A method for detecting diethylstilbestrol based on photothermal probe and lateral chromatography test paper

Abstract

This invention belongs to the field of chemical detection technology, specifically relating to a method for detecting diethylstilbestrol (DES) based on a photothermal probe and lateral chromatography test strip. The method comprises a photothermal material coupled with a DES monoclonal antibody. The detection zone of the lateral chromatography test strip is coated with a PBS solution containing DES antigen, while the control zone is coated with a PBS solution containing goat anti-mouse secondary antibody. This invention combines the photothermal properties of the photothermal material with lateral chromatography test strip technology to achieve sensitive quantitative analysis of DES. In this invention, an 808nm laser is used to irradiate the control and detection zones of the lateral chromatography test strip, and a thermal imager is used to record the temperature changes in each zone. A standard curve is established using the temperature difference and DES concentration to achieve quantitative analysis of DES. This invention can be used for rapid, accurate, intuitive, highly repeatable, and highly stable quantitative detection of samples containing DES.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection technology, specifically relating to a method for detecting diethylstilbestrol based on a photothermal probe and lateral chromatography test paper. 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] Diethylstilbestrol (DES) residues in water resources, soil, and animal-derived foods can enter the human body through the food chain and accumulate there. With the development of detection technologies, several DES detection methods have been developed over the past few decades, including instrumental analysis methods and rapid detection methods. Instrumental analysis methods have advantages such as high detection accuracy and comprehensive data; however, they are inherently limited by the instruments and methods themselves, requiring specific environments and instruments for detection, and the process is relatively cumbersome and costly.

[0004] Among rapid detection methods, sensor-based detection methods and immunological detection methods are widely studied. Sensor-based detection methods utilize electrochemical, thermal, and other sensor devices to detect DES, featuring short detection times and reliable quantitative results. However, in practice, they are often affected by the low specificity of the sensor's constituent elements, resulting in poor repeatability and low detection efficiency. In recent years, immunological detection technology has developed rapidly, especially with the widespread application of lateral chromatography test strip technology, making it a research hotspot in the field of rapid detection. This technology has advantages such as short detection cycle time, simple procedure, reliable quantitative results, and low cost. However, this method mainly relies on visual observation of the color development in the detection and control areas, and the subjectivity of the observer leads to low sensitivity, making quantitative detection impossible.

[0005] Existing technologies combine nanomaterials with special properties with lateral chromatography test strips to improve detection sensitivity. Currently used nanomaterials possess properties including colorimetric, fluorescent, and photothermal characteristics. Utilizing the photothermal effect of nanomaterials and a small infrared thermal imager, the temperature of the detection area can be accurately recorded, and the concentration of the analyte can be calculated. However, the sensitivity, repeatability, and stability of lateral chromatography detection using the photothermal effect are limited by the inherent photothermal properties and stability of the nanomaterials themselves. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention provides a method for detecting diethylstilbestrol (DES) based on a photothermal probe and lateral chromatography test paper. This invention combines the photothermal properties of photothermal materials with lateral chromatography test paper technology to achieve sensitive quantitative analysis of DES. In this invention, an 808nm laser is used to irradiate the control and detection zones of the lateral chromatography test paper, and a thermal imager is used to record the temperature changes in each zone. A standard curve is established using the temperature difference and DES concentration to achieve quantitative analysis of DES. This invention can be used for rapid, accurate, intuitive, highly repeatable, and highly stable quantitative detection of DES-containing samples.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a photothermal probe formed by conjugation of a photothermal material and a diethylstilbestrol monoclonal antibody. The photothermal material includes one or more of purple phosphorus nanosheets and covalent organic framework nanosheets. The monomers forming the covalent organic framework nanosheets include pyromellitic anhydride and 5,10,15,20-tetra(4-aminophenyl)porphyrin.

[0009] Secondly, the present invention provides a method for preparing the photothermal probe described in the first aspect, comprising the following steps:

[0010] S1. Dissolve bovine serum albumin and Tween-20 in ultrapure water to obtain probe storage solution;

[0011] S2. Dilute the purple phosphorus nanosheet solution or the covalent organic framework nanosheet solution, adjust the concentration, and shake.

[0012] S3. Add the PBS solution containing diethylstilbestrol monoclonal antibody to the shaken purple phosphorus nanosheet solution or covalent organic framework solution to obtain a mixed solution, and shake.

[0013] S4. Add 10% skim milk powder solution and 10% Tween-20 solution to the shaken mixture, seal it, and centrifuge after sealing to obtain the precipitate, which is the photothermal probe. The photothermal probe is resuspended in the probe storage solution.

[0014] Thirdly, the present invention provides a lateral chromatography test strip, wherein the detection area of ​​the lateral chromatography test strip is sprayed with a PBS solution of diethylstilbestrol antigen, and the quality control area of ​​the lateral chromatography test strip is sprayed with a PBS solution of goat anti-mouse secondary antibody.

[0015] Fourthly, the present invention provides a method for detecting diethylstilbestrol based on the photothermal probe described in the first aspect and the lateral chromatography test paper described in the third aspect, comprising the following steps:

[0016] A photothermal probe was added to a sample solution containing diethylstilbestrol (DES), and a running buffer was added. The mixture was incubated to obtain a mixed solution. The mixed solution was then loaded onto the sample pad area of ​​a lateral chromatography test strip. After standing, the results were observed and recorded. The control area and the detection area were irradiated with an 808 nm laser, and the temperature changes in each area were recorded using a thermal imager. A standard curve was established using the temperature difference ΔT between the two areas and the DES concentration to achieve quantitative detection of DES.

[0017] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0018] This invention applies high photothermal conversion performance of purple phosphorus nanosheets or covalent organic framework nanosheets to the lateral chromatography detection of diethylstilbestrol. Compared with existing photothermal materials, purple phosphorus nanosheets and covalent organic framework nanosheets have superior laser temperature response and photothermal efficiency, as well as good photothermal stability, effectively improving detection sensitivity and repeatability.

[0019] The diethylstilbestrol detection method based on photothermal probe and lateral chromatography test paper provided by this invention has a low detection limit, with the visual detection limit as low as 3 μg·L⁻¹. -1 The photothermal detection limit is as low as 0.24 μg·L⁻¹. -1 The concentration of DES is between 0.25 and 50 μg·L⁻¹. -1 There is a good linear relationship within the range.

[0020] The diethylstilbestrol (DES) detection method of this invention exhibits significant specificity for DES. The photothermal detection results of the five parallel experiments showed no significant difference, and the repeatability meets the detection requirements. The constructed test strip demonstrates good storage stability. Attached Figure Description

[0021] 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.

[0022] Figure 1 The images show TEM images (A) and particle size distribution (B) of the purple phosphorus nanosheets in Example 1.

[0023] Figure 2 The ultraviolet absorption spectrum (A) and XRD pattern (B) of the purple phosphorus nanosheets in Example 1 are shown.

[0024] Figure 3 The data presented in Example 1 include the temperature change curve (A) under 808 nm laser light, the standard curve of time versus -Ln(θ) (B), data from five photothermal cycles (C), and data at a concentration of 2.5 μg·mL⁻¹. -1 25 μg·mL -150 μg·mL -1 100 μg·mL -1 The ultraviolet absorption spectrum of purple phosphorus nanosheets;

[0025] Figure 4 The ultraviolet absorption spectrum (A) and absorbance value at 450 nm (B) of the purple phosphorus nanosheet photothermal probes in Examples 2-7 are shown in the figure. pH represents different purple phosphorus nanosheet photothermal probes.

[0026] Figure 5 This is a comparison of the sealing effect of the purple phosphorus nanosheet photothermal probes on test paper between Examples 2 and Examples 8 and 9;

[0027] Figure 6 The color development of the purple phosphorus nanosheet photothermal probes on test paper is compared between Example 2 and Examples 10-14;

[0028] Figure 7 This is a comparison chart of different sample loading amounts of purple phosphorus nanosheets onto the photothermal probe in Example 15;

[0029] Figure 8 Comparison of test paper color development for different concentrations of diethylstilbestrol (A) and different concentrations of goat anti-mouse secondary antibody (B) in Example 16;

[0030] Figure 9 The figures (A) show the temperature change curve over time under a fixed irradiation power and (B) show the temperature change graph under different irradiation powers in Example 17, where ΔT is the temperature difference before and after irradiation in the detection area and ΔC is the temperature difference before and after irradiation in the quality control area.

[0031] Figure 10 This is a temperature change graph for different irradiation distances and different thermal imager heights in Example 17, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0032] Figure 11 The images (A) and the standard curve (B) for the detection of diethylstilbestrol at different concentrations in Example 18 are shown. ΔT represents the temperature difference before and after irradiation in the detection area, ΔC represents the temperature difference before and after irradiation in the quality control area, and C represents the concentration of diethylstilbestrol.

[0033] Figure 12 This is a comparison of the detection of α-estradiol, β-estradiol, bisphenol A, bisphenol B, estriol, hexestrol, and esthenone in Example 18, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0034] Figure 13 This is a graph of repeatability test data from Example 18, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0035] Figure 14 This is a graph showing the stability test data in Example 18, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0036] Figure 15 Photographs (A) and (B) of the test strips used in Example 18 to test tap water, milk, and pork;

[0037] Figure 16 TEM image (A) and particle size distribution map (B) of COF nanosheets in Example 19;

[0038] Figure 17 The XRD pattern (A), UV absorption spectrum (B), and IR absorption spectrum (C) of the COF nanosheets in Example 19 are shown, where PDMA is pyromellitic anhydride and TAPP is 5,10,15,20-tetra(4-aminophenyl)porphyrin.

[0039] Figure 18 The temperature change curve (A), the standard curve of time versus -Ln(θ) of COF nanosheets under 808nm laser light (B), and the data of five photothermal cycles (C) in Example 19 are shown.

[0040] Figure 19 Photographs (A) and ultraviolet absorption spectra (B) of the COF nanosheet photothermal probes in Examples 20-24 are shown, with pH representing different purple phosphorus nanosheet photothermal probes.

[0041] Figure 20 In Figure A, the flowability of the COF nanosheet photothermal probes of Examples 20 and 25-27 on the test paper is compared; in Figure B, the flowability of the COF nanosheet photothermal probes of Examples 20 and 28 and 29 on the test paper is compared.

[0042] Figure 21 The color development of the COF nanosheet photothermal probes in Examples 20 and 30-34 on test paper is compared.

[0043] Figure 22 This is a comparison chart of the sample loading amounts of different purple phosphorus nanosheet photothermal probes in Example 35;

[0044] Figure 23 Comparison of test paper color development for different concentrations of diethylstilbestrol (A) and different concentrations of goat anti-mouse secondary antibody (B) in Example 36;

[0045] Figure 24The figures (A) show the temperature change curve over time in Example 37 and the temperature change graph (B) show the temperature change under different irradiation powers, where ΔT is the temperature difference before and after irradiation in the detection area and ΔC is the temperature difference before and after irradiation in the quality control area.

[0046] Figure 25 The temperature change graphs for different irradiation distances (A) and different thermal imager heights (B) in Example 37 are shown, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0047] Figure 26 The images (A) and the standard curve (B) for the detection of diethylstilbestrol at different concentrations in Example 38 are shown. ΔT represents the temperature difference before and after irradiation of the detection area, ΔC represents the temperature difference before and after irradiation of the quality control area, and C represents the concentration of diethylstilbestrol.

[0048] Figure 27 This is a comparison of the detection of α-estradiol, β-estradiol, bisphenol A, bisphenol B, estriol, hexestrol, and esthenone in Example 38, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0049] Figure 28 This is a graph of repeatability test data in Example 38, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0050] Figure 29 This is a graph of stability test data in Example 38, where ΔT is the temperature difference before and after irradiation in the detection area, and ΔC is the temperature difference before and after irradiation in the quality control area.

[0051] Figure 30 Photographs (A) and (B) of the test strips used in Example 38 to test tap water, milk, and pork. Detailed Implementation

[0052] Definition: θ: The photothermal material solution is continuously irradiated with a laser of power I = 0.4W and wavelength 808nm. The initial temperature of the solution is T. 0,mix After the temperature stops rising, use a thermal imager to read the temperature of the photothermal material solution and record it as T. mix Calculate the temperature change ΔT of the photothermal material solution after laser irradiation. max,mix =T mix -T 0,mix Starting from the moment the laser irradiation stops, the temperature difference ΔT' between the photothermal material solution and the ambient temperature is calculated every 10 seconds, and θ = ΔT is introduced.max,mix -ΔT'.

[0053] In a first typical embodiment of the present invention, a photothermal probe is formed by coupling a photothermal material with a diethylstilbestrol monoclonal antibody. The photothermal material includes one or more of purple phosphorus nanosheets and covalent organic framework nanosheets. The monomers forming the covalent organic framework nanosheets include pyromellitic anhydride and 5,10,15,20-tetra(4-aminophenyl)porphyrin.

[0054] A second typical embodiment of the present invention, a method for preparing the photothermal probe described in the first typical embodiment, includes the following steps:

[0055] S1. Dissolve bovine serum albumin and Tween-20 in ultrapure water to obtain probe storage solution;

[0056] S2. Dilute the purple phosphorus nanosheet solution or the covalent organic framework nanosheet solution, adjust the concentration, and shake.

[0057] S3. Add the PBS solution containing diethylstilbestrol monoclonal antibody to the shaken purple phosphorus nanosheet solution or covalent organic framework solution to obtain a mixed solution, and shake.

[0058] S4. Add 10% skim milk powder solution and 10% Tween-20 solution to the shaken mixture, seal it, and centrifuge after sealing to obtain the precipitate, which is the photothermal probe. The photothermal probe is resuspended in the probe storage solution.

[0059] Non-specific binding of antibodies can affect the detection sensitivity of test strips. Blocking solutions, such as serum, skim milk powder, BSA, and gelatin, are commonly used to block the antibodies and eliminate the effects of non-specific binding. Additionally, adding Tween-20 can improve the stability of the detection solution. Tween-20 is a non-ionic surfactant that enhances diffusion, stability, and lubrication.

[0060] In one or more embodiments of this implementation, the method for preparing the purple phosphorus nanosheet solution includes the following steps:

[0061] Purple phosphorus crystal powder was mixed with methanol and ground for 40-60 min. After drying, the mixture was added to ultrapure water and ultrasonically dispersed at 250 W for 15-20 min. The dispersed solution was ultrasonically treated at 500 W for 6-8 h. After centrifugation at 2000-2500 rpm for 4-6 min, the supernatant was collected to obtain a purple phosphorus nanosheet solution.

[0062] The mass-to-volume ratio of the purple phosphorus crystal powder, methanol, and ultrapure water is 19-21 mg: 2 mL: 55-65 mL.

[0063] In one or more embodiments of this implementation, the method for preparing the covalent organic framework solution includes the following steps:

[0064] The mixture of pyromellitic anhydride, 5,10,15,20-tetra(4-aminophenyl)porphyrin, dioxane, and mesitylene was sonicated for 15-20 min in a closed state. After sonication, acetic acid was added and sonicated for 10 s to obtain a mixture. The mixture was frozen with liquid nitrogen and degassed by three freeze-thaw cycles. After degassed, the mixture was heated at 120 °C for 7 days. After filtration and washing, covalent organic framework powder was obtained.

[0065] The covalent organic framework powder was ground for 40-60 min. After drying, the ground covalent organic framework powder was added to ultrapure water and ultrasonically dispersed at 250 W for 15-20 min. The dispersed solution was ultrasonically treated at 500 W for 6-8 h. After centrifugation at 2000-2500 rpm for 4-6 min, the supernatant was collected to obtain the covalent organic framework powder solution.

[0066] The mass-to-volume ratio of the pyromellitic anhydride, 5,10,15,20-tetra(4-aminophenyl)porphyrin, dioxane, mesitylene, and acetic acid is 6-7 mg: 9-11 mg: 0.2-0.3 mL: 0.7-0.8 mL: 0.19-0.21 mL.

[0067] In one or more embodiments of this implementation, the concentration of diethylstilbestrol monoclonal antibody in the PBS solution containing diethylstilbestrol monoclonal antibody is 0.9-1.1 mg / mL. -1 .

[0068] In one or more embodiments of this implementation, the volume ratio of the purple phosphorus nanosheet solution or covalent organic framework nanosheet solution, the PBS solution containing diethylstilbestrol monoclonal antibody, the 10% skim milk powder solution, and the 10% Tween-20 solution is 100:0.4-1:5-20:5-20.

[0069] In one or more embodiments of this implementation, in step S1, the mass-to-volume ratio of bovine serum albumin, Tween-20, and ultrapure water is 95-105 mg: 20-30 μL: 10 mL.

[0070] In one or more embodiments of this implementation, in step S2, the absorbance value at a wavelength of 450 nm is diluted to 1-1.1, the pH is adjusted to 6-11, and the shaking time is 5-10 min.

[0071] In one or more embodiments of this implementation, in step S3, the device oscillates at 36-37°C for 45-60 minutes.

[0072] In one or more embodiments of this implementation, in step S4, the device is sealed at 4°C for 30-40 minutes and centrifuged at 12000 rpm for 20-30 minutes.

[0073] In a third typical embodiment of the present invention, a lateral chromatography test strip is provided, wherein the detection area of ​​the lateral chromatography test strip is sprayed with a PBS solution of diethylstilbestrol antigen, and the quality control area of ​​the lateral chromatography test strip is sprayed with a PBS solution of goat anti-mouse secondary antibody.

[0074] In one or more embodiments of this implementation, the concentration of diethylstilbestrol antigen in the PBS solution of diethylstilbestrol antigen is 0.2-1 mg / mL. -1 The concentration of goat anti-mouse secondary antibody in the PBS solution is 0.1-0.8 mg / mL. -1 .

[0075] A fourth typical embodiment of the present invention provides a method for detecting diethylstilbestrol based on the photothermal probe described in the first typical embodiment and the lateral chromatography test paper described in the third typical embodiment, characterized by comprising the following steps:

[0076] A photothermal probe was added to a sample solution containing diethylstilbestrol (DES), and a running buffer was added. The mixture was incubated to obtain a mixed solution. The mixed solution was then loaded onto the sample pad area of ​​a lateral chromatography test strip. After standing, the results were observed and recorded. The control area and the detection area were irradiated with an 808 nm laser, and the temperature changes in each area were recorded using a thermal imager. A standard curve was established using the temperature difference ΔT between the two areas and the DES concentration to achieve quantitative detection of DES.

[0077] In one or more embodiments of this implementation, the running buffer is an ultrapure aqueous solution containing 0.9-1.1 g / mL sucrose, 0.7-0.9 g / mL bovine serum albumin and 0.9-1.1% Tween-20 (v / v).

[0078] In one or more embodiments of this implementation, the volume ratio of the photothermal probe, sample solution, and running buffer is 3-25:50:10.

[0079] In one or more embodiments of this implementation, the irradiation time of the 808nm laser is 30-80s, the irradiation power is 0.75-1.5W, the irradiation distance is 1-3cm, and the detection height of the thermal imager is 11-15cm.

[0080] Appropriate irradiation time and power can effectively reduce detection time and improve detection efficiency. Excessive irradiation time and power cause the test strip to rise to excessively high temperatures in a short time, resulting in damage to the test strip material and affecting detection accuracy. The temperature rise rate of the test strip is directly affected by the laser irradiation power. Too low a laser power limits the detection sensitivity and range; too high a power leads to a high signal-to-noise ratio, affecting detection accuracy. A suitable irradiation distance can effectively reduce photothermal loss during laser irradiation and minimize the impact of environmental factors on the detection results.

[0081] To enable those skilled in the art to better 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 and comparative examples.

[0082] The running buffer used in this embodiment of the invention is 0.01 mol·L⁻¹. -1 The preparation methods for PBS solution and probe storage solution are as follows:

[0083] (1) Weigh 1.0g sucrose and 800mg BSA, add 10mL ultrapure water to dissolve them completely, then add 100μL Tween-20 to obtain the running buffer;

[0084] (2) Weigh 8.0g NaCl, 0.2g KCl, 3.63g Na2HPO4·12H2O, and 0.24g KH2PO4, dissolve them in 900mL of ultrapure water, adjust the pH to 7.4 with hydrochloric acid, and add water to a final volume of 1L to obtain 0.01mol·L⁻¹ -1 PBS solution, store at room temperature for later use;

[0085] (3) Take 100 mg BSA and 25 μL Tween-20, add 10 mL of ultrapure water to dissolve and obtain probe storage solution, and store at 4 °C for later use.

[0086] Example 1

[0087] Weigh 20.0 mg of purple phosphorus crystal powder and place it in an agate mortar. Add 2 mL of methanol and grind thoroughly for 40 min to make it homogeneous. Dry it in an oven and add it to 60 mL of ultrapure water. Ultrasonize at 250 W for 15 min to fully disperse the material in the ultrapure water. Ultrasonize at 500 W for 6 h and centrifuge at 2000 rpm for 5 min. Take the supernatant to obtain the purple phosphorus nanosheet solution and store it at 4℃ for later use.

[0088] like Figure 1 As shown in Figure A, the purple phosphorus nanosheets exhibit a layered structure and good dispersibility. Further analysis using a nanoparticle size and Zeta potential analyzer was conducted to determine the particle size distribution of the purple phosphorus nanosheets. Figure 1As shown in B, its particle size distribution ranges from 100 to 400 nm, with an average particle size of approximately 213 nm.

[0089] The purple phosphorus nanosheet solution was diluted to a concentration of 12.5 μg·mL. -1 25 μg·mL -1 50 μg·mL -1 100 μg·mL -1 And measure the ultraviolet absorption spectrum, such as Figure 2 As shown in Figure A, the purple phosphorus nanosheets exhibit broad absorption in the 400–850 nm range, indicating their high photothermal conversion potential. Furthermore, the absorbance value significantly increases with increasing material concentration. Figure 2 As shown in B, the PXRD pattern of purple phosphorus nanosheets is compared with that of purple phosphorus crystals. The diffraction peaks at 16.21°, 26.35°, 30.67°, 52.15°, and 56.8° correspond to the crystal planes (004), (006), (008), (152), and (534) in the purple phosphorus crystal, respectively.

[0090] like Figure 3 As shown in Figure A, purple phosphorus nanosheets were irradiated with an 808 nm laser, and their temperature changes were recorded. Figure 3 As shown in Figure B, a standard curve of time versus -Ln(θ) during temperature change was plotted. Calculations showed that the photothermal conversion efficiency was 31.1%. Five photothermal cycle experiments were conducted on the purple phosphorus nanosheets, as shown... Figure 3 As shown in Figure C, the temperature changes were similar during the five photothermal cycles, indicating good photothermal stability. Different concentrations of purple phosphorus nanosheets were irradiated with an 808 nm laser, as shown in Figure C. Figure 3 The concentration of D shown in the figure is 100 μg·mL. -1 The purple phosphorus nanosheets can be heated by about 15°C within 300 seconds.

[0091] Example 2

[0092] The purple phosphorus nanosheet solution from Example 1 was diluted to a absorbance of 1.0 at 450 nm. 400 μL of the diluted purple phosphorus nanosheet solution was then added to a 0.1 mol / L solution. -1 Prepare a potassium carbonate solution, adjust the pH to 8, and shake for 5 minutes. Add the DES antibody to a 0.01 mol·L⁻¹ solution. -1 PBS solution diluted to 1 mg / mL -1Add 1.6 μL of DES antibody diluent to the purple phosphorus nanosheet solution (corresponding to a DES antibody concentration of 4 μg / mL), and shake at 37°C for 45 min. After shaking, add 40 μL of 10% skim milk powder and 40 μL of 10% Tween-20 to the solution (final concentration is 1% skim milk powder and 1% Tween-20), and block at 4°C for 30 min. After blocking, centrifuge at 12000 rpm for 20 min at 4°C, remove the supernatant, and resuspend the purple phosphorus nanosheet photothermal probe precipitate in 80 μL of probe storage solution for later use.

[0093] Example 3

[0094] Unlike Example 2, the pH was adjusted to 6.

[0095] Example 4

[0096] Unlike Example 2, the pH was adjusted to 7.

[0097] Example 5

[0098] Unlike Example 2, the pH was adjusted to 9.

[0099] Example 6

[0100] Unlike Example 2, the pH was adjusted to 10.

[0101] Example 7

[0102] Unlike Example 2, the pH was adjusted to 11.

[0103] The pH environment during antibody conjugation with purple phosphorus nanosheets significantly affects the conjugation efficiency and post-conjugation dispersibility. Too low a pH can lead to material aggregation, while too high a pH reduces the conjugation efficiency. Therefore, finding a suitable pH environment is crucial. The UV absorption spectra of the photothermal probes prepared at different pH values ​​in Examples 2-7 are shown below. Figure 4 As shown in A, the absorbance value at 450nm is as follows: Figure 4 As shown in B, the material exhibits the best antibody-coupled effect when the pH is 8.0.

[0104] Example 8

[0105] Unlike Example 4, 20 μL of 10% skim milk powder and 20 μL of 10% Tween-20 were added (the final concentration was 0.5% skim milk powder and 0.5% Tween-20).

[0106] Example 9

[0107] Unlike Example 2, 80 μL of 10% skim milk powder and 80 μL of 10% Tween-20 were added (the final concentration was 2% skim milk powder and 2% Tween-20).

[0108] like Figure 5 As shown, when the concentration of skim milk powder is 1% and the concentration of Tween-20 is 1%, the sealing is good and the background color is light. Therefore, 1% Tween-20 and 1% skim milk powder are selected as the best sealing liquid components.

[0109] Example 10

[0110] Unlike Example 2, no DES antibody diluent was added.

[0111] Example 11

[0112] Unlike Example 2, 0.8 μL of DES antibody diluent was added to the purple phosphorus nanosheet solution (corresponding to a DES antibody concentration of 2 μg / mL).

[0113] Example 12

[0114] Unlike Example 2, 2.4 μL of DES antibody diluent was added to the purple phosphorus nanosheet solution (corresponding to a DES antibody concentration of 6 μg / mL).

[0115] Example 13

[0116] Unlike Example 2, 3.2 μL of DES antibody diluent was added to the purple phosphorus nanosheet solution (corresponding to a DES antibody concentration of 8 μg / mL).

[0117] Example 14

[0118] Unlike Example 2, 4 μL of DES antibody diluent was added to the purple phosphorus nanosheet solution (corresponding to a DES antibody concentration of 10 μg / mL).

[0119] Load 80 μL of negative test sample (50 μL PBS buffer, 10 μL running buffer, 20 μL of purple phosphorus nanosheet photothermal probe from Example 2 and 10-14) onto each test strip. Observe the color development of the detection area of ​​each test strip after 10 minutes. Figure 6 As shown, when the antibody concentration is 4 μg / mL, the color development in the control area and the detection area is clear. When the antibody concentration continues to increase, the color deepening in the detection area no longer increases significantly.

[0120] Example 15

[0121] 5, 10, 15, 20, and 25 μL of the purple phosphorus nanosheet detection probe from Example 2 were taken respectively and mixed with 50 μL of PBS buffer and 10 μL of running buffer to prepare test solutions. The test solutions were then added to the test strips to examine their colorimetric performance. The results are as follows: Figure 7As shown, when the sample loading amount of the purple phosphorus nanosheet detection probe is 15 μL, the color of the test strip is clear and the background is clean. Therefore, 15 μL is selected as the optimal sample loading amount for the purple phosphorus nanosheet photothermal probe.

[0122] Example 16

[0123] Diethylstilbestrol antigen was diluted to concentrations of 0.5, 0.6, 0.7, and 0.8 mg / mL using PBS buffer. -1 The diluted diethylstilbestrol antigen was sprayed onto the test strip detection area using a coating applicator, while the secondary antibody concentration in the test strip control area was 0.5 mg / mL. -1 After spraying, the sample was dried at 37°C. After drying, 80 μL of negative test sample (50 μL PBS buffer, 10 μL running buffer, and 20 μL of the purple phosphorus nanosheet photothermal probe from Example 2) was loaded onto each strip. After 10 minutes, the color development of the detection area on each test strip was observed. The group with uniform and clear color development in the detection area represented the optimal concentration of diethylstilbestrol antigen. Figure 8 As shown in A, the optimal concentration of diethylstilbestrol antigen in the detection area is 0.4 mg·mL⁻¹. -1 .

[0124] The goat anti-mouse secondary antibody was diluted with PBS buffer to concentrations of 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. -1 The diluted secondary antibody was sprayed onto the quality control area of ​​the test strip using a coating applicator, while the optimal concentration of diethylstilbestrol antigen (DES) in the test strip detection area was fixed at 0.8 mg / mL. -1 After the test strips were coated, they were dried at 37°C. After drying, 80 μL of negative test sample (50 μL PBS buffer, 10 μL running buffer, and 20 μL of the purple phosphorus nanosheet photothermal probe from Example 2) was loaded onto each strip. After 10 minutes, the color development of the detection area on each test strip was observed. The group with uniform and clear color development in the control area represented the optimal secondary antibody concentration. Figure 8 As shown in B, the optimal concentration of goat anti-mouse secondary antibody in the quality control area is 0.8 mg / mL. -1 .

[0125] Example 17

[0126] The photothermal probe using purple phosphorus nanosheets from Example 2 was used, and the concentration of diethylstilbestrol antigen in the detection area was 0.4 mg / mL. -1 The concentration of goat anti-mouse secondary antibody coating in the quality control area was 0.8 mg / mL. -1 The test strips were used for testing.

[0127] The detection area is illuminated with a laser of fixed power, and the real-time temperature is recorded every 10 seconds using a thermal imager. Figure 9 As shown in A in the diagram. After 30 seconds of irradiation, the temperature of the test strip reached equilibrium with the environment, and the temperature change slowed down within the dynamic range. Therefore, 30 seconds was chosen as the optimal irradiation time.

[0128] Select lasers with power levels of 0.75W, 1W, 1.25W, and 1.5W to irradiate the test strips. Figure 9 As shown in B, the temperature difference reaches its maximum value when the laser power is 1W. Therefore, 1W is selected as the optimal power for laser irradiation.

[0129] like Figure 10 As shown in Figure A, the temperature difference between the quality control area and the detection area is most significant when the irradiation distance is 1.5 cm. Therefore, 1.5 cm is chosen as the optimal irradiation distance. Figure 10 As shown in B, the temperature difference between the quality control area and the detection area is most significant when the thermal imager height is 12cm. Therefore, 12cm is selected as the optimal thermal imager height.

[0130] Example 18

[0131] The photothermal probe using purple phosphorus nanosheets from Example 2 was used, and the concentration of diethylstilbestrol antigen in the detection area was 0.4 mg·mL⁻¹. -1 The concentration of goat anti-mouse secondary antibody coating in the quality control area was 0.8 mg / mL. -1 The test strips were used for detection. The irradiation time was 30 seconds, the irradiation power was 1W, and the irradiation distance was 1.5cm. The height of the thermal imager was 12cm.

[0132] DES was diluted to concentrations of 0.05, 0.125, 0.25, 0.75, 1.5, 3.0, 6.0, 12.5, 25, and 50 μg·L⁻¹, respectively. -1 The standard solution was tested, and the DES was qualitatively detected by visual inspection after 15 minutes. The test strip detection area and quality control area were irradiated with an 808nm laser, and the temperature difference corresponding to each concentration of DES was recorded by a thermal imager to plot the standard curve.

[0133] like Figure 11 As shown in A, the limit of detection by the naked eye is 25 μg·L⁻¹. -1 Record the temperature difference between the control area and the detection area for 10 blank tests and calculate the average value. Then subtract three times the standard deviation of this average value to calculate the photothermal detection limit as 1.95 μg / L. -1 .like Figure 11 As shown in B, the DES concentration is between 3 and 50 μg / L. -1 There is a good linear relationship within the range.

[0134] Seven estrogens—α-estradiol, β-estradiol, bisphenol A, bisphenol B, estriol, hexestrol, and estrone—were selected for non-target analyte determination. The standard solutions prepared for the assays were at a concentration of 25 μg / L. -1 These are then combined with photothermal probes for detection. For example... Figure 12As shown, when detecting other estrogens, the detection area showed obvious color development, and the temperature difference between the detection area and the control area was less than 5°C. The results indicate that this method has significant specificity for DES.

[0135] For a concentration of 25 μg·L -1 Five parallel experiments were conducted on the DES. For example... Figure 13 As shown, there was no significant difference between the visual and photothermal detection results of the five parallel experiments, proving that the repeatability of the detection method meets the detection requirements.

[0136] Stability tests were conducted on test strips stored at 4℃ for 20 days. The results are as follows: Figure 14 As shown, after storage at 4℃ for a certain period of time, the detection stability of the test strip decreased to some extent. After 20 days of storage, the final detection efficiency was 76% of the initial detection efficiency. This indicates that the prepared test strip has good storage stability.

[0137] DES recovery experiments were conducted using tap water, milk, and pork. Different concentrations of DES (7.5, 15, and 30 μg·L⁻¹) were added to the three actual samples. -1 ) to conduct tests, such as Figure 15 As shown in Figure A. Due to the influence of complex matrices in actual samples, such as ions in tap water, proteins in milk, and fats in pork products, the established detection method shows different detection results in the three actual samples compared to PBS solution. Figure 15 As shown in Figure B, the recoveries of DES in the three actual samples ranged from 82.2% to 115.2%, with coefficients of variation ranging from 2.0% to 10.8%. This indicates that the method has good feasibility and applicability in the detection of actual samples.

[0138] Example 19

[0139] Weigh 6.5 mg of pyromellitic anhydride and 10.0 mg of 5,10,15,20-tetra(4-aminophenyl)porphyrin into a large-bore tube. Add 0.25 mL of dioxane and 0.75 mL of trimethylbenzene. Seal the tube with sealing film and sonicate for 15 min to obtain a uniform dispersion. Then add 0.2 mL of acetic acid and sonicate for 10 s. Rapidly freeze the tube in liquid nitrogen and degas it through three freeze-thaw cycles. Freeze in liquid nitrogen for 2 min, solidify, connect to a double-row tube, vacuum for 3 min, close the valve, thaw in ethanol or water, freeze again for 2 min, vacuum for 5 min, repeat three times. Seal the large-bore tube under frozen conditions. Then heat at 120 °C for 7 days. After the reaction is complete, filter out the covalent organic framework (COF) powder and wash with anhydrous ethanol. Place the powder in an agate mortar and grind for 40 min to make it homogeneous, then dry in an oven. Then, it was added to 60 mL of ultrapure water and sonicated at 250 W for 15 min to fully disperse the material in the ultrapure water. Subsequently, it was sonicated at 500 W for 6 h, centrifuged at 2000 rpm for 5 min, and the supernatant was collected to obtain the COF nanosheet solution, which was then stored in a refrigerator at 4 °C for later use.

[0140] like Figure 16 As shown in Figure A, the COF nanosheets exhibit a layered structure and good dispersibility. Figure 16 As shown in B, the COF nanosheets have a particle size range of 100–300 nm, with an average diameter of approximately 198 nm.

[0141] like Figure 17 As shown in Figure A, the XRD pattern of the COF nanosheets only shows a peak at 3.32° corresponding to the (100) crystal plane. Figure 17 As shown in Figure B, the ultraviolet absorption spectrum of COF nanosheets shows an absorption peak at 450 nm and exhibits broad absorption in the range of 300–800 nm. Figure 17 As shown in C, COF nanosheets at 1660 cm⁻¹ -1 An amide bond formed by the dehydration condensation of porphyrin and mesitylene appears at the point.

[0142] like Figure 18 As shown in Figure A, COF nanosheets were irradiated with an 808 nm laser, and their temperature response was recorded. A standard curve of time versus -Ln(θ) during the temperature response process was established, as shown in Figure A. Figure 18 As shown in B, its photothermal conversion efficiency is calculated to be 51.17%. Figure 18 As shown in C, five photothermal cycling experiments were conducted on COF nanosheets. The temperature changes were similar during the five photothermal cycles, indicating that they have good photothermal stability.

[0143] Example 20

[0144] The COF nanosheet solution from Example 19 was diluted to a absorbance of 1.0 at 450 nm. 400 μL of the diluted COF nanosheet solution was then added to a 0.1 mol·L⁻¹ solution. -1 Prepare a potassium carbonate solution, adjust the pH to 8, and shake for 5 minutes. Add the DES antibody to a 0.01 mol·L⁻¹ solution. -1 PBS solution diluted to 1 mg / mL -1 Add 3.2 μL of DES antibody diluent to the purple phosphorus nanosheet solution (corresponding to a DES antibody concentration of 8 μg / mL), and shake at 37°C for 45 min. After shaking, add 40 μL of 10% skim milk powder and 60 μL of 10% Tween-20 to the solution (final concentration is 1% skim milk powder and 1.5% Tween-20), and block at 4°C for 30 min. After blocking, centrifuge at 12,000 rpm for 20 min at 4°C, remove the supernatant, and resuspend the COF nanosheet photothermal probe precipitate in 80 μL of probe storage solution for later use.

[0145] Example 21

[0146] Unlike Example 20, the pH was adjusted to 7.

[0147] Example 22

[0148] Unlike Example 20, the pH was adjusted to 9.

[0149] Example 23

[0150] Unlike Example 20, the pH was adjusted to 10.

[0151] Example 24

[0152] Unlike Example 20, the pH was adjusted to 11.

[0153] The COF nanosheet photothermal probes obtained in Examples 20-24 are shown in the photographs of their resuspended in probe storage solution. Figure 19 As shown in A. (As in...) Figure 19 As shown in B, COF nanosheets conjugated with antibodies showed the best effect at pH 8.0.

[0154] Example 25

[0155] Unlike Example 20, 40 μL of 10% skim milk powder and 20 μL of 10% Tween-20 were added (the final concentration was 1% skim milk powder and 0.5% Tween-20).

[0156] Example 26

[0157] Unlike Example 20, 40 μL of 10% skim milk powder and 40 μL of 10% Tween-20 were added (the final concentration was 1% skim milk powder and 1% Tween-20).

[0158] Example 27

[0159] Unlike Example 20, 40 μL of 10% skim milk powder and 80 μL of 10% Tween-20 were added (the final concentration was 1% skim milk powder and 2% Tween-20).

[0160] Example 28

[0161] Unlike Example 20, 20 μL of 10% skim milk powder and 60 μL of 10% Tween-20 were added (the final concentration was 0.5% skim milk powder and 1.5% Tween-20).

[0162] Example 29

[0163] Unlike Example 20, 80 μL of 10% skim milk powder and 60 μL of 10% Tween-20 were added (the final concentration was 2% skim milk powder and 1.5% Tween-20).

[0164] The fluidity of the COF nanosheet photothermal probes obtained in Examples 20 and 25-27 was compared, and the results are as follows: Figure 20 As shown in A, the test strip exhibits good flowability at a Tween-20 concentration of 1.5%. Flowability was compared between Examples 20 and 28-29, as shown... Figure 20 As shown in B, when the concentration of skim milk powder is 1%, the test strip has good fluidity and the background color is light.

[0165] Example 30

[0166] Unlike Example 20, no DES antibody diluent was added.

[0167] Example 31

[0168] Unlike Example 20, 0.8 μL of DES antibody dilution solution was added to the COF nanosheet solution (corresponding to a DES antibody concentration of 2 μg·mL). -1 ).

[0169] Example 32

[0170] Unlike Example 20, 1.6 μL of DES antibody diluent was added to the COF nanosheet solution (corresponding to a DES antibody concentration of 4 μg·mL). -1 ).

[0171] Example 33

[0172] Unlike Example 20, 2.4 μL of DES antibody diluent was added to the COF nanosheet solution (corresponding to a DES antibody concentration of 6 μg·mL). -1 ).

[0173] Example 34

[0174] Unlike Example 20, 4 μL of DES antibody diluent was added to the COF nanosheet solution (corresponding to a DES antibody concentration of 10 μg / mL). -1 ).

[0175] 75 μL of negative test sample (60 μL PBS buffer, 10 μL running buffer, and 5 μL COF nanosheet photothermal probes from Examples 20, 30-34) was loaded onto each test strip. The color development of the detection area on each test strip was observed after 10 minutes. Results are as follows: Figure 21 As shown, when the antibody concentration in the COF detection probe is 8 μg·mL -1 Initially, the control area and the detection area showed clear color development. However, as the antibody concentration continued to increase, the color deepening in the detection area no longer showed a significant improvement. Therefore, 8 μg / mL was selected. -1 The optimal antibody-conjugated concentration.

[0176] Example 35

[0177] 3, 5, 7, and 9 μL of the COF nanosheet photothermal probe from Example 20 were mixed with 60 μL of PBS buffer and 10 μL of running buffer to prepare test solutions. The test solutions were then added to test strips to assess their colorimetric performance. The results are as follows: Figure 22 As shown, when the sample loading amount of the COF nanosheet photothermal probe is 5 μL, the color of the test strip is clear and the background is clean. Therefore, 5 μL is selected as the optimal sample loading amount for the COF nanosheet photothermal probe.

[0178] Example 36

[0179] The original coating concentration was diluted to 0.1, 0.2, 0.3, and 0.4 mg / mL using PBS buffer. -1 The diluted coating agent was sprayed onto the test area of ​​the test strip using a coating apparatus. The secondary antibody concentration in the control area of ​​the test strip was 0.5 mg / mL. -1 After the test strips were coated, they were dried at 37°C. After drying, 75 μL of negative test sample (60 μL PBS buffer, 10 μL running buffer, and 5 μL COF nanosheet photothermal probe from Example 20) was loaded onto each strip. After 10 minutes, the color development of the detection area on each test strip was observed. The strip with uniform and clear color development in the detection area was considered to have the optimal coating concentration. Figure 23 As shown in A, the optimal concentration of diethylstilbestrol antigen in the detection area is 0.4 mg·mL. -1 .

[0180] The goat anti-mouse secondary antibody was diluted with PBS buffer to concentrations of 0.7, 0.8, 0.9, and 1.0 mg / mL, respectively. -1 The diluted secondary antibody was applied to the control area of ​​the test strip using a coating applicator, and the test area of ​​the test strip was coated with the original optimum concentration of 0.4 mg / mL. -1 After spraying, the sample was dried at 37°C. After drying, 75 μL of negative test sample (60 μL PBS buffer, 10 μL running buffer, and 5 μL COF nanosheet photothermal probe from Example 20) was loaded onto each strip. After 10 minutes, the color development of the test area on each strip was observed. The group with uniform and clear color development in the control area represented the optimal secondary antibody concentration. Figure 23 As shown in B, the optimal concentration of goat anti-mouse secondary antibody in the quality control area is 0.8 mg / mL. -1 .

[0181] Example 37

[0182] The COF nanosheet photothermal probe used in Example 20 had a diethylstilbestrol antigen concentration of 0.4 mg / mL in the detection region. -1 The concentration of goat anti-mouse secondary antibody in the quality control area was 0.8 mg / mL. -1 The test strips were used for testing.

[0183] Diethylstilbestrol (DES) solutions of 1.5 μg / mL, 3 μg / mL, and 6 μg / mL were added to the test strips, and the detection area was irradiated with a laser of fixed power. The real-time temperature was recorded every 10 seconds using a thermal imager. Figure 24 As shown in A, after 40 seconds of irradiation, the temperature of the test strip reached equilibrium, and the temperature change slowed down within the dynamic range.

[0184] Choose lasers with power levels of 1.5W, 1.75W, 2W, and 2.25W to irradiate the test strips. Figure 24 As shown in B, when the laser power is increased to 2W, T0 / T no longer increases. Therefore, 2W is selected as the optimal power for laser irradiation.

[0185] like Figure 25 As shown in Figure A, the temperature difference between the detection area and the quality control area is most significant after an irradiation time of 1.5 cm. Therefore, 1.5 cm is chosen as the optimal irradiation distance. Figure 25 As shown in Figure B, the temperature difference between the detection area and the quality control area is most significant when the thermal imager height is 12cm. Therefore, 12cm is selected as the optimal thermal imager height.

[0186] Example 38

[0187] The COF nanosheet photothermal probe used in Example 20 had a diethylstilbestrol antigen concentration of 0.4 mg / mL in the detection region. -1The concentration of goat anti-mouse secondary antibody in the quality control area was 0.8 mg / mL. -1 The test strips were used for detection. The irradiation time was 40 seconds, the irradiation power was 2W, and the irradiation distance was 1.5cm. The height of the thermal imager was 12cm.

[0188] DES was diluted to concentrations of 0.05, 0.125, 0.25, 0.75, 1.5, 3.0, 6.0, 12.5, and 25 μg·L⁻¹. -1 The standard solution was tested using test strips. After 15 minutes, DES could be qualitatively detected visually. Once the test strips were dry, the detection and control areas were irradiated with an 808nm laser, and the temperature difference corresponding to each DES concentration was recorded using a thermal imager to plot the standard curve. Figure 26 As shown in A, the limit of detection by the naked eye is 3 μg·L⁻¹. -1 The detection limit of the photothermal test strip is calculated by subtracting three times the standard deviation of the temperature difference between the control area and the detection area in 10 blank tests. The temperature differences between the control area and the detection area in 10 blank tests were recorded and the average value was calculated, then subtracted three times the standard deviation of that average value. The calculated photothermal detection limit is 0.24 μg·L⁻¹. -1 .like Figure 26 As shown in B, the DES concentration ranges from 0.25 to 50 μg·L⁻¹. -1 There is a good linear relationship within the range.

[0189] Seven estrogens—α-estradiol, β-estradiol, bisphenol A, bisphenol B, estriol, hexestrol, and estrone—were selected for assay. The standard solutions prepared during the assay had a concentration of 50 μg / L. -1 .like Figure 27 As shown, when detecting DES, the detection area showed no color development, and the temperature difference between the detection area and the control area was greater than 15℃. When detecting other estrogens, the detection area showed obvious color development, and the temperature difference between the detection area and the control area was approximately 5℃. The results indicate that this method has significant specificity for DES.

[0190] For a concentration of 1.5 μg·L -1 Five parallel experiments were conducted on the DES. For example... Figure 28 As shown, there was no significant difference in the photothermal detection results of the five parallel experiments, proving that the repeatability of the detection method meets the detection requirements.

[0191] Stability analysis was performed on test strips stored at 4°C for 20 days. For example... Figure 29 As shown, the test strip's detection stability decreased to some extent after being stored at 4℃ for a certain period of time. After 20 days of storage, its detection efficiency decreased to 73% of the initial value. This indicates that the prepared test strip has good storage stability.

[0192] DES recovery experiments were conducted using tap water, milk, and pork. Different concentrations of DES (1.5, 5, and 12.5 μg·L⁻¹) were added to the three actual samples. -1 Perform the test. For example... Figure 30 As shown in A, due to the influence of complex matrices in actual samples, such as ions in tap water, proteins in milk, and fats in pork products, the detection effect of the established detection method differs in the three actual samples compared to that in PBS solution. Figure 30 As shown in Figure B, the recovery rate of DES in the three actual samples was 89.2%–118.7%, and the coefficient of variation was 2.4%–12.0%, indicating that the method has good feasibility and applicability in the detection of actual samples.

[0193] 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 diethylstilbestrol based on a photothermal probe and lateral chromatography test paper, characterized in that, Includes the following steps: A photothermal probe was added to a sample solution containing diethylstilbestrol (DES), and a running buffer was added. The mixture was incubated to obtain a mixed solution. The mixed solution was then loaded onto the sample pad area of ​​a lateral chromatography test strip. After standing, the results were observed and recorded. The control area and the detection area were irradiated with an 808 nm laser, and the temperature changes in each area were recorded using a thermal imager. A standard curve was established using the temperature difference ΔT between the two areas and the DES concentration to achieve quantitative detection of DES. The photothermal probe is formed by conjugation of a photothermal material and a diethylstilbestrol monoclonal antibody, wherein the photothermal material is purple phosphorus nanosheets. The preparation method of the purple phosphorus nanosheet solution includes the following steps: Purple phosphorus crystal powder was mixed with methanol and ground for 40-60 min. After drying, the mixture was added to ultrapure water and ultrasonically dispersed at 250W for 15-20 min. The dispersed solution was ultrasonically treated at 500W for 6-8 h. After centrifugation at 2000-2500 rpm for 4-6 min, the supernatant was collected to obtain a purple phosphorus nanosheet solution. The mass-to-volume ratio of the purple phosphorus crystal powder, methanol, and ultrapure water is 19-20 mg: 2 mL: 55-65 mL. The detection zone of the lateral chromatography test strip is coated with a PBS solution containing diethylstilbestrol antigen, and the control zone of the lateral chromatography test strip is coated with a PBS solution containing goat anti-mouse secondary antibody; the concentration of diethylstilbestrol antigen in the PBS solution containing diethylstilbestrol antigen is 0.2-1 mg / mL, and the concentration of goat anti-mouse secondary antibody in the PBS solution containing goat anti-mouse secondary antibody is 0.1-0.8 mg / mL.

2. The method for detecting diethylstilbestrol as described in claim 1, characterized in that, The preparation method of the photothermal probe includes the following steps: S1. Dissolve bovine serum albumin and Tween-20 in ultrapure water to obtain probe storage solution; S2. Dilute the purple phosphorus nanosheet solution, adjust the concentration, and shake. S3. Add the PBS solution containing diethylstilbestrol monoclonal antibody to the shaken purple phosphorus nanosheet solution to obtain a mixed solution, and shake. S4. Add 10% skim milk powder solution and 10% Tween-20 solution to the shaken mixture, seal, and centrifuge after sealing to obtain the precipitate, which is the photothermal probe. The photothermal probe is resuspended in the probe storage solution.

3. The method for detecting diethylstilbestrol as described in claim 2, characterized in that, The concentration of diethylstilbestrol monoclonal antibody in the PBS solution containing diethylstilbestrol monoclonal antibody was 0.9-1.1 mg / mL. -1 ; The volume ratio of the purple phosphorus nanosheet solution, the PBS solution containing diethylstilbestrol monoclonal antibody, the 10% skim milk powder solution, and the 10% Tween-20 solution was 100:0.4-1:5-20:5-20.

4. The method for detecting diethylstilbestrol as described in claim 2, characterized in that, In step S1, the mass-to-volume ratio of bovine serum albumin, Tween-20, and ultrapure water is 95-105 mg: 20-30 μL: 10 mL. In step S2, dilute to an absorbance of 1-1.1 at a wavelength of 450 nm, adjust the pH to 6-11, and shake for 5-10 min. In step S3, oscillate at 36-37 ℃ for 45-60 min; In step S4, the container is sealed at 4 ℃ for 30-40 min and then centrifuged at 12000 rpm for 20-30 min.

5. The method for detecting diethylstilbestrol as described in claim 1, characterized in that, The running buffer is an ultrapure aqueous solution containing 0.9-1.1 g / mL sucrose, 0.7-0.9 g / mL bovine serum albumin and 0.9-1.1% Tween-20 (v / v). The volume ratio of the photothermal probe, sample solution, and running buffer is 3-25:50:10; The irradiation time of the 808 nm laser is 30-80 s, the irradiation power is 0.75-1.5 W, the irradiation distance is 1-3 cm, and the detection height of the thermal imager is 11-15 cm.

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