Test paper for detecting oestradiol and method for preparing the same

Abstract

The application discloses a test paper for detecting oestrogen and a preparation method thereof, which comprises the following steps: (1) preparing an As2MoO6@NiS compound, and adsorbing a to-be-detected substance antibody, i.e., a first antibody, on the surface of the As2MoO6@NiS compound to obtain a photothermal signal probe; (2) assembling a test paper strip, and fixing a to-be-detected substance antigen and a second antibody on a detection area and a quality control area respectively; (3) mixing the photothermal signal probe with a to-be-detected sample liquid, and inserting a sample pad end of the test paper strip into the mixture to perform chromatography; and (4) detecting a photothermal signal: irradiating the detection area with a near-infrared LED, collecting temperature by using a thermal imaging or temperature measuring device, and performing quantitative analysis according to a linear relationship between the temperature of the detection area and the concentration of the to-be-detected substance of the sample. 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 particularly relates to a test paper for detecting estriol and a preparation method thereof. Background Art

[0002] Lateral flow immunoassay (LFIA) has the characteristics of fast analytical performance, good selectivity, low cost, small sample volume requirement, easy large-scale production and stable long-term storage. It is widely used in the design of portable analytical detection sensors.

[0003] In traditional LFIA, colloidal gold nanoparticles are often used as photothermal signal probes. Qualitative or semi-quantitative detection can be performed directly by observation with the naked eye. However, colorimetric LFIAs based on colloidal gold have drawbacks such as low sensitivity, narrow linear range, and lack of quantitative analysis. To improve the sensitivity of analytical detection, researchers have developed fluorescent LFIAs by using various fluorescent nanoparticles as probes. However, these fluorescent substances are subject to interference from 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 (LSPR) effect of nanoparticles has been discovered and re-applied to LFIAs as a photothermal signal probe. Compared to colorimetric methods, this method offers tens to hundreds of times higher sensitivity, leading to the increased interest and exploration of photothermal LFIAs. This detection method, which relies on temperature changes caused by the photothermal effect, offers low background signals, high sensitivity, and can be performed on a variety of substrates with different colors, showing strong application potential. Currently, new nanomaterials with higher photothermal conversion efficiencies are needed as photothermal signal probes to create a more pronounced thermal contrast and further enhance the performance of photothermal LFIAs. 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 application thereof. The test strips are used to detect estriol, thereby realizing a detection method with a lower detection limit, a wider detection range and better specificity, and a higher sensitivity than other photothermal detection test strips.

[0006] In one aspect, 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, which are overlapped and affixed horizontally from top to bottom on a PVC base plate; wherein the nitrocellulose membrane is used to separate and detect analytes from other substances in the sample, the sample pad is used for sample loading, and the absorption pad is used to absorb excess liquid; and the PVC base plate provides physical support for the test strip;

[0008] The nitrocellulose membrane includes a detection area, namely the T area, and a quality control area, namely the C area; wherein the antigen of the test object, namely the test molecule-protein conjugate, is fixed on the T area; and the secondary antibody is fixed on the C area, which 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 mixture;

[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 analyte antigen is estriol with fewer antigenic sites or only a single antigenic site, including but not limited to aflatoxins, fusarium toxins 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 of As2MoO6 nanoparticles to 5-10 mL of deionized water containing 10-20 mg of 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 of NiS and stirring overnight; collecting a 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] Prepare As2MoO6@NiS-mAb mixture of As2MoO6@NiS composite material, antibody to be tested, and bovine serum albumin;

[0018] Construct the test strip: drip or spray the antigen 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. After drying, store in a vacuum bag for future use;

[0019] The photothermal signal probe, i.e., the As2MoO6@NiS-mAb mixture, is stored in a sealed container in the form of a solution or lyophilized powder; or the photothermal signal probe, i.e., the As2MoO6@NiS-mAb mixture, 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 of As2MoO6 nanoparticles to 5-10 mL of deionized water containing 10-20 mg of 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. The specific method is: take the As2MoO6@NiS composite material in a centrifuge tube, add a weak alkaline solution to adjust the pH of the system to 6-8, oscillate and mix, and then add the antibody to be tested. After oscillating and reacting at room temperature for 30-60 minutes, bovine serum albumin BSA is added for blocking, and the reaction is oscillated 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 an As2MoO6@NiS-mAb mixed solution, which is refrigerated for later use.

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

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

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

[0027] On the other hand, the photothermal temperature of the detection zone is negatively correlated with the estriol content to be measured, 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 strip 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 test strip main structure.

[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. The test strip was placed in the test strip and the colorimetric results were read after 15-30 minutes for semi-quantitative analysis. The colorimetric results were photographed and grayscale analyzed 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 membrane of the test strip main structure.

[0037] Specifically, the method involves drying the photothermal signal probe, namely the As2MoO6@NiS-mAb mixture, onto a conjugate pad, which is then inserted between the sample pad and the NC membrane. The sample to be tested is mixed with running buffer and placed on the test strip. After 10 minutes, the test strip is dried and then exposed to a near-infrared LED light source for excitation. Thermal imaging or temperature measurement equipment is used to obtain temperature readings 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 embodiment, 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 photothermal test strip for detecting estriol of the present invention works as follows: the quality control zone (zone C) serves 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 migrate toward the absorbent paper under capillary action. When there is no estriol in the sample, the As2MoO6@NiS-mAb is captured by the estriol-BSA in the T zone, and the T zone appears purple to the naked eye. Under laser excitation, the As2MoO6@NiS-mAb produces an LSPR effect, causing the temperature to rise. Excess As2MoO6@NiS-mAb is captured by the secondary antibody in the C zone, causing the C zone to also appear purple. When the sample contains estriol, estriol binds to some of the As2MoO6@NiS-mAb, thereby reducing the total amount of As2MoO6@NiS-mAb captured in the T zone. The LSPR effect of the As2MoO6@NiS-mAb in the T zone decreases 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 has at least 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 convenient storage, or it can be fixed on the binding pad of the test strip for easy portability.

[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, uses a high photothermal conversion efficiency, micro-sized As2MoO6@NiS composite material as a T-zone fixed photothermal signal probe, is excited by a near-infrared LED light source, and is combined with 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.

[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 Transmission electron microscopy image (A) of the As2MoO6@NiS composite material in the photothermal signal probe of the test strip of the present invention, photothermal conversion efficiency (B), Ni element energy spectrum (C) and 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 are shown;

[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 based on the following examples. However, it is readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are merely illustrative of 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 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 using a hydrothermal method. 0.5g of Na2MoO4·2H2O and 1.5g of Bi(NO3)3·5H2O were dissolved in 5-10mL of ethylene glycol, respectively. After mixing, 35mL of ethanol was added. After stirring for 0.5h, the solution was transferred to a 50mL autoclave and heated at 180°C for 10h. The precipitate after centrifugation was washed with deionized water and ethanol and dried at 80°C for 10h. The resulting powder was then calcined at 450°C (heating rate of 2°C / min) for 2h to obtain As2MoO6 nanocrystals.

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

[0057] 1.0 mg of As2MoO6 nanoparticles was added to 5 mL of deionized water containing 10 mg of polyethylene glycol (MW = 5000-10000), ultrasonicated for 20 minutes, and stirred overnight; 1 mL of the above mixture was added to 1 mL of an aqueous solution containing 1.0 mg of 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 element 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 material's light-to-heat conversion efficiency. The light-to-heat conversion efficiency is calculated based on the temperature drop of the material after 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, and 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 is the material's absorbance in the infrared region. Calculations show that the As2MoO6@NiS composite material has a photothermal conversion efficiency of 78%, compared to 60% for As2MoO6. This makes the As2MoO6@NiS composite material superior to 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 mixture.

[0064] 1 mL of As2MoO6@NiS was placed in a centrifuge tube. 4 μL of 0.2 mol / L K2CO3 solution was added to adjust the pH of the system. After vortexing, 5 μL of 1 mg / mL estriol antibody was added. After mixing, the reaction was allowed to oscillate at room temperature for 45 minutes. After completion of the reaction, 100 μL of 5% BSA (m / m) was added for blocking for 1 hour. The tube was centrifuged at 12,000 rpm for 30 minutes, the supernatant removed, and the tube was redissolved in 100 μL of buffer (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain the As2MoO6@NiS-mAb mixture, which was stored at 4°C until 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 the 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 base plate, and the sample pad and the absorbent pad are overlapped on the left and right ends of the NC membrane respectively, so that they cover the NC membrane by about 2mm. The assembled large card is cut into 3mm wide strips to obtain blank test strips. Figure 1 Add 0.5 μL of the T zone solution and 0.5 μL of the L zone solution to the T zone and C zone respectively. Place the test strips after spotting in an oven at 37°C for 60 minutes and store in a vacuum bag until use.

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

[0072] The NC membrane was affixed to the center of a PVC substrate. The right end of the conjugate pad overlapped the left end of the NC membrane by approximately 2 mm. The sample pad overlapped the left end of the conjugate pad by approximately 2 mm. The absorbent pad overlapped the right end of the NC membrane by approximately 2 mm. The assembled large card was cut into 3 mm wide strips to create blank test strips. A mixture of 10 μL of As2MoO6@NiS composite material, analyte antibody, and bovine serum albumin (BSA), i.e., the As2MoO6@NiS-mAb mixture, was immobilized on the conjugate pad. 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, respectively. The sample-applied test strips were dried in an oven at 37°C for 60 minutes and stored in a vacuum bag until ready for use.

[0073] Example 4 Working Curve Drawing

[0074] Prepare 10 mL of 1 mg / mL estriol standard solution in acetonitrile and dilute it 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. 80 μL of the test solution was mixed with 10 μL As2MoO6@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 minutes, and 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 3 .

[0075] The results showed that in the photothermal mode, the temperature of the negative T zone was the highest and the concentration of estriol was 10 5 When the concentration of β-catenin was less than 10 μg / mL, the temperature of T zone dropped to the lowest, and this concentration was the detection limit of 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 for 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] Prepare 10 mL of 1 mg / mL estriol, estradiol, estrone, diethylstilbestrol, and ethinylestradiol standard solutions with acetonitrile, and dilute to a concentration of 120 ng / mL with 10 mM PBS for use as test solutions.

[0086] 5. Sample testing

[0087] 80 μL of the test solution was mixed with 10 μL of As2MoO6@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 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 weak responses to the four substances, 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 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. 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. The As2MoO6@NiS composite material with high photothermal conversion efficiency and small size is used as the T-zone fixed photothermal signal probe, and is excited by a near-infrared laser light source. The photothermal signal is collected in combination with an intelligent terminal and infrared thermal imaging accessories, which effectively removes the base color of the sample liquid and the fluorescence background interference of the test strip, 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 limiting 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 (NC membrane), and an absorption pad, which are overlapped and affixed horizontally from top to bottom on a PVC base plate; the nitrocellulose membrane includes a detection area (T area) and a quality control area (C area); wherein the antigen 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, which 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 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 tested, i.e., the primary antibody, can be derived from mouse, rat, or rabbit; the secondary antibody can include goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody, or donkey anti-rabbit secondary antibody.

3. A test paper for detecting estriol according to claim 1, characterized in that, The analyte antigen is estriol having only a single antigenic site.

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

5. 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 mixture of As2MoO6@NiS composite material, antibody to be tested, and bovine serum albumin; Construct the test strip: drip or spray the antigen 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. After drying, store in a vacuum bag for future use; The photothermal signal probe, i.e., the As2MoO6@NiS-mAb mixture, is stored in a sealed container in the form of a solution or lyophilized powder; or the photothermal signal probe, i.e., the As2MoO6@NiS-mAb mixture, is dried on a conjugate pad and the conjugate pad is inserted between the sample pad and the NC membrane for use.

6. The method according to claim 5, characterized in that The specific method for preparing the photothermal signal probe As2MoO6@NiS composite material includes: adding 1-2 mg of As2MoO6 nanoparticles to 5-10 mL of deionized water containing 10-20 mg of polyethylene glycol, ultrasonicating for 20-30 minutes, and 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 of NiS and stirring overnight; centrifuging to collect a dark gray powder and drying it.

7. A test paper for detecting estriol according to claim 6, characterized in that, As2MoO6 nanoparticles were synthesized by hydrothermal method.

8. A test paper for detecting estriol according to claim 6, characterized in that, The polyethylene glycol has a MW of 5000-10000.

9. The method according to claim 5, characterized in that The method for preparing an As2MoO6@NiS-mAb mixed solution of an As2MoO6@NiS composite material, an antibody to be tested, and bovine serum albumin includes 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, then adding the antibody to be tested, oscillating and reacting at room temperature for 30-60 minutes, adding bovine serum albumin (BSA) for blocking, oscillating and reacting at room temperature for 1-1.5 hours, removing the supernatant after centrifugation, and redissolving the remaining substance in a buffer solution to obtain an As2MoO6@NiS-mAb mixed solution, which is refrigerated for later use.

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

11. A test paper for detecting estriol according to any one of claims 1 to 4, or a use of a test paper for detecting estriol prepared according to the method according to any one of claims 5 to 10, characterized in that: Used to detect estriol.

12. The use according to claim 11, 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 membrane of the main structure of the test strip.

13. The use according to claim 12, characterized in that Specifically include: 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 were placed in the running buffer and mixed for 3-10 minutes, and then placed in the test strip; after 15-30 minutes, the colorimetric results were read and semi-quantitative analysis was performed; a colorimetric result photo was obtained and grayscale analysis was 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 membrane of the test paper main structure, the following steps are specifically performed: The sample to be tested is mixed with the running buffer 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 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.

14. The use according to claim 11, characterized in that The photothermal temperature of the test area of ​​the test paper is negatively correlated with the estriol content to be measured, 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 strip results and always appears purple.

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