Test paper for detecting estriol and preparation method thereof

Abstract

The preparation method comprises the following steps: (1) preparing an As2MoO6 (at) NiS compound, and adsorbing an antibody of an object to be detected, namely a primary antibody, on the surface of the As2MoO6 (at) NiS compound to obtain a photo-thermal signal probe; (2) assembling the test strip, and respectively fixing an antigen and a secondary antibody of an object to be detected 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 invention belongs to the technical field of small molecule analysis and detection, and specifically relates to a test paper for detecting estriol 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. They are widely used in the design of portable analytical detection sensors.

[0003] In traditional LFIA, colloidal gold nanoparticles are usually used as photothermal signal probes. The naked eye can directly observe for qualitative or semi-quantitative detection. However, the colorimetric LFIA based on colloidal gold has defects such as low sensitivity, narrow linear range and lack of quantitative analysis. In order to improve the sensitivity of analytical detection, researchers have developed fluorescent LFIA by using various fluorescent nanoparticles as probes. However, these fluorescent substances are interfered by autofluorescence, resulting in a low signal-to-noise ratio. In addition, expensive and complex instruments are usually used for signal acquisition, which also reduces its applicability for on-site detection.

[0004] In recent years, the surface plasmon resonance effect (LSPR) of nanoparticles has been discovered and used again in LFIA in the form of photothermal signal probes. Compared with the colorimetric mode, the sensitivity has increased by dozens to hundreds of times. Photothermal LFIA has begun to attract the attention and exploration of researchers. 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 more obvious thermal contrast to further improve the performance of photothermal LFIA. Summary of the invention

[0005] In order to solve the technical problems in the related art, the present invention provides an As2MoO6@NiS composite material with high photothermal conversion efficiency for preparing photothermal test strips, a preparation method and an application thereof. The test strips are used to detect estriol to achieve a detection method with a lower detection limit, a wider detection range and better specificity, which is more sensitive than other photothermal detection test strips.

[0006] On the one hand, the present invention provides a photothermal test strip for detecting estriol, which is used to detect estriol. The test strip comprises a test strip body and a photothermal signal probe.

[0007] The test strip body comprises: a sample pad, a nitrocellulose membrane (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, the absorption pad is used for absorbing excess liquid, and the PVC bottom plate provides physical support for the test strip;

[0008] The nitrocellulose membrane comprises a detection area, namely, a T area, and a quality control area, namely, a C area; wherein the antigen of the object to be detected, namely, the molecule-protein conjugate to be detected, is fixed on the T area; and the 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;

[0009] The photothermal signal probe comprises a mixture of an As2MoO6@NiS composite material, an antibody to be detected, i.e., a primary antibody, and bovine serum albumin, i.e., an As2MoO6@NiS-mAb mixed solution;

[0010] 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.

[0011] As an optional embodiment, the antigen to be detected is estriol with fewer antigenic sites or only a single antigenic site, including but not limited to aflatoxin, fusarium toxin and other fungal toxins, fungicides, antibiotics, hormones and other pesticide residues.

[0012] As an optional embodiment, the antibody to be detected, i.e., the primary antibody, may be derived from, but not limited to, mouse, rat, and rabbit.

[0013] As an optional embodiment, the secondary antibody includes but is not limited to goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody, and donkey anti-rabbit secondary antibody.

[0014] As an optional embodiment, the preparation method of the As2MoO6@NiS composite material includes: adding 1-2 mg As2MoO6 nanoparticles to 5-10 mL deionized water dissolved with 10-20 mg polyethylene glycol, ultrasonicating for 20-30 minutes, stirring overnight to obtain a mixed solution; adding 1 mL of the mixed solution to 1 mL of an aqueous solution containing 1-2 mg NiS and stirring overnight; collecting dark gray powder by centrifugation and drying.

[0015] On the other hand, a method for preparing a photothermal test strip for detecting estriol is provided, the method for preparing the photothermal test strip comprising:

[0016] Preparation of As2MoO6@NiS composite materials;

[0017] preparing an As2MoO6@NiS-mAb mixture of an As2MoO6@NiS composite material, an antibody to be tested, and bovine serum albumin;

[0018] Construct the test paper: drip or spray the antigen of the object to be tested on the T area, drip or spray the secondary antibody against the source of the primary antibody on the C area, i.e., the anti-primary antibody, and store it in a vacuum bag after drying for future use;

[0019] The photothermal signal probe, i.e., the As2MoO6@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 As2MoO6@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.

[0020] As an optional embodiment, the specific method for preparing the photothermal signal probe As2MoO6@NiS composite material is: add 1-2 mg As2MoO6 nanoparticles to 5-10 mL deionized water containing 10-20 mg polyethylene glycol, ultrasonicate for 20-30 minutes, and stir overnight; add 1 mL of the above mixture to 1 mL of NiS aqueous solution and stir overnight; collect the dark gray powder by centrifugation and dry it for further use.

[0021] As an optional embodiment, the polyethylene glycol has a MW of 5000-10000.

[0022] As an optional embodiment, As2MoO6 nanoparticles are synthesized by a hydrothermal method.

[0023] As an optional embodiment, the As2MoO6@NiS-mAb mixed solution of the As2MoO6@NiS composite material, the antibody to be tested, and bovine serum protein is prepared by the following specific method: taking the As2MoO6@NiS composite material in a centrifuge tube, adding a weak alkaline solution to adjust the pH of the system to 6-8, oscillating and mixing, and then adding the antibody to be tested, after oscillating reaction at room temperature for 30-60 minutes, adding bovine serum albumin BSA for blocking, oscillating reaction at room temperature for 1-1.5 hours, removing the supernatant after centrifugation, and then redissolving the remaining substance in a buffer solution to obtain an As2MoO6@NiS-mAb mixed solution, and refrigerating for later use.

[0024] As an optional embodiment, the buffer includes phosphate buffer, borate buffer or carbonate buffer.

[0025] As an optional embodiment, the antigen to be detected is dripped or sprayed on the T zone, specifically: a 5-20 mM PBS solution containing 0.5-10 mg / mL of the antigen to be detected is dripped or sprayed on the T zone.

[0026] As an optional embodiment, the C area is dripped or sprayed with a secondary antibody targeting the primary antibody source, i.e., anti-primary antibody, specifically: a 5-20 mM PBS solution containing 0.05-10 mg / mL of the secondary antibody is dripped or sprayed on the C area.

[0027] On the other hand, the photothermal temperature of the detection zone is negatively correlated with the estriol content to be detected, and the color intensity is negatively correlated with the estriol content in the sample, specifically:

[0028] When the sample does not contain estriol, the temperature of the T zone is high;

[0029] When the sample contained estriol, the temperature of the T zone decreased;

[0030] The quality control area serves as a reference for verifying the validity of the test paper results and always appears purple.

[0031] On the other hand, an application of a photothermal test strip for detecting estriol is provided, which is used for detecting estriol.

[0032] As an optional embodiment, the photothermal signal probe is used independently 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 of the main structure of the test strip.

[0033] The photothermal signal probe is used in a form independent of the main structure of the photothermal test strip, specifically including:

[0034] The sample to be tested and the As2MoO6@NiS-mAb solution were placed in the running buffer and mixed for 3-10 minutes, and then the test strip was placed in the test strip. After 15-30 minutes, the colorimetric result was read for semi-quantitative analysis. A colorimetric result photo was obtained, and grayscale analysis was performed using image processing software for quantitative analysis.

[0035] After the test strip is dried, place it under a near-infrared LED light source for excitation, and use thermal imaging or temperature measurement equipment to obtain the temperature result.

[0036] Another application of a photothermal test strip for detecting estriol is to detect estriol, wherein the photothermal signal probe is dried on a conjugate pad, and the conjugate pad is inserted between a sample pad and an NC film of a main structure of the test strip for use.

[0037] Specifically, the photothermal signal probe, i.e., the As2MoO6@NiS-mAb mixed solution, is dried on the conjugate pad and the conjugate pad is inserted between the sample pad and the NC membrane. The sample to be tested is mixed with the running buffer and placed in the test strip. After 10 minutes, the test strip is dried and placed under a near-infrared LED light source for excitation. The temperature result is obtained using thermal imaging or temperature measurement equipment for quantitative analysis.

[0038] As an optional embodiment, the running buffer is a PBS solution with a pH of 6.5-8.0 and a total concentration of 10 mM, and the following component contents are in mass percentage, specifically comprising: 0-15% sucrose, 1-10% BSA, and 0.15-1% Tween-20.

[0039] As an optional implementation, the thermal imaging or temperature measurement equipment includes but is not limited to mobile phone infrared thermal imaging analysis accessories, infrared thermal imagers or temperature measuring guns.

[0040] As an optional implementation, the smart display terminal includes but is not limited to a computer and a smart phone.

[0041] As an optional implementation, the thermal imaging device collects photothermal imaging photos and outputs and displays them through a smart display terminal connected thereto.

[0042] The detection principle of the photothermal test strip for detecting estriol 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 strip results, and always appears purple. After the sample solution and As2MoO6@NiS-mAb are pre-mixed, they move toward the absorbent paper under capillary action. When there is no estriol in the sample, As2MoO6@NiS-mAb is captured by estriol-BSA in the T area, and the T area is visible to the naked eye as purple. Under the excitation of the laser, As2MoO6@NiS-mAb produces an LSPR effect, and the temperature rises. Excess As2MoO6@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 estriol, estriol combines with part of the As2MoO6@NiS-mAb, thereby reducing the total amount of As2MoO6@NiS-mAb captured in the T area, and the LSPR effect of the As2MoO6@NiS-mAb in the T area is reduced or even disappears, and the temperature drops. As the concentration of estriol in the sample increases, less and less As2MoO6@NiS-mAb is captured in the T zone, and the temperature of the T zone gradually decreases, which is inversely proportional to the analyte concentration.

[0043] The present invention at least has the following beneficial effects:

[0044] (1) The As2MoO6@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. It can be used independently of the photothermal test strips for easy storage, or it can be fixed on the binding pad of the test strip for easy carrying.

[0045] (2) The detection method provided by the present invention includes a photothermal test strip. The photothermal test strip for detecting estriol is based on the plasma resonance effect, with a high photothermal conversion efficiency and a tiny size As2MoO6@NiS composite material as a T-zone fixed photothermal signal probe, near-infrared LED light source excitation, combined with a smart 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.

[0046] (3) The inspection method provided by the present invention has a lower detection limit, a wider detection range and better specificity. It is more sensitive than other photothermal detection test strips and is suitable for the rapid detection of estriol. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram of the photothermal test strip of the present invention;

[0048] Figure 2 The transmission electron microscope image (A) of the As2MoO6@NiS composite material in the photothermal signal probe of the test strip of the present invention, the photothermal conversion efficiency (B), the Ni element energy spectrum (C) and the Mo element energy spectrum (D);

[0049] Figure 3 The response of the test strip of the present invention to water samples with different concentrations of estriol in the photothermal mode and the working curve;

[0050] Figure 4 The figure shows the response of the test strip of the present invention to estriol and four other estrogens. DETAILED DESCRIPTION

[0051] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0052] The following examples provide a full and detailed description of the preparation of the entire test strip and the use of the detection device.

[0053] Example 1 Preparation of raw materials

[0054] 1.1 Preparation of As2MoO6 nanocrystals

[0055] As2MoO6 nanoparticles were synthesized by hydrothermal method. 0.5g Na2MoO4·2H2O and 1.5g Bi(NO3)3·5H2O were dissolved in 5-10mL 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 As2MoO6 nanocrystals.

[0056] 1.2 Preparation of As2MoO6@NiS composites and characterization of their structural properties

[0057] 1.0 mg As2MoO6 nanoparticles were added to 5 mL deionized water containing 10 mg polyethylene glycol (MW = 5000-10000), ultrasonicated for 20 minutes, and stirred overnight; 1 mL of the above mixture was added to 1 mL of aqueous solution containing 1.0 mg NiS and stirred overnight; the dark gray powder was collected by centrifugation and dried for further use.

[0058] The transmission electron microscopy, photothermal conversion efficiency and elemental energy spectrum results of As2MoO6@NiS composite materials are as follows: Figure 2 ,in, Figure 2 A shows the close bonding between As2MoO6 and NiS in the As2MoO6@NiS composite material.

[0059] Figure 2 B is the temperature rise and fall curve used to calculate the light-to-heat conversion efficiency of the material. The light-to-heat conversion efficiency is calculated based on the temperature drop of the material after the radiation is turned off. The calculation formula is as follows:

[0060]

[0061] η represents the light-to-heat conversion efficiency, A is the surface area of ​​the container, ΔT max,mix is the maximum temperature drop of the material solution, ΔT max,H2O is the maximum temperature drop of solvent water, I is the light source power, A λ It is the absorbance of the material in the infrared region. Calculation shows that the As2MoO6@NiS composite material has a photothermal conversion efficiency of 78%, while the photothermal conversion efficiency of As2MoO6 is 60%. The photothermal conversion efficiency of As2MoO6@NiS composite material is better than that of As2MoO6.

[0062] Figure 2 C and 2D give the energy spectra of element Ni and element Mo respectively, proving that the material does contain these two elements.

[0063] 1.3 As2MoO6@NiS and antibody adsorption: Prepare a mixture of As2MoO6@NiS composite material, antibody to be tested, and bovine serum albumin, i.e., As2MoO6@NiS-mAb.

[0064] Take 1mL As2MoO6@NiS in a centrifuge tube, add 4μL 0.2mol / L K2CO3 solution to adjust the pH value of the system, shake and mix, then add 5μL 1mg / mL estriol 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 (20mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain As2MoO6@NiS-mAb mixed solution, which is stored at 4℃ for later use.

[0065] 1.4 Preparation of the test zone (T zone) solution

[0066] Estriol-BSA was diluted to 0.6 mg / mL with 10 mM PBS solution.

[0067] 1.5 Preparation of Quality Control Zone (Zone C) Solution

[0068] Dilute goat anti-mouse secondary antibody to 0.4 mg / mL in 10 mM PBS solution.

[0069] Example 2 Preparation of test strips

[0070] according to Figure 1 The NC membrane is pasted in the middle of the PVC bottom plate, and the sample pad and the absorbent pad are overlapped on the left and right ends of the NC membrane, so that they cover the NC membrane for about 2mm. The assembled large card is cut into 3mm wide strips to obtain blank test strips. Figure 1 0.5 μL of the T zone solution and 0.5 μL of the C zone solution were added to the T zone and C zone. The test strips were placed in an oven and dried at 37°C for 60 min, and then stored in a vacuum bag for later use.

[0071] Example 3 Preparation of a Test Strip Containing a Binding Pad

[0072] Paste the NC membrane in the middle of the PVC base plate, overlap the right end of the conjugate pad on the left end of the NC membrane, and make it cover the NC membrane by about 2mm. The sample pad overlaps the left end of the conjugate pad, and makes it cover the conjugate pad by about 2mm. The absorbent pad overlaps the right end of the NC membrane, and makes it cover the NC membrane by about 2mm. Cut the assembled large card into 3mm wide strips to obtain a blank test strip. A mixture of 10μL As2MoO6@NiS composite material, antibody to be tested, and bovine serum albumin, i.e., As2MoO6@NiS-mAb mixture, is fixed on the conjugate pad. Add 0.5μL T zone solution and 0.5μL C zone solution to the T zone and C zone respectively. Place the test strip after spotting in an oven, dry it at 37℃ for 60min, and store it in a vacuum bag for later use.

[0073] Example 4 Working curve drawing

[0074] Prepare 10 mL of 1 mg / mL estriol 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 As2MoO6@NiS-mAb and 10μL running buffer (10mM PBS solution, containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) in a centrifuge tube for 10min, and then the test strip was inserted into the centrifuge tube. After the test strip was dried, 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 3 .

[0075] The results showed that in the photothermal mode, the negative T zone had the highest temperature and the estriol 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.

[0076] Example 5 Specificity of detecting estriol

[0077] The specificity verification of the As2MoO6@NiS composite photothermal quantitative test strip of estriol includes the following steps:

[0078] 1. Preparation of test paper materials

[0079] Same as Example 1

[0080] 2. Preparation of test strips

[0081] Same as Example 2

[0082] 3. Working curve drawing

[0083] Same as Example 3

[0084] 4. Specificity

[0085] Use acetonitrile to prepare 10 mL of 1 mg / mL estriol, estradiol, estrone, diethylstilbestrol, and ethinyl estradiol standard solutions, dilute with 10 mM PBS to a concentration of 120 ng / mL, and use as the test solution.

[0086] 5. Sample testing

[0087] 80 μL of the test solution was mixed with 10 μL As2MoO6@NiS-mAb and 10 μL 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 4 Compared with the blank, estradiol, estrone, diethylstilbestrol, and ethinylestradiol had very weak effects on the T-zone temperature of the test paper, while estriol 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.

[0088] It can be seen from the above embodiments that the As2MoO6@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 provided by the present invention includes photothermal test strips. The photothermal test strips for detecting estriol are based on the plasma resonance effect, and the As2MoO6@NiS composite material with high photothermal conversion efficiency and tiny size is used as the T-zone fixed photothermal signal probe, near-infrared laser light source excitation, combined with smart terminals 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 strips, thereby improving 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.

[0089] In summary, the contents of this specification should not be understood as limitations of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A test paper for detecting estriol, characterized in that: The test paper comprises a photothermal signal probe and a test strip body; The photothermal signal probe comprises a mixture of an As2MoO6@NiS composite material, an antibody to be detected, i.e., a primary antibody, and bovine serum albumin, i.e., an As2MoO6@NiS mixed solution; 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; the nitrocellulose membrane comprises a detection area, i.e., a T area, and a quality control area, i.e., a C area; wherein the antigen of the object to be tested, i.e., the molecule-protein conjugate to be tested, 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; 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.

2. A test paper for detecting estriol according to claim 1, characterized in that, The antibody to be detected, i.e., the primary antibody, may be derived from, but is not limited to, mouse, rat, and rabbit; the secondary antibody may be derived from, but is not limited to, goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody, and donkey anti-rabbit secondary antibody; Preferably, the antigen to be detected is estriol having only a single antigenic site, fungal toxins including aflatoxin and fusarium toxin, or pesticide residues including fungicides, antibiotics, and hormones.

3. A test paper for detecting estriol according to claim 1, characterized in that, The preparation method of the As2MoO6@NiS composite material comprises: adding 1-2 mg As2MoO6 nanoparticles into 5-10 mL deionized water containing 10-20 mg polyethylene glycol, ultrasonicating for 20-30 minutes, stirring overnight to obtain a mixed solution; adding 1 mL of the mixed solution into 1 mL of an aqueous solution containing 1-2 mg NiS and stirring overnight; collecting dark gray powder by centrifugation and drying to obtain the result.

4. A method for preparing a test paper for detecting estriol, characterized in that: The method comprises: Preparation of photothermal signal probe As2MoO6@NiS composite material; Prepare As2MoO6@NiS-mAb mixed solution of As2MoO6@NiS composite material, antibody to be tested and bovine serum albumin; Construct the test paper: drip or spray the antigen of the object to be tested on the T area, drip or spray the secondary antibody against the source of the primary antibody on the C area, i.e., the anti-primary antibody, and store it in a vacuum bag after drying for future use; The photothermal signal probe, i.e., the As2MoO6@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 As2MoO6@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.

5. The method according to claim 4, characterized in that The specific method for preparing the photothermal signal probe As2MoO6@NiS composite material comprises: adding 1-2 mg As2MoO6 nanoparticles into 5-10 mL deionized water containing 10-20 mg polyethylene glycol, ultrasonicating for 20-30 minutes, stirring overnight to obtain a mixed solution; adding 1 mL of the mixed solution into 1 mL of an aqueous solution containing 1-2 mg NiS and stirring overnight; collecting dark gray powder by centrifugation and drying to obtain a mixed solution; Preferably, the As2MoO6 nanoparticles are synthesized by a hydrothermal method; Preferably, the polyethylene glycol has a MW of 5000-10000; 6. The method according to claim 4, characterized in that The method for preparing an As2MoO6@NiS-mAb mixed solution of an As2MoO6@NiS composite material, an antibody to be detected and bovine serum protein comprises the following steps: placing the As2MoO6@NiS composite material in a centrifuge tube, adding a weak alkaline solution to adjust the pH of the system to 6-8, oscillating and mixing, and then adding the antibody to be detected, oscillating and reacting at room temperature for 30-60 minutes, adding bovine serum albumin (BSA) to block, oscillating and reacting at room temperature for 1-1.5 hours, removing the supernatant after centrifugation, and then redissolving the remaining substances in a buffer solution to obtain an As2MoO6@NiS-mAb mixed solution, and refrigerating and storing for later use.

7. The method according to claim 4, characterized in that The T zone is dripped or sprayed with the antigen to be tested, specifically including: dripping or spraying a 5-20mM PBS solution containing 0.5-10mg / mL of the antigen to be tested on the T zone; the C zone is dripped or sprayed with a secondary antibody targeting the source of the primary antibody, i.e., anti-primary antibody, specifically including: dripping or spraying a 5-20mM PBS solution containing 0.05-10mg / mL of the secondary antibody on the C zone.

8. An application of a test paper for detecting estriol prepared according to any one of claims 1 to 3 or claims 4 to 7, characterized in that: Used to detect estriol.

9. The use according to claim 8, characterized in that: 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 film of the main structure of the test strip. Preferably, the method specifically includes: 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 As2MoO6@NiS-mAb solution are placed in a running buffer and mixed for 3-10 minutes, and then placed in the test strip; after 15-30 minutes, the colorimetric result is read and semi-quantitative analysis is performed; a colorimetric result photo is obtained, and grayscale analysis is performed using image processing software 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: The sample to be tested is mixed with the running buffer solution, and placed in the test strip. After 20-40 minutes, the colorimetric result is read, and a colorimetric result photo is obtained for semi-quantitative analysis. Grayscale analysis is performed using image processing software for quantitative analysis. After the test strip is dried, it 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 but is not limited to a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager or a temperature measuring gun, and the intelligent display terminal includes but is not limited to a computer or a smart phone. The thermal imaging device collects photothermal imaging photos and outputs and displays them through the intelligent display terminal connected thereto.

10. The use according to claim 8, characterized in that: The photothermal temperature of the test area of ​​the test paper is negatively correlated with the estriol content to be tested, and the color intensity is negatively correlated with the estriol content in the sample, specifically: When the sample does not contain estriol, the T zone appears purple and the temperature is high; When the sample contains estriol, 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.

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