A highly sensitive screening method for thyroid hormone disruptors

Abstract

The present application relates to a kind of thyroid hormone interferent high-sensitivity screening method, belong to new pollutant screening method technical field.The present application is based on the biological recognition performance of polypeptide and the peroxidase-like activity of nano gold combined with polypeptide material, based on this, a kind of thyroid hormone interferent screening method under medium alkaline condition can guarantee the activity of polypeptide and also can play the enzyme activity of nano gold.The dopamine can be catalyzed by nano gold under medium alkaline condition and quickly generate red product.Once interferent combines specific recognition polypeptide, occupies the enzyme activity recognition site of nano gold, reduces enzyme activity, in dopamine color system, different absorbance values are shown, so as to show the thyroid hormone interference toxic effect, reach the purpose of recognizing thyroid hormone interferent.The method does not need to use a large amount of organic solvent, and toxicity evaluation of thyroid hormone interferent can be realized by absorbance determination, with the advantages of simple preparation, low cost, high sensitivity.

Description

Technical Field

[0001] This invention relates to the technical field of new pollutant screening methods, and more specifically, to a highly sensitive screening method for thyroxine interferons. Background Technology

[0002] Thyroxine disruptors are a class of exogenous compounds that interfere with thyroxine levels. They are widely distributed in the environment and can enter the human body through various pathways, leading to abnormal thyroxine levels and thus affecting bodily functions. Studies have shown that exposure to thyroxine disruptors is associated with poor brain development and cognitive deficits in offspring, and the effects are irreversible (Salazar, P., et al. Environmental Research, 2021, 200, 111345.). In addition, some studies have pointed out that the known interference effects of thyroxine disruptors account for only a very small part of the total interference effect, and a large number of unknown disruptors exist in the environment (Hamers, T., et al. Environmental Health Perspectives, 2020, 128(1), 017015.). Therefore, the detection of thyroxine disruptors is very important.

[0003] Thyroxine disruptors primarily interfere with thyroid hormones through transport toxicity. When they enter the bloodstream, their structure is similar to thyroid hormones, allowing them to compete with thyroid transport proteins for binding and transport to target organs, thus causing abnormal thyroid hormone levels and health damage. Common screening methods for thyroid hormone disruptors utilize structural similarity, identifying them through specific binding sites, such as thyroxine transport protein competitive binding assays and thyroxine receptor competitive binding assays (OECD. OECD Series on Testing and Assessment, 2006; Vol. ENV / JM / MONO (2006) 24; Ren, XM, et al. Environ Health Perspectives, 2020, 128(10), 107008.). However, these methods require expensive proteins that are difficult to import, and their sensitivity is insufficient for analyzing trace amounts of disruptors in human samples. Chinese invention patents 201811282189 and 202210294197 both disclose the use of biomimetic recognition technology. Based on the structural similarity between thyroxine interfering agents and thyroxine, thyroxine is used as a template to prepare thyroxine molecularly imprinted polymers, providing specific recognition sites for thyroxine interfering agents and reducing costs. However, this process involves a large amount of organic solvents and a long and complex preparation cycle. Therefore, we need a cost-effective, simple, rapid, and highly sensitive method for detecting thyroxine interfering agents. Summary of the Invention

[0004] This invention provides a highly sensitive screening method for thyroxine interfering substances. It utilizes the specific recognition performance of polypeptide segments for thyroxine interfering substances and the rapid self-polymerization and color development of dopamine under moderately alkaline enzymatic catalysis to construct a highly sensitive method for identifying thyroxine interfering substances. This invention significantly reduces the amount of organic solvent used, simplifies preparation, and accelerates determination; it has the advantages of low cost and low background interference, and its detection sensitivity is improved by at least two orders of magnitude, enabling its application to the identification and analysis of trace interfering substances. This solves the technical problem of low detection sensitivity in existing thyroxine interfering substance detection technologies, which are unsuitable for trace detection.

[0005] According to the present invention, a highly sensitive screening method for thyroxine interferons is provided, comprising the following steps:

[0006] (1) Add the gold nanoparticle dispersion to the peptide solution to form a gold nanoparticle-peptide material. The amino acid sequence of the peptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0007] (2) The gold nanoparticle-peptide material obtained in step (1) was incubated with different concentrations of the test compound and then spread on a filter membrane. Then, a moderately alkaline buffer solution, hydrogen peroxide solution and dopamine solution were added to the filter membrane in sequence. Under the enzyme-like catalysis of the gold nanoparticle, dopamine and hydrogen peroxide could stably generate a red substance under moderately alkaline conditions. Then, the absorbance value A470nm at the peak value of 470nm and the absorbance value A750nm at the background value of 750nm were measured respectively. ΔA = A470nm - A750nm. The dose-response curve was obtained by fitting the ΔA of each concentration of the test compound to the ΔA of the blank control as the ordinate and the logarithm of the concentration of the corresponding test compound as the abscissa. The different concentrations of the test compound could cause the dose-response curve to show two plateau periods.

[0008] (3) Take the absolute value of the difference between the ordinates corresponding to the two plateau periods on the dose-effect curve obtained in step (2) as the total interference inhibition effect. Mark 20% of the total interference inhibition effect on the ordinate corresponding to the dose-effect curve as IR20, and mark 50% of the total interference inhibition effect on the ordinate corresponding to the dose-effect curve as IR50. ΔIR = IR20 - IR50. If ΔIR ≥ 0.01, the test compound is a thyroid hormone interferon. If ΔIR < 0.01, the test compound is a non-thyroid hormone interferon.

[0009] Preferably, the incubation temperature is 20–60°C and the incubation time is 5–40 minutes.

[0010] Preferably, the buffer solution is a Tris solution with a pH of 7.5 to 10.

[0011] Preferably, the concentration of the dopamine solution is 0.1–3.0 mg / mL.

[0012] Preferably, the molar ratio of hydrogen peroxide in the hydrogen peroxide solution to dopamine in the dopamine solution is (1-4.6):1.

[0013] Preferably, the nano-gold dispersion is prepared by mixing tetrachloroauric acid solution and bovine serum albumin solution, and then reducing it with sodium borohydride.

[0014] Preferably, the concentration of the polypeptide solution is 62.5–500 μM.

[0015] Preferably, the test compounds of different concentrations are at least five different concentrations.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0017] (1) This invention combines the biorecognition properties of peptides with the peroxidase-like activity of gold nanoparticles, based on gold nanoparticles. A toxicity assessment method under moderately alkaline conditions is constructed that ensures both peptide activity and the enzyme activity of gold nanoparticles. Under moderately alkaline conditions, dopamine can be rapidly catalyzed by gold nanoparticles to generate a red product. Once the interfering substance binds to the specifically recognizing peptide, it occupies the enzyme activity recognition site of the gold nanoparticles, reducing enzyme activity and exhibiting different absorbance values ​​in the dopamine colorimetric system, thus demonstrating the toxic effect of thyroxine interference and achieving the purpose of identifying thyroxine interfering substances. Compared with the competitive binding experiment of thyroxine transporters, this invention has the advantages of low cost and low background interference. Its detection sensitivity is improved by at least two orders of magnitude for most compounds, and it can be applied to the identification and analysis of trace interfering substances in the population.

[0018] (2) On the one hand, peptides with thiol groups can spontaneously bind to gold nanoparticles without the need for additional reagents, making preparation simple; moreover, binding to gold nanoparticles can prolong the half-life of peptides, which is beneficial for peptide preservation and application. In addition, gold nanoparticles have enzyme-like activity, which can catalyze substrates and achieve signal conversion. Here, the enzyme-like activity of gold nanoparticles converts the binding signal of difficult-to-detect interfering substances to peptides into an easily detectable absorbance signal of dopamine color development. Traditional competitive binding experiments involve mixing compounds with proteins and probes to measure fluorescence polarization values, which may be subject to background signal interference from compounds. In this invention, unbound compounds are washed off before the enzyme reaction begins, and the gold nanoparticle peptide materials are all retained on the filter membrane. Finally, only the absorbance value of the product after the reaction is measured, which is not affected by proteins and compounds, greatly reducing background signal interference.

[0019] (3) Since the application range of peptides is mainly under moderately alkaline conditions, while the commonly used enzyme activity reaction system is under acidic conditions, it does not meet the detection requirements. The self-polymerization colorimetric reaction of dopamine under moderately alkaline conditions provides a good application environment, and dopamine can rapidly polymerize and develop color under the catalysis of enzyme reaction, which has good application prospects. This method is simple and fast, greatly reduces the amount of organic reagents used, and can also effectively improve the sensitivity of the method. Attached Figure Description

[0020] Figure 1 This is a flowchart of the detection method for thyroid hormone interferon.

[0021] Figure 2 This is a comparison of the sensitivity of the detection methods for thyroxine interferon and the competitive binding assays for thyroxine transporters.

[0022] Figure 3 This study compares the detection sensitivity of thyroxine interferon detection methods and thyroid transporter protein competitive binding assays in population samples. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0024] This invention provides a highly sensitive method for detecting thyroid hormone interferons, as detailed below:

[0025] 1. Preparation of materials for detecting thyroxine interferon

[0026] After washing and dispersing the nano-gold suspension, it was incubated with a certain concentration of peptide solution at room temperature for 12 hours, washed, and then stored at 4℃.

[0027] In some embodiments, the preparation method of the nano-gold dispersion is as follows: 4 mL of 200 nM tetrachloroauric acid solution and 1 mL of 10 μM bovine serum albumin solution are mixed for 5 minutes, and then 1 mg of 1 mg / mL sodium borohydride is added for reduction.

[0028] Preferably, the concentration of the polypeptide solution is 62.5–500 μM.

[0029] 2. Establishment and application of detection methods for thyroid hormone interferons

[0030] A certain amount of the obtained gold nanopeptide material and compound were incubated at a certain temperature for a certain time and then spread evenly on a filter membrane. Tris buffer, hydrogen peroxide solution of a certain concentration, and dopamine solution were added sequentially. Under the enzyme-like catalysis of gold nanoparticles, dopamine and hydrogen peroxide could stably generate a red substance with an absorption peak at 470 nm under moderately alkaline conditions. After a certain reaction time, the absorbance values ​​at 470 nm (peak value) and 750 nm (background value) were measured. ΔA was calculated.

[0031] ΔA = A470nm - A750nm. If the compound is a thyroxine disruptor, the peptide can specifically recognize and bind to it, thereby occupying the enzyme active site of the gold nanoparticles bound to the peptide, thus reducing the peroxidase-like activity of the gold nanoparticles, resulting in a reduction in the final red substance, a lighter color, and a lower absorbance value at 470nm. If it is a non-thyroxine disruptor, it cannot bind to the peptide, and the absorbance value of the final red product remains unchanged. Next, the interference effect of the thyroxine disruptor recognition material on thyroxine and its structural analogs is calculated. The ratio is the ratio of the compound's ΔA to the ΔA of the blank control (the compound is replaced with 10% dimethyl sulfoxide). A dose-response curve is fitted with the logarithm of the compound concentration on the x-axis and the ratio on the y-axis to calculate ΔIR. Based on the dose-response curve, there is a plateau at both low and high disruptor concentrations; the difference in the ratio corresponding to these two plateaus is considered the total inhibitory effect. Figure 1As shown, a 20% inhibitory effect is equivalent to 20% of the total inhibitory effect, and a 50% inhibitory effect is equivalent to 50% of the total inhibitory effect. The difference between the ratios corresponding to these two inhibitory effects is ΔIR, i.e., ΔIR = IR20 - IR50. The software first directly provides the IC20 (the concentration value corresponding to a 20% inhibitory effect) and IC50 values ​​(the concentration value corresponding to a 50% inhibitory effect). Then, based on this, the IR20 and IR50 values ​​corresponding to IC20 and IC50 are found from the series of data provided by the software. The difference between the ratios corresponding to these two inhibitory effects is ΔIR, i.e., ΔIR = IR20 - IR50. ΔIR ≥ 0.01 indicates a thyroid hormone interferon; otherwise, it is considered a non-interferon.

[0032] Preferably, the amount of the gold nanopeptide material is 75–450 μg.

[0033] Preferably, the incubation temperature of the compound is 20–60°C.

[0034] Preferably, the incubation time for the compound is 5 to 40 minutes.

[0035] Preferably, the pH value of the Tris solution is 7.5 to 10.

[0036] Preferably, the concentration of dopamine is 0.1–3.0 mg / mL.

[0037] Preferably, the molar ratio of hydrogen peroxide to dopamine is 1:2 to 2.3:1.

[0038] Preferably, the reaction time of the system is 5 to 120 minutes.

[0039] Preferably, the dimethyl sulfoxide content is 1% to 100%.

[0040] The following are specific embodiments.

[0041] Example 1: Preparation of a material for recognizing thyroxine interference

[0042] (1) Screening of key recognition peptide segments

[0043] The specific recognition sites (7 polypeptides) of thyroid transporter protein and thyroxine receptor protein were obtained by screening from the protein database (https: / / www.rcsb.org / ), namely FTKIITPAITRVVD, MEIMSLRAAVR, SETLTLNGEM, MIGACHASRFL, SSKTLKKWNRL, ILERSTRSILF, and IAALLSPYSYSTTA, and are designated as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0044] (2) Preparation of materials for recognizing thyroxine interference

[0045] Bovine serum albumin solution (4 mL, 10 μM) was mixed with tetrachloroauric acid solution (1 mL, 24.3 mM) and stirred for 5 minutes. Then, sodium borohydride solution (1 mL, 1 mg / mL) was added, and the mixture was reacted in the dark for 30 minutes to obtain a gold nanoparticle suspension. After washing, the gold nanoparticle suspension (7.5 mL, 1.5 mg / mL) was reacted with a peptide solution (MIGACHASRFL) (2.5 mL, 1 mM) at room temperature for 12 hours. After washing, the mixture was stored at 4 °C.

[0046] Example 2: Detection Method and Application of Thyroxine Interferon

[0047] like Figure 1 As shown in the flowchart, the obtained thyroxine interferon recognition material (200 μL, 300 μg) and the compound were incubated with shaking at 40 °C for 30 minutes, then spread evenly on a filter membrane. Tris buffer (80 μL, pH = 8.0), hydrogen peroxide solution (10 μL, 158 mM), and dopamine solution (10 μL, 2 mg / mL) were added sequentially. After reacting for 30 minutes, the absorbance values ​​at 470 nm and 750 nm were measured, and ΔA was calculated. ΔA = A470nm - A750nm. Next, the interference effect of the thyroxine interferon recognition material on thyroxine and its structural analogs was calculated. The interference effect is the ratio of the ΔA of the compound to the ΔA of the blank control (the compound was replaced with 10% dimethyl sulfoxide). Meanwhile, we compared the detection sensitivity of the thyroxine transporter competitive binding assay method described in this paper. Here, a warning value is used to represent this sensitivity. When the ratio of a compound at a certain concentration point begins to exceed three standard deviations of the blank control, it indicates that the compound has an interfering effect; the corresponding lowest concentration point is the warning value. For example... Figure 2 As shown, the alert values ​​of this method are below 2 μM for most compounds, and the detection sensitivity for most compounds is improved by at least two orders of magnitude compared to the competitive binding assay for thyroid transport proteins.

[0048] Example 3: Comparison of detection sensitivity of thyroxine interferon detection method and thyroxine transporter competitive binding assay method in actual human samples.

[0049] Urine samples collected from the population were pretreated and reconstituted with dimethyl sulfoxide (DMSO). Each sample was divided into four equal portions, with three portions containing low (100 nM / known interferon), medium (10 μM / known interferon), and high (1 mM / known interferon) mixed standard solutions of 40 known interferons, respectively. The interference effects were detected using both this method and the thyroxine transporter competitive binding assay. Figure 3 As shown, each set of images, from left to right, represents the original urine extract, low-concentration spiked (100 nM / known interfering agent), medium-concentration spiked (10 μM / known interfering agent), and high-concentration spiked (1 mM / known interfering agent). As the compound concentration increases, the interference effect exhibited by this method becomes increasingly stronger, while the thyroid transporter competitive binding assay remains essentially unchanged. Even with the original urine extract, the interference effect demonstrated by this invention is stronger than that of the thyroid transporter competitive binding assay. This proves that this method can be applied to identify the interference effect of mixed compounds in human samples, and its sensitivity is far superior to that of the thyroid transporter competitive binding assay.

[0050] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A highly sensitive screening method for thyroxine interferons, characterized in that, Includes the following steps: (1) Add the gold nanoparticle dispersion to the peptide solution to form a gold nanoparticle-peptide material. The amino acid sequence of the peptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:

7. (2) The gold nanoparticle-peptide material obtained in step (1) was incubated with different concentrations of the test compound and then spread on a filter membrane. Then, a moderately alkaline buffer solution, hydrogen peroxide solution and dopamine solution were added to the filter membrane in sequence. Under the enzyme-like catalysis of the gold nanoparticle, dopamine and hydrogen peroxide could stably generate a red substance under moderately alkaline conditions. Then, the absorbance value A470nm at the peak value of 470nm and the absorbance value A750nm at the background value of 750nm were measured respectively. ΔA = A470nm - A750nm. The dose-response curve was obtained by fitting the ΔA of each concentration of the test compound to the ΔA of the blank control as the ordinate and the logarithm of the concentration of the corresponding test compound as the abscissa. The different concentrations of the test compound could cause the dose-response curve to show two plateau periods. (3) Take the absolute value of the difference between the ordinates corresponding to the two plateau periods on the dose-effect curve obtained in step (2) as the total interference inhibition effect. Mark 20% of the total interference inhibition effect on the ordinate corresponding to the dose-effect curve as IR20, and mark 50% of the total interference inhibition effect on the ordinate corresponding to the dose-effect curve as IR50. ΔIR = IR20 - IR50. If ΔIR ≥ 0.01, the test compound is a thyroid hormone interferon. If ΔIR < 0.01, the test compound is a non-thyroid hormone interferon.

2. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The incubation temperature is 20–60°C, and the time is 5–40 minutes.

3. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The buffer solution is a Tris solution with a pH of 7.5–10.

4. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The concentration of the dopamine solution is 0.1–3.0 mg / mL.

5. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The molar ratio of hydrogen peroxide in the hydrogen peroxide solution to dopamine in the dopamine solution is (1-4.6):

1.

6. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The nano-gold dispersion was prepared by mixing tetrachloroauric acid solution and bovine serum albumin solution, and then reducing it with sodium borohydride.

7. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The concentration of the polypeptide solution is 62.5–500 μM.

8. The highly sensitive screening method for thyroxine interferons as described in claim 1, characterized in that, The number of test compounds at different concentrations is at least five.

Images