Test paper for detecting ethinylestradiol and preparation method thereof

Abstract

The preparation method comprises the following steps: (1) preparing a Sb2WO6-coated NiS compound, and adsorbing an ethinylestradiol antibody, namely a primary antibody, on the surface of the Sb2WO6-coated NiS compound to obtain a photo-thermal signal probe; (2) assembling the test strip, and respectively fixing an ethinylestradiol antigen and a secondary antibody on the detection area and the quality control area; (3) mixing the photo-thermal signal probe with a to-be-detected sample solution, and inserting the sample pad end of the test strip into the mixed solution for chromatography; and (4) detecting a photo-thermal signal: irradiating the detection area by using a near-infrared LED, collecting the temperature by using thermal imaging or temperature measuring equipment, and carrying out quantitative analysis according to the linear relationship between the temperature of the detection area and the concentration of the sample to-be-detected substance. The detection mode is flexible, the linear range is wide, the sensitivity is high, and the specificity is good.

Description

Technical Field

[0001] The present invention relates to the technical field of small molecule analysis and detection, and in particular to a test paper for detecting ethinyl estradiol and a preparation method thereof. Background Art

[0002] Lateral flow immunochromatographic test strips (LFIA) have the characteristics of fast analytical performance, good selectivity, low cost, small sample volume requirement, easy large-scale production and stable long-term storage, and are widely used in the design of portable analytical detection sensors. Photothermal LFIA, which uses photothermal signal probes in LFIA, has a sensitivity that is tens to hundreds of times higher than the colorimetric mode. The signal of this type of detection method is caused by temperature changes due to the photothermal effect, with low background signals and sensitive sensing. It can be performed on various substrates of different colors and has great application potential. At present, new nanomaterials with higher photothermal conversion efficiency are still needed as photothermal signal probes to form a more obvious thermal contrast to further improve the performance of photothermal LFIA.

[0003] Sb2WO6 is a compound with special photoelectric properties, which shows good application prospects in the fields of photocatalysis and photothermal conversion. For example, a study constructed a three-dimensional bionic bird's nest-shaped nanocomposite material by in-situ composite Sb2WO6 and D-fructose (D-Fru). The composite material exhibited excellent photothermal conversion performance and could achieve efficient solar photothermal conversion and water evaporation. In addition, NiS is a sulfide semiconductor material with a narrow band gap and a high light absorption coefficient, so it also has certain application potential in the fields of photocatalysis and photothermal conversion. However, compared with Sb2WO6, there are fewer direct studies on NiS in photothermal conversion. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a test paper for detecting ethinyl estradiol and a preparation method thereof. The Sb2WO6@NiS composite material is used to prepare a photothermal test paper strip for detecting ethinyl estradiol. The test paper strip of the present invention is used to detect ethinyl estradiol, and the detection limit is as low as 10 -1 pg / mL.

[0005] The technical solution of the present invention is as follows:

[0006] The first object of the present invention is to provide a Sb2WO6@NiS composite material, wherein the preparation method of the composite material comprises the following steps:

[0007] (1) adding Sb2WO6 nanoparticles into a polyethylene glycol aqueous solution, performing ultrasonic dispersion and stirring reaction to obtain a mixed solution;

[0008] (2) The mixed solution obtained in step (1) is added to the NiS aqueous solution, stirred for reaction, and centrifuged and dried after the reaction is completed to obtain the composite material.

[0009] In one embodiment of the present invention, in step (1), one or more of the following conditions are met:

[0010] The weight average molecular weight of polyethylene glycol is 5000-10000;

[0011] The concentration of polyethylene glycol aqueous solution is 1-4 mg / mL;

[0012] The mass ratio of Sb2WO6 nanoparticles to polyethylene glycol is 1:10-1:5;

[0013] The ultrasonic dispersion conditions are as follows: ultrasonic power is 120-600W, and ultrasonic time is 20-30min;

[0014] The stirring reaction conditions are: room temperature, 500-1000rpm, 10-15h

[0015] In step (2), one or more of the following conditions are met:

[0016] The concentration of NiS aqueous solution is 0.1-1% (containing 5% nitric acid);

[0017] The volume ratio of the mixed solution to the NiS aqueous solution is 1:5-5:1.

[0018] The stirring reaction conditions are: 80-95°C, 10-15h.

[0019] In one embodiment of the present invention, 1-2 mg of Sb2WO6 nanoparticles are added to 5-10 mL of deionized water containing 10-20 mg of polyethylene glycol (MW = 5000-10000), ultrasonicated for 20-30 minutes, and stirred overnight; 1 mL of the above mixture is added to 1 mL of NiS aqueous solution and stirred overnight; the dark gray powder is collected by centrifugation and dried for further use.

[0020] In one embodiment of the present invention, Sb2WO6 nanoparticles are synthesized by a hydrothermal method and have a particle size of 10-100 nm.

[0021] The second object of the present invention is to provide an application of the above-mentioned Sb2WO6@NiS composite material for preparing a photothermal signal probe.

[0022] The third object of the present invention is to provide a photothermal signal probe prepared from the above-mentioned Sb2WO6@NiS composite material, comprising Sb2WO6@NiS composite material, ethinyl estradiol antibody and bovine serum albumin.

[0023] In one embodiment of the present invention, the method for preparing the photothermal signal probe is as follows: the Sb2WO6@NiS composite material is mixed with a test antibody and bovine serum albumin to obtain a Sb2WO6@NiS-mAb mixed solution.

[0024] In one embodiment of the present invention, a Sb2WO6@NiS composite material is placed in a centrifuge tube, a weak alkaline solution is added to adjust the pH of the system to 6-8, the mixture is shaken and mixed, and then ethinyl estradiol antibody is added. After shaking reaction at room temperature for 30-60 minutes, bovine serum albumin BSA is added for blocking, and the mixture is shaken and reacted at room temperature for 1-1.5 hours. After centrifugation, the supernatant is removed, and the remaining substance is redissolved in a buffer solution to obtain a Sb2WO6@NiS-mAb mixed solution, which is refrigerated for later use.

[0025] In one embodiment of the present invention, the buffer is a phosphate buffer, a borate buffer or a carbonate buffer.

[0026] A fourth object of the present invention is to provide a test strip containing the above-mentioned photothermal signal probe, wherein the test strip comprises the photothermal signal probe and a test strip body;

[0027] The photothermal signal probe is prepared by modifying a Sb2WO6@NiS composite nanomaterial with an ethinyl estradiol polyclonal antibody, and is used in a form independent of the main structure of the photothermal test strip or in a form dried on a conjugate pad;

[0028] The test strip body comprises a PVC bottom plate, a sample pad, a nitrocellulose membrane (NC membrane), and an absorption pad.

[0029] In one embodiment of the present invention, the test strip body comprises: a sample pad, a nitrocellulose membrane, i.e., an NC membrane, and an absorption pad are overlapped and pasted in sequence from top to bottom in a horizontal direction on a PVC bottom plate; wherein the nitrocellulose membrane is used to separate and detect analytes from other substances in the sample, the sample pad is used for loading samples, the absorption pad is used to absorb excess liquid, and the PVC bottom plate provides physical support for the test strip; the nitrocellulose membrane includes a detection area, i.e., a T area, and a quality control area, i.e., a C area; wherein, ethinyl estradiol antigen, i.e., ethinyl estradiol-protein conjugate, is fixed on the T area; and a secondary antibody is fixed on the C area, and the secondary antibody is a secondary antibody directed against the source of the primary antibody, i.e., an anti-primary antibody;

[0030] The photothermal signal probe is used in a form independent of the main structure of the photothermal test strip, or the photothermal signal probe is dried on a conjugate pad and the conjugate pad is inserted between the sample pad and the NC membrane for use.

[0031] In one embodiment of the present invention, the ethinyl estradiol antibody, i.e., the primary antibody, is derived from at least one of mouse, rat, and rabbit; the secondary antibody comprises at least one of goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody, and donkey anti-rabbit secondary antibody.

[0032] A fifth object of the present invention is to provide a method for preparing the above-mentioned test strip, comprising the following steps:

[0033] Preparation of Sb2WO6@NiS composite materials;

[0034] Preparation of Sb2WO6@NiS-mAb mixture of Sb2WO6@NiS composite material, ethinyl estradiol antibody, and bovine serum albumin;

[0035] Construct the test strip: drip or spray ethinyl estradiol antigen on the T area, drip or spray the secondary antibody against the source of the primary antibody on the C area, i.e., anti-primary antibody, and store in a vacuum bag after drying for future use;

[0036] The photothermal signal probe, i.e., the Sb2WO6@NiS-mAb mixed solution, is stored in a sealed container in the form of a solution or a lyophilized powder; or the photothermal signal probe, i.e., the Sb2WO6@NiS-mAb mixed solution, is dried on a conjugate pad and the conjugate pad is inserted between the sample pad and the NC membrane for use.

[0037] In one embodiment of the present invention, the T zone is dripped or sprayed with ethinyl estradiol antigen, specifically: a 5-20mM PBS solution containing 0.5-10mg / mL ethinyl estradiol antigen is dripped or sprayed on the T zone; the C zone is dripped or sprayed with a secondary antibody targeting the source of the primary antibody, i.e., an anti-primary antibody, specifically: a 5-20mM PBS solution containing 0.05-10mg / mL secondary antibody is dripped or sprayed on the C zone.

[0038] The sixth object of the present invention is to provide an application of the above-mentioned test strip for detecting ethinyl estradiol; the photothermal temperature of the detection zone is negatively correlated with the ethinyl estradiol content to be measured, and the color intensity is negatively correlated with the ethinyl estradiol content in the sample, specifically:

[0039] When the sample does not contain ethinyl estradiol, the T zone appears purple and the temperature is high;

[0040] When the sample contains ethinyl estradiol, the T zone appears light purple or even disappears, and the temperature decreases;

[0041] The quality control area serves as a reference for verifying the validity of the test paper results and always appears purple.

[0042] A sixth object of the present invention is to provide a test strip for detecting ethinyl estradiol, comprising a photothermal signal probe and a test strip body;

[0043] The preparation method of the photothermal signal probe is as follows: Sb2WO6@NiS composite material is mixed with the test object antibody and bovine serum albumin to obtain a Sb2WO6@NiS-mAb mixed solution.

[0044] The test strip body comprises: a sample pad, a nitrocellulose membrane, i.e., an NC membrane, and an absorption pad are overlapped and pasted in sequence from top to bottom in the horizontal direction on a PVC bottom plate; wherein, the nitrocellulose membrane is used to separate and detect the analyte from other substances in the sample, the sample pad is used for loading the sample, and the absorption pad is used to absorb excess liquid, and the PVC bottom plate provides physical support for the test strip; the nitrocellulose membrane includes a detection area, i.e., a T area, and a quality control area, i.e., a C area; wherein, ethinyl estradiol antigen, i.e., ethinyl estradiol-protein conjugate, is fixed on the T area; a secondary antibody is fixed on the C area, and the secondary antibody is a secondary antibody directed against the source of the primary antibody, i.e., anti-primary antibody; the primary antibody is a mouse-derived ethinyl estradiol polyclonal antibody; and the secondary antibody is a sheep anti-mouse antibody.

[0045] The photothermal signal probe is used in a form independent of the main structure of the photothermal test strip, or the photothermal signal probe is dried on a conjugate pad and the conjugate pad is inserted between the sample pad and the NC membrane for use.

[0046] In one embodiment of the present invention, the method for detecting ethinyl estradiol with a test strip comprises the following steps:

[0047] When the photothermal signal probe is used in a form independent of the main structure of the photothermal test strip, it specifically includes:

[0048] The sample to be tested and the Sb2WO6@NiS-mAb solution were mixed in the running buffer, and then placed in the test strip. After 10-15 minutes, the test strip was taken out. After the test strip was dried, the photothermal analysis results were read for quantitative analysis.

[0049] When the photothermal signal probe is dried on the conjugate pad and the conjugate pad is inserted between the sample pad and the NC film of the test paper main structure for use, the method specifically includes:

[0050] Mix the sample to be tested with the running buffer, then put it into the test strip, take it out after 10-15 minutes, and read the photothermal analysis results after the test strip is dried for quantitative analysis;

[0051] After the test strip is dried, the method for reading the photothermal analysis result is as follows: the test strip is placed under a near-infrared LED light source for excitation, and the temperature result is obtained using a thermal imaging or temperature measuring device and an intelligent display terminal. The thermal imaging or temperature measuring device includes at least one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, and a temperature measuring gun. The intelligent display terminal includes at least one of a computer and a smart phone. The thermal imaging device collects photothermal imaging photos and outputs and displays them through the intelligent display terminal connected thereto.

[0052] In one embodiment of the present invention, the running buffer is a PBS solution with a pH of 6.5 to 8.0 and a total concentration of 10 mM, and the following components are contained in mass percentages, specifically comprising: 0 to 15% sucrose, 1 to 10% BSA, and 0.15 to 1% Tween-20.

[0053] In one embodiment of the present invention, the wavelength of the near-infrared LED light source is 980 nm.

[0054] The detection principle of the photothermal test strip for detecting ethinyl estradiol of the present invention is as follows: the quality control area (area C) is used as a reference for verifying the validity of the test paper results, and always appears purple. After the sample solution and Sb2WO6@NiS-mAb are pre-mixed, they move toward the absorbent paper under capillary action. When there is no ethinyl estradiol in the sample, Sb2WO6@NiS-mAb is captured by ethinyl estradiol-BSA in the T area, and the T area is visible to the naked eye as purple. Under the excitation of the laser, Sb2WO6@NiS-mAb produces an LSPR effect, and the temperature rises. Excess Sb2WO6@NiS-mAb will be captured by the secondary antibody in the C area, so that the C area also appears purple; when the sample contains ethinyl estradiol, ethinyl estradiol combines with part of the Sb2WO6@NiS-mAb, thereby reducing the total amount of Sb2WO6@NiS-mAb captured in the T area, and the LSPR effect of the Sb2WO6@NiS-mAb in the T area is reduced or even disappears, and the temperature drops. As the concentration of ethinyl estradiol in the sample increased, less and less Sb2WO6@NiS-mAb was captured in the T zone, and the temperature of the T zone gradually decreased, which was inversely proportional to the analyte concentration.

[0055] The beneficial technical effects of the present invention are:

[0056] The Sb2WO6@NiS composite material provided by the present invention is used in the preparation and application of photothermal test strips. There is no need to change the conventional structure of the test strips. It can be used as a photothermal signal probe and can be used independently of the photothermal test strips for convenient storage, or it can be fixed on the binding pad of the test strip for easy carrying.

[0057] The detection method including the photothermal test strip provided by the present invention is based on the plasma resonance effect, and the photothermal test strip for detecting ethinyl estradiol uses a Sb2WO6@NiS composite material with high photothermal conversion efficiency and microsize as a T-zone fixed photothermal signal probe, near-infrared LED light source excitation, and combines an intelligent terminal and infrared thermal imaging accessories to realize the collection of photothermal signals, effectively remove the base color of the sample liquid and the fluorescence background interference of the test strip, thereby improving the signal-to-noise ratio.

[0058] The detection method provided by the present invention has a detection limit of 10 -1 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a schematic diagram of the photothermal test strip of the present invention;

[0060] Figure 2(A) scanning electron microscope image, (B) transmission electron microscope image and (C) X-ray diffraction image of the Sb2WO6@NiS composite material in the photothermal signal probe of the test strip of the present invention;

[0061] Figure 3 The photothermal temperature rise curves of Sb2WO6@NiS composite material, Sb2WO6 and NiS in the photothermal signal probe of the test strip of the present invention;

[0062] Figure 4 The response of the test strip of the present invention to water samples with different concentrations of ethinyl estradiol in the photothermal mode and the working curve are shown;

[0063] Figure 5 The figure shows the response of the test strip of the present invention to ethinyl estradiol and four other estrogens. DETAILED DESCRIPTION

[0064] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0065] Example 1 Preparation of raw materials

[0066] 1.1 Preparation of Sb2WO6 nanocrystals

[0067] Sb2WO6 nanoparticles were synthesized by hydrothermal method. 0.5g Na2WO4·2H2O and 1.5g Bi(NO3)3·5H2O were dissolved in 5mL ethylene glycol respectively. After mixing, 35mL ethanol was added. After stirring for 0.5h, the solution was transferred to a 50mL autoclave and heated at 180℃ for 10h. The precipitate after centrifugation was washed with deionized water and ethanol and dried at 80℃ for 10h. Then, the obtained powder was calcined at 450℃ (2℃ / min heating rate) for 2h to obtain Sb2WO6 nanocrystals.

[0068] 1.2 Preparation of Sb2WO6@NiS composites and characterization of their structural properties

[0069] 1 mg of Sb2WO6 nanoparticles was added to 5 mL of deionized water containing 10 mg of polyethylene glycol (MW = 7000), ultrasonicated at 200 W for 200 min, and stirred for 10 h; 1 mL of the above mixture was added to 1 mL of NiS aqueous solution and stirred overnight; the dark gray powder was collected by centrifugation and dried for further use.

[0070] The SEM, TEM and XRD characteristics of Sb2WO6@NiS composites were determined. Figure 2 As shown; the photothermal heating curve is as follows Figure 3 As shown, the Sb2WO6@NiS composite material has the strongest heating ability, which is better than Sb2WO6 and NiS.

[0071] 1.3 Sb2WO6@NiS and antibody adsorption: Prepare a mixture of Sb2WO6@NiS composite material, ethinyl estradiol antibody, and bovine serum albumin, namely, a mixture of Sb2WO6@NiS-mAb.

[0072] Take 1mL Sb2WO6@NiS in a centrifuge tube, add 4μL 0.2Wl / L K2CO3 solution to adjust the pH value of the system, shake and mix, then add 5μL 1mg / mL ethinyl estradiol antibody, mix and shake at room temperature for 45min. After the reaction, add 100μL BSA (m / m=5%) to block for 1h, centrifuge at 12000r / min for 30min, remove the supernatant, and redissolve in 100μL buffer (20mol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain Sb2WO6@NiS-mAb mixed solution, which is stored at 4℃ for later use.

[0073] 1.4 Preparation of the test zone (T zone) solution

[0074] Ethinylestradiol-BSA was diluted to 0.6 mg / mL using 10 mM PBS solution at pH 7.4.

[0075] 1.5 Preparation of Quality Control Zone (Zone C) Solution

[0076] Dilute the goat anti-mouse secondary antibody to 0.4 mg / mL in 10 mM PBS solution at pH 7.4.

[0077] Example 2 Preparation of test strips

[0078] according to Figure 1 The NC membrane was pasted in the middle of the PVC bottom plate, and the sample pad and the absorbent pad were overlapped on the left and right ends of the NC membrane, so that they covered the NC membrane by about 2 mm. The assembled large card was cut into 3 mm wide strips, and the detection line (T zone) and the quality control line (C zone) were made on the NC membrane. The two lines were separated by 4 mm to obtain a blank test strip. 0.5 μL of T zone solution and 0.5 μL of C zone solution were dropped in the T zone and C zone respectively. The test strips after spotting were placed in an oven, dried at 37°C for 60 min, and stored in a vacuum bag for later use.

[0079] Example 3 Preparation of a Test Strip Containing a Binding Pad

[0080] Test strips according to Figure 1The binding mode consists of a sample pad, a nitrocellulose membrane (NC), a binding pad and a PVC base plate; the NC membrane is pasted in the middle of the PVC base plate, and a detection line (T zone) and a quality control line (C zone) are made on the NC membrane, with a spacing of 4 mm between the two lines; then the right end of the binding pad is overlapped on the left end of the NC membrane, so that it covers the NC membrane by about 2 mm, the sample pad is overlapped on the left end of the binding pad, so that it covers the binding pad by about 2 mm, and the absorbent pad is overlapped on the right end of the NC membrane, so that it covers the NC membrane by about 2 mm; the assembled large card is cut into 3 mm wide paper strips, and a mixture of 10 μL Sb2WO6@NiS composite material, ethinyl estradiol antibody, and bovine serum albumin, i.e., Sb2WO6@NiS-mAb mixture, is fixed on the binding pad, and 0.5 μL T zone solution and 0.5 μL C zone solution are added to the T zone and C zone, respectively. The test strips after spotting are placed in an oven, dried at 37°C for 60 min, and stored in a vacuum bag for later use.

[0081] Example 4 Working curve drawing

[0082] Prepare 10 mL of 1 mg / mL ethinyl estradiol standard solution in acetonitrile and dilute to a concentration of 10 with 10 mM PBS. -2 pg / mL, 10 -1 pg / mL, 10 0 pg / mL, 10 1 pg / mL, 10 2 pg / mL, 10 3 pg / mL, 10 4 pg / mL, 10 5 pg / mL, as the test solution. 80μL of the test solution was mixed with 10μL of Sb2WO6@NiS-mAb and 10μL of running buffer (10mM PBS solution, containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) in a centrifuge tube, and then the test strip was inserted into the centrifuge tube. After 10 minutes, the test strip was taken out and the test strip was dried with a near-infrared LED (power 1.36W / cm 2 ) for 3 minutes, and use a mobile phone and infrared thermal imaging accessories to monitor temperature changes. The results of the photothermal mode are as follows Figure 4 As shown; the working curve is y = 1.983x + 0.377 (R 2 =0.9934).

[0083] The results showed that in the photothermal mode, the negative T zone had the highest temperature and the ethinyl estradiol concentration was 10 5 pg / mL, the temperature of the T zone dropped to the lowest, and this concentration was the detection limit of the photothermal mode. Therefore, the detection limit of the constructed dual-mode test paper can reach 10 -1 pg / mL.

[0084] Example 5 Detection of ethinyl estradiol

[0085] The specificity verification of the Sb2WO6@NiS composite photothermal quantitative test strip of ethinyl estradiol includes the following steps:

[0086] 1. Preparation of test paper materials

[0087] Same as Example 1.

[0088] 2. Preparation of test strips

[0089] Same as Example 3.

[0090] 3. Working curve drawing

[0091] Same as Example 4.

[0092] 4. Specificity

[0093] Use acetonitrile to prepare 10 mL of 1 mg / mL ethinyl estradiol, estradiol, estrone, estriol, and diethylstilbestrol standard solutions, dilute with 10 mM PBS to a concentration of 120 ng / mL, and use as test solutions.

[0094] 5. Sample testing

[0095] 80 μL of the test solution was mixed with 10 μL of Sb2WO6@NiS-mAb and 10 μL of running buffer (10 mM PBS solution, containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) in a centrifuge tube for 10 min. Then the test strip was inserted into the centrifuge tube. After the test strip was dried, a near-infrared LED (power 1.36 W / cm 2 ) for 3 minutes, and use a mobile phone and infrared thermal imaging accessories to monitor temperature changes. The results of the photothermal mode are as follows Figure 5 Compared with the blank, estradiol, estrone, estriol, and diethylstilbestrol had very weak effects on the T-zone temperature of the test paper, while ethinyl estradiol had a very significant effect on the T-zone temperature of the test paper, indicating that the test paper had a weak response to the four, that is, the test paper had good specificity.

[0096] It can be seen from the above embodiments that the Sb2WO6@NiS composite material provided in the present application is used in the photothermal test strips for preparing the photothermal test strips. There is no need to change the conventional structure of the test strips. It can be used as a photothermal signal probe, and can be used independently of the photothermal test strips for convenient storage, or fixed on the binding pad of the test strips for use, which is convenient to carry. The detection method comprising the photothermal test strips provided by the present invention is based on the plasma resonance effect. The photothermal test strips for detecting ethinyl estradiol are fixed with the Sb2WO6@NiS composite material with high photothermal conversion efficiency and tiny size as the T-zone photothermal signal probe, excited by a near-infrared laser light source, and combined with the smart terminal and infrared thermal imaging accessories to realize the collection of photothermal signals, effectively removing the base color of the sample liquid and the fluorescence background interference of the test strips to improve the signal-to-noise ratio. The inspection method has a lower detection limit, a wider detection range and better specificity, and is more sensitive than other photothermal detection test strips.

[0097] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A test paper for detecting ethinyl estradiol, characterized in that: The test paper comprises a photothermal signal probe and a test strip body; The photothermal signal probe is prepared by modifying a Sb2WO6@NiS composite nanomaterial with an ethinyl estradiol polyclonal antibody, and is used in a form independent of the main structure of the photothermal test strip or in a form dried on a conjugate pad; The test strip body comprises a PVC bottom plate, a sample pad, a nitrocellulose membrane (NC membrane), and an absorption pad.

2. The test paper for detecting ethinyl estradiol according to claim 1, characterized in that The preparation method of the Sb2WO6@NiS composite material comprises the following steps: (1) adding Sb2WO6 nanoparticles into a polyethylene glycol aqueous solution, performing ultrasonic dispersion and stirring reaction to obtain a mixed solution; (2) The mixed solution obtained in step (1) is added to the NiS aqueous solution, stirred for reaction, and centrifuged and dried after the reaction is completed to obtain the composite material.

3. The test paper for detecting ethinyl estradiol according to claim 2, characterized in that, In step (1), one or more of the following conditions are met: The weight average molecular weight of polyethylene glycol is 5000-10000; The concentration of polyethylene glycol aqueous solution is 1-4 mg / mL; The mass ratio of Sb2WO6 nanoparticles to polyethylene glycol is 1:10-1:5; The ultrasonic dispersion conditions are as follows: ultrasonic power is 120-600W, and ultrasonic time is 20-30min; The stirring reaction conditions are: room temperature, 500-1000 rpm, 10-15 h; In step (2), one or more of the following conditions are met: The concentration of NiS aqueous solution is 0.1-1%, containing 5% nitric acid; The volume ratio of the mixed solution to the NiS aqueous solution is 1:5-5:

1. The stirring reaction conditions are: 80-95°C, 10-15h.

4. A photothermal signal probe prepared from the Sb2WO6@NiS composite material of claim 1, characterized in that: Contains Sb2WO6@NiS composite material, ethinyl estradiol antibody and bovine serum albumin.

5. The photothermal signal probe according to claim 4, characterized in that: The Sb2WO6@NiS composite material was mixed with ethinyl estradiol antibody and bovine serum albumin to obtain a Sb2WO6@NiS-mAb mixed solution.

6. A test strip containing the photothermal signal probe according to claim 4, characterized in that: The test strip includes a photothermal signal probe and a test strip body; The test strip body comprises: a sample pad, a nitrocellulose membrane, namely, an NC membrane, and an absorption pad are overlapped and pasted in sequence from top to bottom in the horizontal direction on a PVC bottom plate; wherein the nitrocellulose membrane is used to separate and detect analytes from other substances in the sample, the sample pad is used for loading samples, and the absorption pad is used to absorb excess liquid, and the PVC bottom plate provides physical support for the test strip; the nitrocellulose membrane includes a detection area, namely, a T area, and a quality control area, namely, a C area; wherein, ethinyl estradiol antigen, namely, ethinyl estradiol-protein conjugate, is fixed on the T area; and a secondary antibody is fixed on the C area, and the secondary antibody is a secondary antibody directed against the source of the primary antibody, namely, an anti-primary antibody; The photothermal signal probe is used in a form independent of the main structure of the photothermal test strip, or the photothermal signal probe is dried on a conjugate pad and the conjugate pad is inserted between the sample pad and the NC membrane for use.

7. The test strip according to claim 6, characterized in that: The ethinyl estradiol antibody, ie, the primary antibody, is derived from at least one of mouse, rat, and rabbit.

8. The test strip according to claim 6, characterized in that: The secondary antibody comprises at least one of goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody and donkey anti-rabbit secondary antibody.

9. An application of the test strip according to claim 6, characterized in that: Used to detect ethinyl estradiol; the photothermal temperature of the detection area is negatively correlated with the ethinyl estradiol content to be measured, and the color intensity is negatively correlated with the ethinyl estradiol content in the sample, specifically: When the sample does not contain ethinyl estradiol, the T zone appears purple and the temperature is high; When the sample contains ethinyl estradiol, the T zone appears light purple or even disappears, and the temperature decreases; The quality control area serves as a reference for verifying the validity of the test paper results and always appears purple.

10. The use according to claim 9, characterized in that: The method for detecting ethinyl estradiol with a test strip comprises the following steps: When the photothermal signal probe is used in a form independent of the main structure of the photothermal test strip, it specifically includes: The sample to be tested and the Sb2WO6@NiS-mAb solution were mixed in the running buffer, and then placed in the test strip. After 10-15 minutes, the test strip was taken out. After the test strip was dried, the photothermal analysis results were read for quantitative analysis. When the photothermal signal probe is dried on the conjugate pad and the conjugate pad is inserted between the sample pad and the NC film of the test paper main structure for use, the method specifically includes: Mix the sample to be tested with the running buffer, then put it into the test strip, take it out after 10-15 minutes, and read the photothermal analysis results after the test strip is dried for quantitative analysis; After the test strip is dried, the method for reading the photothermal analysis result is as follows: the test strip is placed under a near-infrared LED light source for excitation, and the temperature result is obtained using a thermal imaging or temperature measuring device and an intelligent display terminal. The thermal imaging or temperature measuring device includes at least one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, and a temperature measuring gun. The intelligent display terminal includes at least one of a computer and a smart phone. The thermal imaging device collects photothermal imaging photos and outputs and displays them through the intelligent display terminal connected thereto.

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