Electrochemical method for qualitative screening and quantitative detection of estrogen compounds based on ligand receptor effect
By using human estrogen receptor α (hERα) as a biorecognition element, E2-HRP conjugates were prepared and the electrode surface was modified, solving the problem of the difficulty in rapidly detecting estrogen-effect compounds in the prior art, and realizing a simple and efficient detection of estrogen compounds.
Patent Information
- Application Number
- CN202411050215.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-01
- Publication Date
- 2026-02-03
AI Technical Summary
Existing technologies are not quick and easy to detect compounds with estrogenic effects in the environment, and traditional methods are complex, costly, and cannot effectively identify a variety of target compounds.
Using human estrogen receptor α (hERα) as a biorecognition element, estradiol horseradish peroxidase conjugate (E2-HRP) was prepared through ligand-receptor interaction and competitively bound to Ni-NTA complex to modify the electrode surface for electrochemical detection.
It enables the identification and detection of all compounds with estrogenic effects. The detection steps are simple, time-saving, and have low detection limits, making it suitable for on-site testing and applicable to the detection of estrogenic compounds in the environment, food, and pharmaceuticals.
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Figure CN121453879A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of electrochemical biosensor technology, specifically relating to an electrochemical method for qualitative screening and quantitative detection of estrogen compounds based on ligand-receptor interactions. Background Technology
[0002] Estrogens are widely present in the environment, including water, soil, and household products. They exert their estrogen-related effects primarily by interfering with estrogen-related metabolism in animals and humans through the mimicking of natural estrogen's receptor agonist effects, thereby regulating the transcription of estrogen receptors and modulating their quantity and sensitivity. This interference can ultimately harm the health of organisms. Existing research has shown that estrogen is associated with obesity, abnormal thyroid secretion, sperm abnormalities, gonadal developmental abnormalities, estrogen-dependent tumors (such as breast cancer, endometrial cancer, neuroblastoma, and melanoma), and neurobehavioral disorders (such as autism spectrum disorder and attention deficit hyperactivity disorder). Traditional methods for detecting estrogen include chromatography / mass spectrometry, enzyme-linked immunosorbent assay (ELISA), and capillary electrophoresis. While these methods offer high precision, they often suffer from drawbacks such as complex operation, high cost, large equipment size, and the need for specialized operators. Therefore, there is an urgent need to develop simple, rapid, and effective new methods to screen for and detect substances with estrogenic effects in the environment.
[0003] Electrochemical sensors are widely used in food safety, healthcare, and environmental monitoring due to their advantages such as high sensitivity, rapid response, low cost, simple operation, and ease of miniaturization. Currently, electrochemical biosensors used for estrogen detection mainly utilize enzymes, aptamers, antibodies, and functional nucleic acids as biorecognition elements. Such studies typically only analyze single targets and cannot detect compounds outside the target. Estrogen receptors are target proteins in organisms with a high affinity for estrogen. Compounds with estrogenic effects need to bind to estrogen receptors to form receptor-ligand complexes to produce the corresponding physiological effects. To date, no research has been reported on designing an electrochemical method for detecting estrogen-like compounds using human estrogen receptor α (hERα) as the recognition element. Summary of the Invention
[0004] Therefore, the purpose of this invention is to provide an electrochemical method for detecting estrogen-like compounds based on ligand receptor interaction. This invention uses estrogen receptors as biorecognition elements, which can identify and detect all compounds with estrogenic effects, and has advantages such as simple detection steps, short detection time, and low detection limit.
[0005] The objective of this invention is achieved through the following methods:
[0006] This invention provides an electrochemical method for qualitative screening and quantitative detection of estrogen compounds based on ligand-receptor interactions, comprising the following steps:
[0007] (1) The carboxyl group of beta-estradiol 17-hemisuccinate was activated, and horseradish peroxidase (HRP) solution was added after activation. The mixture was incubated together to prepare estradiol horseradish peroxidase conjugate (E2-HRP). The estradiol horseradish peroxidase conjugate was purified by dialysis and the volume was adjusted to a concentration of 0.1–2.0 mg / ml. -1 ;
[0008] (2) The human estrogen receptor α solution (hERα) and Ni agarose resin solution (Ni-NTA) were mixed and incubated in a centrifuge tube to allow the human estrogen receptor α to bind to the surface of Ni-NTA and form a Ni-NTA-hERα complex.
[0009] (3) Add a certain volume of the estradiol horseradish peroxidase conjugate purified in step (1) and a certain volume of the target analyte solution to a centrifuge tube containing the Ni-NTA-hERα complex in step (2) and incubate together so that E2-HRP competes with the target analyte to bind to hERα on the surface of Ni-NTA in the centrifuge tube. After incubation, centrifuge and take the supernatant. The supernatant contains unbound free E2-HRP.
[0010] (4) The supernatant obtained in step (3) is dropped onto the surface of the working electrode and dried to obtain an electrode with E2-HRP on the surface;
[0011] (5) Add Nafion solution dropwise to the electrode surface covered with E2-HRP obtained in step (4), and let it dry to obtain the working electrode modified with E2-HRP@Nafion.
[0012] (6) The working electrode, counter electrode, and reference electrode obtained in step (5) are placed together in an electrolyte solution containing tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical testing.
[0013] Based on the above technical solution, the estrogenic compounds refer to compounds that have estrogenic effects on organisms such as humans and animals, including but not limited to estrone, estradiol, estriol, nonylphenol, and bisphenol A.
[0014] Based on the above technical solution, in step (1), EDC and NHS are used to activate beta-estradiol 17-hemisuccinate, and the molar ratio of EDC, NHS and beta-estradiol 17-hemisuccinate is 1-5:1-5:1.
[0015] Based on the above technical solution, further, the reaction solution used for activation in step (1) is a mixed solution of methanol and PBS buffer, and the activation time is 1 to 3 hours.
[0016] Based on the above technical solution, further, in step (1), the molar ratio of horseradish peroxidase to activated beta-estradiol 17-hemisuccinate is 1-20:1, and the incubation time is 3-5 h; the dialysis process is carried out in PBS buffer solution at pH 7.4, the maximum molecular weight allowed to pass through the dialysis bag used in the dialysis process is 20000 Da, and the volume is adjusted based on the concentration of horseradish peroxidase.
[0017] Based on the above technical solution, further, the human estrogen receptor α described in step (2) has a His tag.
[0018] Based on the above technical solution, further, in step (2), the concentration of the human estrogen receptor α solution is 0.5-5 mg / mL, and the incubation time is 1-3 h.
[0019] Based on the above technical solution, further, in step (3), the volume ratio of the purified estradiol horseradish peroxidase conjugate, the target analyte solution and the Ni-NTA-hERα complex is 1:20:2 to 1:30:5.
[0020] Based on the above technical solution, further, in step (3), the co-incubation time is 0.5 to 2 hours, the centrifugation speed is 2000 to 4000 rpm, and the centrifugation time is 1 to 5 minutes.
[0021] Based on the above technical solution, the working electrode mentioned in step (4) includes, but is not limited to, glassy carbon electrode, gold electrode, platinum electrode, and screen-printed electrode, and the volume of the added clear liquid is 1 to 10 μL.
[0022] Based on the above technical solution, further, the concentration of the Nafion solution mentioned in step (5) is 0.1-1%, and the volume of dropwise addition is 1-5 μL.
[0023] Based on the above technical solution, further, the reference electrode in step (6) is an Ag-AgCl electrode, the counter electrode is a platinum wire electrode, and the electrolyte solution is a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution containing 0.2 mM TMB and 4% (v:v) H2O2.
[0024] In another aspect, the present invention provides the application of the above-mentioned electrochemical method for detecting estrogen compounds based on ligand receptor action in the detection of estrogen compounds in the environment, food, and pharmaceuticals, as well as its application in the screening and quantitative detection of estrogen activity in chemicals.
[0025] Based on the above technical solution, further, the electrochemical method is used to detect the degree of current change of a series of estrogen compounds (e.g., estradiol with a concentration range of 0.01 to 50 nM), and a standard curve is obtained between the concentration of the estrogen compound and the degree of current change. The degree of current change obtained from detecting the test solution is substituted into the standard curve to determine the content or equivalent concentration (calculated as estradiol) of the estrogen compound in the test sample solution.
[0026] Based on the above technical solution, the estrogenic activity of the sample or chemical to be tested can be quantitatively assessed by converting it into an equivalent concentration of estradiol.
[0027] Based on the above technical solution, the concentration range of the estrogen compounds is further 0.01–50 nM.
[0028] Based on the above technical solution, it can be further applied to the screening of known / unknown estrogenic compounds in various samples, the screening of estrogenic effects of various chemicals (whether they have estrogenic effects) and the assessment of effect intensity.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] This invention uses estrogen receptors as biorecognition elements to identify and detect all compounds with estrogenic effects. It has advantages such as simple detection steps, short detection time, and low detection limit. It does not require complicated pretreatment processes for actual samples, making it suitable for on-site detection and has a very broad application prospect. Attached Figure Description
[0031] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.
[0032] Figure 1 This is a schematic diagram illustrating the principle of the electrochemical biosensor of the present invention;
[0033] Figure 2 This is an electrochemical DPV signal diagram obtained by detecting different concentrations of E2 in Example 3;
[0034] Figure 3 This is a linear relationship graph of the E2 concentration signal in Example 3;
[0035] Figure 4 This is a comparison diagram of the electrochemical DPV signals obtained from detecting other compounds in Example 4.
[0036] Figure 5 This is a graph showing the reproducibility results of the detection method of the present invention. Detailed Implementation
[0037] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.
[0038] The solution components involved in the examples are as follows:
[0039] The TMB substrate solution was a 50 mM citrate-disodium hydrogen phosphate buffer solution at pH 5.0 containing 0.2 mM TMB and 0.04% (v:v) H2O2. The beta-estradiol 17-hemisuccinate (E2-COOH) stock solution was prepared to 2 mM with methanol, then diluted to 1 mM with pH 5.5 PBS buffer, and the carboxyl groups on its structure were activated with EDC and NHS. EDC and NHS were dissolved separately in pH 5.5 PBS buffer at a concentration of 12 mM each. The human estrogen receptor α (hERα) HRP solution was prepared with pH 7.4 PBS buffer at a concentration of 10 mg / mL. -1 The concentration of PBS buffer at pH 5.5 and pH 7.4 was 10 mM.
[0040] Example 1: Preparation of E2-HRP Couplings
[0041] First, take 400 μL of E2-COOH solution and add 100 μL each of EDC and NHS solution. After 2 hours, slowly add 400 μL of horseradish peroxidase (HRP) solution directly to the mixed solution and incubate for 4 hours to allow HRP to fully react with E2-COOH to form an E2-HRP conjugate. After the conjugate is formed, it needs to be purified using dialysis (the dialysis membrane used during dialysis has a pore size limited to small molecules with a molecular weight below 20,000 Da). Dialyze in 1 L of PBS buffer for 4 hours, then use a pipette to remove the remaining liquid in the dialysis tube and bring the volume to 4 ml, so that the final concentration of E2-HRP is 1 mg / ml (based on HRP concentration). After the conjugate is prepared, it should be sealed and stored in a light-protected container at 4°C.
[0042] Example 2: Pretreatment of Working Electrode
[0043] First, polish the surface of the glassy carbon electrode on a clean chamois using alumina slurry with particle sizes of 0.3μm and 0.05μm, polishing it to a mirror finish. Then, ultrasonically clean the electrode for three minutes in ultrapure water, anhydrous ethanol, and ultrapure water in sequence. Finally, remove the electrode and dry it with nitrogen gas before use.
[0044] Example 3: Detection of typical estrogen E2
[0045] First, 3 μL of hERα (1 mg / mL) and 2 μL of Ni agarose resin (Ni-NTA) were pipetted into a centrifuge tube and incubated together at 4°C for 2 hours. This process utilizes the His tag inherent in the protein and the Ni-NTA tag. 2+ The interaction between the two allows hERα to bind to the Ni-NTA surface, forming a Ni-NTA-hERα complex. After incubation, 2 μL of 1 mg / mL E2-HRP conjugate and 50 μL of beta-estradiol (E2) solution were added to the solution system simultaneously, and incubated at 4 °C for 1 hour to allow E2-HRP and E2 to fully compete for binding ERα, forming a Ni-NTA-hERα-E2 complex. Then, the mixture was centrifuged at 3000 rpm for 3 minutes, and 3 μL of the supernatant was taken out and used to modify the surface of the pretreated glassy carbon working electrode. After the electrode surface dried, 2 μL of 0.5% (v:v) Nafion solution was added to fix E2-HRP. After the modified working electrode was completely dry, the electrode system, including the working electrode, reference electrode, and counter electrode, was placed in a citric acid buffer solution containing 0.2 mM TMB and 0.04% (v:v) H2O2 to test the electrochemical DPV signal.
[0046] Graphs of E2 detection signals at different concentrations are shown below. Figure 2 The linear relationship between E2 concentration and current signal is shown in the graph. Figure 3 The figure clearly shows that the DPV current signal gradually decreases with increasing estradiol concentration, and the DPV current signal and estradiol concentration have a good linear relationship in the range of 0.04–40 nM.
[0047] Example 4
[0048] To further evaluate the reliability of the electrochemical biosensor system, estrogen effect screening tests were conducted on two actual samples: tap water and mineral water. Tap water samples were obtained from a laboratory, while mineral water samples were purchased from a supermarket (Nongfu Spring mineral water). Equal volumes of PBS buffer solution were added to both tap water and mineral water samples, mixed thoroughly, and then blank and enhanced experiments were performed. The experimental results are shown in Table 1, with recoveries ranging from 92% to 106%.
[0049] Table 1. Results of spiked test on tap water and mineral water samples
[0050]
[0051] To evaluate the reproducibility of the detection method of this invention, a 10 nM E2 solution was tested repeatedly 7 times. The results are shown in [Figure number missing]. Figure 5 The results showed that the RSD was 2.3%, indicating that the detection method of the present invention has good reproducibility.
[0052] Example 5: Detection of other hormone compounds
[0053] First, 3 μL of hERα (1 mg / mL) and 2 μL of Ni-NTA were pipetted into a centrifuge tube and incubated together at 4°C for 2 hours. This process utilizes the His tag inherent in the protein and the Ni-NTA tag. 2+ The interaction between them allows hERα to bind to the Ni-NTA surface, forming a Ni-NTA-hERα complex. After incubation, 2 μL of 1 mg / mL E2-HRP conjugate and 50 μL of estrone (E1) (or estriol (E3), bisphenol A (BPA), testosterone (Te), triiodothyronine (T3)) solution (where E1, E3, and BPA are estrogens, and Te and T3 are androgens) are added to the solution system. The system is incubated at 4°C for 1 hour to allow E2-HRP to fully compete with E1 (or E3, BPA, Te, T3) for binding to ERα, forming a Ni-NTA-hERα-E1 (or E3, BPA, Te, and T3) complex. Then, the system is centrifuged at 3000 rpm for 3 minutes. After centrifugation, 3 μL of the supernatant is taken out and used to modify the surface of the pretreated glassy carbon working electrode. After the electrode surface is dried, 2 μL of 0.5% (v:v) Nafion solution is added to fix E2-HRP. After the modified working electrode is completely dried, the electrode system, including the working electrode, reference electrode, and counter electrode, is placed in a citric acid buffer solution containing 0.2 mM TMB and 0.04% (v:v) H2O2 to test the electrochemical DPV signal.
[0054] A comparison of signals obtained from detecting other compounds (estrogens, androgens) is shown in the figure. Figure 4 .from Figure 4 The detection results clearly show that the electrochemical biosensor of the present invention can distinguish estrogen compounds well and can rank the estrogenic effects of estrogens (estrogen intensity: E2>E1>E3>BPA).
[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. An electrochemical method for qualitative screening and quantitative detection of estrogen compounds based on ligand-receptor interactions, characterized in that, Includes the following steps: (1) The carboxyl group of beta-estradiol 17-hemisuccinate was activated, and horseradish peroxidase (HRP) solution was added after activation. The mixture was incubated together to prepare estradiol-horseradish peroxidase conjugate (E2-HRP). The estradiol-horseradish peroxidase conjugate was purified by dialysis and the volume was adjusted to a concentration of 0.1–2.0 mg / ml. -1 ; (2) The human estrogen receptor α solution (hERα) and Ni agarose resin solution (Ni-NTA) were mixed and incubated in a centrifuge tube to allow the human estrogen receptor α to bind to the surface of Ni-NTA and form a Ni-NTA-hERα complex. (3) Add a certain volume of the estradiol horseradish peroxidase conjugate purified in step (1) and a certain volume of the target analyte solution to a centrifuge tube containing the Ni-NTA-hERα complex in step (2) and incubate together so that E2-HRP competes with the target analyte to bind to hERα on the surface of Ni-NTA in the centrifuge tube. After incubation, centrifuge and take the supernatant. The supernatant contains unbound free E2-HRP. (4) The supernatant obtained in step (3) is dropped onto the surface of the working electrode and dried to obtain an electrode with E2-HRP on the surface; (5) Add Nafion solution dropwise to the electrode surface covered with E2-HRP obtained in step (4), and let it dry to obtain the working electrode modified with E2-HRP@Nafion. (6) The working electrode, counter electrode, and reference electrode obtained in step (5) are placed together in an electrolyte solution containing tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical testing.
2. The electrochemical method according to claim 1, characterized in that, The estrogenic compounds mentioned above refer to compounds that have estrogenic effects on organisms such as humans and animals, including but not limited to estrone, estradiol, estriol, nonylphenol, and bisphenol A.
3. The electrochemical method according to claim 1, characterized in that, In step (1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate beta-estradiol 17-hemisuccinate. The molar ratio of EDC, NHS and beta-estradiol 17-hemisuccinate was 1–5:1–5:
1. The reaction solution used for activation was a mixture of methanol and PBS buffer, and the activation time was 1–3 h. The molar ratio of horseradish peroxidase to activated beta-estradiol 17-hemisuccinate was 1–20:1, and the incubation time was 3–5 h. The dialysis process was carried out in PBS buffer solution at pH 7.
4. The maximum molecular weight allowed to pass through the dialysis bag used in the dialysis process was 20,000 Da. The volume was adjusted based on the concentration of horseradish peroxidase.
4. The electrochemical method according to claim 1, characterized in that, The human estrogen receptor α described in step (2) has a His tag; the concentration of the human estrogen receptor α solution is 0.5-5 mg / mL, and the incubation time is 1-3 h.
5. The electrochemical method according to claim 1, characterized in that, In step (3), the volume ratio of the purified estradiol-horseradish peroxidase conjugate, the target analyte solution, and the Ni-NTA-hERα complex is 1:20:2 to 1:30:5; the co-incubation time is 0.5 to 2 h, the centrifugation speed is 2000 to 4000 rpm, and the centrifugation time is 1 to 5 min.
6. The electrochemical method according to claim 1, characterized in that, The working electrode mentioned in step (4) includes, but is not limited to, glassy carbon electrode, gold electrode, platinum electrode, and screen-printed electrode, and the volume of the added supernatant is 1 to 10 μL; the Nafion solution mentioned in step (5) has a concentration of 0.1 to 1% and a volume of 1 to 5 μL; the reference electrode mentioned in step (6) is an Ag-AgCl electrode or a saturated calomel electrode, the counter electrode is a platinum wire electrode, and the electrolyte solution is a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution containing 0.2 mM TMB and 4% (v:v) H2O2.
7. The application of the electrochemical method for detecting estrogen compounds based on ligand-receptor interaction as described in any one of claims 1-6 in the detection of estrogen compounds in the environment / food / pharmaceuticals, and in the screening and quantitative detection of estrogen activity in chemicals.
8. The application according to claim 7, characterized in that, The electrochemical method described above detects the degree of current change of a series of estrogen compounds (e.g., estradiol with a concentration range of 0.01–50 nM) at a gradient concentration, and obtains a standard curve between the concentration of the estrogen compound and the degree of current change. The degree of current change obtained from testing the solution to be tested is substituted into the standard curve to determine the content or equivalent concentration (calculated as estradiol) of the estrogen compound in the sample solution to be tested.
9. The application according to claim 7 or 8, characterized in that, The estrogenic activity of the sample or chemical being tested can be quantitatively assessed by converting it into an equivalent concentration of estradiol.