pH was between 4 and 7, but decreased with increasing pH. When pH > 7, the recovery rate dropped sharply with increasing pH. This may be because the number of -OH groups in the water increases significantly with increasing pH, interacting with estradiol and competing for estradiol molecules bound to the magnetic MOFs material. Therefore, to ensure the quality of the detection, the pH of the environmental water sample should be adjusted to between 5 and 7 before the experiment.

[0053] Actual sample spiked recovery experiment:

[0054] A series of estradiol standard solutions were prepared using environmental water samples. The estradiol was detected using two methods: direct detection with an ELISA kit and detection with a magnetic MOFs enrichment-ELISA kit. The results are shown in Table 1.

[0055] Experimental testing showed that the method in this embodiment improved the detection limit of the estradiol ELISA kit (Meizheng) from 1 pmol / L as stated in the kit instructions to 0.02 pmol / L by 50 times. For low concentrations (0.1 pmol / L and 0.5 pmol / L) of estradiol, the ELISA kit could not detect it directly, but the value obtained by the magnetic MOFs enrichment-ELISA kit was more accurate. For medium concentrations (2 pmol / L), the values ​​obtained by the ELISA kit and the magnetic MOFs enrichment-ELISA kit were similar and both were relatively accurate. For high concentrations of estradiol solution (20 pmol / L), the ELISA kit was accurate, but the magnetic MOFs enrichment-ELISA kit was inaccurate.

[0056] For low-concentration estradiol solutions, the reasons may be: 1. The partition coefficient of estradiol between magnetic MOF particles and water is low, especially for low-concentration estradiol solid-phase extraction, the loss ratio is relatively large; 2. When eluting impurities with 2% acetonitrile aqueous solution, some estradiol is eluted from the surface of magnetic MOF particles, resulting in loss.

[0057] For high-concentration estradiol samples (20 pmol / L), the values ​​measured by the magnetic MOFs enrichment-ELISA detection kit are inaccurate. This may be because the sample has undergone MOFs enrichment, resulting in an excessively high concentration of estradiol in the test solution, which is already in the latter part of the standard curve of the ELISA detection kit. At this point, the slope of the detection signal relative to the concentration is too low, making accurate quantification impossible.

[0058] In summary, the method using the magnetic MOFs enrichment-ELISA kit is suitable for samples with low estradiol concentrations, which is close to the actual estradiol content in environmental water samples. The method described in this embodiment is applicable to the determination of estradiol in environmental water samples.

[0059] Table 1. Detection results of estradiol in water samples using ELISA kits and magnetic MOF enrichment-ELISA kits.

[0060]

[0061] Specificity test:

[0062] Structural and functional analogues of estradiol (E2), namely bisphenol A (BPA), diethylstilbestrol (DES), estriol (E3), and estradiol (E1), were prepared at concentrations of 0.1, 0.5, and 2 pmol / L, respectively. These were detected using a magnetic MOFs enrichment-ELISA kit. The recovery rate was plotted on the ordinate. The results are shown below. Figure 2As shown in the figure, the recovery rate of this method for estradiol was significantly higher than that for the other four structural and functional analogs, demonstrating that the ELISA kit has good specificity for estradiol detection. The method also showed a high response value for high concentrations (2 pmol / L) of interfering substances. Among the four interfering substances, the method responded differently to different interfering substances. For example, at the same concentration, the method showed a lower response to bisphenol A and a higher response to diethylstilbestrol. In practice, when measuring estradiol in environmental water samples, although these interfering substances do not show a high response to the estradiol ELISA kit, if the absolute concentration is too high, it can easily cause significant deviations in the detection values. Therefore, in the detection of estradiol and its analogs in actual environmental water samples, it is best to use other ELISA kits in conjunction with the assay before data analysis.

[0063] Elution repeatability test:

[0064] The synthesized magnetic MOFs were used for a single enrichment pretreatment, followed by three immersions in acetone for further enrichment experiments with estradiol. Quantitative analysis was then performed using an ELISA kit. Recovery rates were determined for samples with low (0.1 pmol / L) and high (2 pmol / L) concentrations of estradiol, and the results are shown below. Figure 3 With increasing usage, the recovery rate of estradiol significantly decreased, especially for high-concentration analytes (2 pmol / L). The decline in recovery rate was even more pronounced with repeated use, possibly because the acetone eluent damaged the magnetic MOF structure and dissolved some of the organic coating on the particle surface. When the analyte concentration was low, the physical adsorption effect due to the large surface area of ​​the magnetic particles was significant. However, for high-concentration analytes (2 pmol / L), the adsorption capacity of the magnetic particle surface area alone tended to saturate, resulting in a decrease in recovery rate. Therefore, for the accuracy of the detection experiment, this magnetic MOF material is only allowed to be used once and reuse is not recommended.

[0065] Accuracy analysis:

[0066] Estradiol solutions of 0.1 pmol / L and 2 pmol / L were prepared and repeatedly detected using magnetic MOFs enrichment-ELISA. The results showed high parallelism. Figure 4 As shown. The relative standard deviation was 25% for low-concentration samples (0.1 pmol / L) and 8% for high-concentration samples (2 pmol / L), indicating good detection accuracy. Experiments showed that when the sample volume was 1 L, the estradiol concentration in environmental water samples was between 0.02 and 5 pmol / L, and the magnetic MOFs enrichment-ELISA detection method performed well within the quantification range. If the sample concentration was higher than this range, direct detection using an ELISA kit was used.

[0067] The above description is merely a preferred embodiment of the present invention and is 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, improvements, etc., 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 detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA), characterized in that, Estradiol in the sample was enriched using a magnetic organometallic framework solid-phase extractant, and then the enriched estradiol was detected by enzyme-linked immunosorbent assay (ELISA). The preparation method of the magnetic organometallic framework solid-phase extractant includes carrying out a solvothermal reaction of mercaptoacetic acid-modified Fe3O4 particles, zirconium salt and 2-amino-terephthalic acid at 110~130 °C for 22~26 h to obtain the extractant. The mass ratio of the thioglycolic acid-modified Fe3O4 particles, zirconium salt, and 2-amino-terephthalic acid is 1:7~9:5~7; The magnetic organometallic framework solid-phase extractant is added to the sample to be tested, and the pH is adjusted to 4-7; the concentration of estradiol in the sample to be tested is 0.02-2 pmol / L.

2. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 1, characterized in that, The mass ratio of the thioglycolic acid-modified Fe3O4 particles, zirconium salt, and 2-amino-terephthalic acid is 1:7.5~8.5:5.5~6.

5.

3. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 1, characterized in that, The solvent for the solvothermal reaction is N,N-dimethylformamide.

4. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 1, characterized in that, The preparation process of Fe3O4 particles modified with mercaptoacetic acid is as follows: Fe3O4 particles are added to a solution of mercaptoacetic acid and dispersed evenly, and then modified at room temperature to obtain the final product.

5. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 4, characterized in that, The method for achieving uniform dispersion is ultrasonic treatment.

6. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 4, characterized in that, The modification treatment time is 10~14 h.

7. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 4, characterized in that, The ratio of Fe3O4 particles to mercaptoacetic acid is 15~25:

1.

8. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 1, characterized in that, The Fe3O4 particles are prepared by adding ferrous salt and ferric salt to deoxygenated water, adding ammonia, heating and stirring the reaction under an inert atmosphere, and then magnetically separating the mixture.

9. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 8, characterized in that, The reaction temperature is 70~90 ℃.

10. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 8, characterized in that, The molar ratio of ferrous salt to ferric salt is 1:1.8~2.

2.

11. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 1, characterized in that, The enrichment process is as follows: the magnetic organometallic framework solid-phase extractant is added to the sample to be tested and shaken for extraction, followed by a first magnetic separation. Estradiol in the solid obtained from the first magnetic separation is eluted with an eluent, and then a second magnetic separation is performed. The eluent from the second magnetic separation is collected.

12. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 11, characterized in that, The eluent used was acetone.

13. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 11, characterized in that, Shaking is applied during the elution process.

14. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 11, characterized in that, Impurities are washed with an acetonitrile aqueous solution during the first magnetic separation process.

15. The method for detecting estradiol using organometallic framework enrichment combined with enzyme-linked immunosorbent assay (ELISA) as described in claim 14, characterized in that, The mass concentration of the acetonitrile aqueous solution is 1-3%.

16. The application of the detection method according to any one of claims 1 to 15 in monitoring the concentration of estradiol in surface water.

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