Test paper for detecting hexestrol and preparation method thereof

Abstract

The invention discloses test paper for detecting hexestrol and a preparation method of the test paper, and belongs to the technical field of small molecule analysis and detection. The test paper for detecting the hexethylstilbestrol comprises a test paper main body and a photo-thermal signal probe, wherein the test paper main body comprises a sample pad, a nitrocellulose membrane, namely an NC membrane and an absorption pad which are sequentially overlapped and adhered on a PVC (Polyvinyl Chloride) bottom plate from top to bottom along the horizontal direction; the NC membrane comprises a detection area, namely a T area, and a quality control area, namely a C area; wherein hexestrol-BSA (Bovine Serum Albumin) is fixed on the T region; a second antibody is fixed on the C area; and the photo-thermal signal probe is an As2MoO6 (at) NiS-mAb mixed solution. When the test paper is used for detecting the hexestrol, 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 hexane 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. The photothermal type 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, the background signal is low, the sensing is sensitive, and it can be performed on various substrates of different colors, and has great application potential. However, the photothermal conversion efficiency of the photothermal signal probe in the prior art is low, especially the response to hexane estradiol is poor, which in turn affects its detection sensitivity to hexane estradiol. Summary of the invention

[0003] [Technical issues]

[0004] The existing photothermal signal probes have low photothermal conversion efficiency, especially poor response to hexane estradiol, resulting in low hexane estradiol detection sensitivity and poor quantitative effect on the hexane estradiol target.

[0005] [Technical solution]

[0006] In order to solve the defects and shortcomings of the prior art, the purpose of the present invention is to provide a test paper for detecting hexane estradiol and a preparation method thereof. The test paper uses As2MoO6@NiS composite material with high photothermal conversion efficiency as a signal probe, and when used to detect hexane estradiol, it has a lower detection limit, a wider detection range, better specificity and higher sensitivity.

[0007] As a metal sulfide, NiS has certain potential in photothermal conversion, but the photothermal performance of NiS is usually affected by factors such as its own particle size, specific surface area and morphology, and is especially easily affected by the materials with which it is composited. As2MoO6, as an inorganic compound, has specific optical and thermal properties; the present invention uses a composite material obtained by combining As2MoO6 and NiS, and the results show that the composite material exhibits a high photothermal conversion efficiency and can be used for specific detection of hexaneestrol.

[0008] In order to achieve the above purpose, the technical solutions provided are as follows:

[0009] The first object of the present invention is to provide a test paper for detecting hexane estradiol, wherein the test paper comprises a test paper body and a photothermal signal probe.

[0010] 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;

[0011] The nitrocellulose membrane includes a detection area, namely, a T area, and a quality control area, namely, a C area; wherein hexane estradiol-BSA 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;

[0012] The photothermal signal probe comprises a mixture of As2MoO6@NiS composite material, hexane estradiol antibody (i.e., primary antibody), and bovine serum albumin (i.e., As2MoO6@NiS-mAb mixed solution);

[0013] The photothermal signal probe is used in a form independent of the test paper main body structure, 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.

[0014] In one embodiment, the hexane estradiol antibody, ie, the primary antibody (hexane estradiol antibody), is derived from mice, rats, and rabbits.

[0015] In one embodiment, the secondary antibody comprises any one of goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody, and donkey anti-rabbit secondary antibody.

[0016] In one embodiment, the preparation of hexestrol-BSA is as follows: (1) first preparing hexestrol carboxymethyl ether: 100-120 mg hexestrol, 400-500 mg KOH and 6-10 mL DMSO are dissolved by 100-400W ultrasound for 5 min, 100 mg bromoacetic acid (pre-dissolved in 1 mL DMSO) is added under magnetic stirring to carry out nucleophilic substitution reaction, and after reacting for 1.5-2 h, about 50 mL ice water is added to terminate the reaction; extraction is repeated 3 times with 8-10 mL ethyl acetate to collect unreacted hexestrol, the aqueous phase is collected in a clean beaker, 2 mol / L HCl is added dropwise to the aqueous phase under stirring, and a white precipitate is subsequently formed, and the precipitate is repeatedly centrifuged with ultrapure water (9000 rpm, 15 min) to wash until the pH value of the supernatant is about 7.0; the precipitate after centrifugation is vacuum freeze-dried to obtain a white powder, which is hexestrol-CME;

[0017] (2) Preparation of estradiol-BSA: 3-4 mg of estradiol-CME, 5-7 mg of NHS and 6-8 mg of EDC·HCl were weighed into a 10 mL centrifuge tube, 0.5-1.0 mL of DMSO was added, and the mixture was stirred gently for 10-20 h. 15-20 mg of BSA was weighed and dissolved in 4-6 mL of carbonate buffer (50 mmol / L, pH 9.6), and the activated estradiol-CME solution was slowly added dropwise to the BSA solution for coupling reaction. The mixture was stirred for 10-20 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag for dialysis to remove impurities. The dialysis solution was 8-12 mmol / L PBS (pH 7.4) at a dialysis temperature of 4°C. The dialysis solution was replaced every 10-15 h for 3 consecutive days. After the dialysis was completed, the solution in the dialysis bag was divided and stored in a freezer. The resulting solution was the estradiol-BSA solution.

[0018] In one embodiment, the preparation of the As2MoO6@NiS composite material includes: adding As2MoO6 nanocrystals to deionized water dissolved with polyethylene glycol, ultrasonicating, stirring overnight to obtain a mixed solution; then adding the mixed solution to an equal volume of a mixed aqueous solution of nickel chloride with a mass fraction of 0.1%-1% (containing 5wt% nitric acid) and sodium sulfide with a molar ratio of 2:1-1:2 and stirring overnight; collecting dark gray powder by centrifugation and drying to obtain.

[0019] In one embodiment, the mass volume ratio of the As2MoO6 nanocrystals, polyethylene glycol and water is

[0020] 1~2:10~20:5~10; mg:mg:ml.

[0021] In one embodiment, the polyethylene glycol has a MW of 5000-10000.

[0022] In one embodiment, the ultrasonic conditions are: ultrasonic power is 120-600W, 20-30 minutes.

[0023] In one embodiment, the specific preparation method of the As2MoO6@NiS-mAb mixed solution 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, then add hexane estradiol antibody, oscillate and react at room temperature for 30-60 minutes, add bovine serum albumin BSA for blocking, oscillate and react at room temperature for 1-1.5 hours, remove the supernatant after centrifugation, and then dissolve the remaining substance in a buffer solution to obtain an As2MoO6@NiS-mAb mixed solution.

[0024] In one embodiment, the buffer comprises one or more of a phosphate buffer, a borate buffer or a carbonate buffer.

[0025] The second object of the present invention is to provide a method for preparing the test paper for detecting hexane estradiol, the method for preparing the test paper comprising the following steps:

[0026] (1) Paste the NC membrane in the middle of the PVC base plate, overlap the sample pad and the absorbent pad on the left and right ends of the NC membrane, and press the NC membrane with 2-3 mm. Cut the assembled large card into strips with a width of 3-5 mm, and make a test line (T zone) and a quality control line (C zone) on the NC membrane, respectively. The two lines are 4-6 mm apart.

[0027] (2) dripping or spraying hexane estradiol-BSA on the T area, and dripping or spraying the secondary antibody against the source of the primary antibody, i.e., the anti-primary antibody, on the C area. After drying, the test strip is stored in a vacuum bag for later use;

[0028] (3) 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.

[0029] In one embodiment, a 5-20 mmol / L PBS solution containing 0.5-10 mg / mL hexane estradiol-BSA is dripped or sprayed on the T zone; a secondary antibody targeting the primary antibody source, i.e., anti-primary antibody, is dripped or sprayed on the C zone, specifically: a 5-20 mmol / L PBS solution containing 0.05-10 mg / mL secondary antibody is dripped or sprayed on the C zone.

[0030] The third object of the present invention is to provide a method for qualitatively detecting hexaneestrol based on the above-mentioned test paper, comprising:

[0031] Place the sample to be tested and the As2MoO6@NiS-mAb mixed solution in the running buffer and mix for 3-10 minutes, then put it into the test strip body. After 15-30 minutes, take it out and dry it. Then, place the dried test strip under a near-infrared LED light source for excitation, and use thermal imaging or temperature measurement equipment to obtain the temperature for interpretation;

[0032] The photothermal temperature of the detection zone is negatively correlated with the hexane estradiol content to be measured, and the color intensity is negatively correlated with the hexane estradiol content in the sample, specifically:

[0033] When the sample does not contain hexaneestrol, the temperature of the T zone is high;

[0034] When the sample contained hexaneestrol, the temperature of the T zone decreased;

[0035] The quality control area serves as a reference for verifying the validity of the test paper results and always appears purple.

[0036] The fourth object of the present invention is to provide a method for quantitatively detecting hexaneestrol based on the above-mentioned test paper, comprising:

[0037] The sample to be tested and the As2MoO6@NiS-mAb mixed solution are placed in the running buffer and mixed for 3-10 minutes, and then the test strip body is placed in the test strip. After 15-30 minutes, the test strip is taken out and dried, and then the dried test strip is placed under a near-infrared LED light source for excitation. The temperature result is obtained using thermal imaging or temperature measurement equipment, and quantitative analysis is performed based on the quantitative relationship model.

[0038] In one embodiment, the method for quantitatively detecting hexaneestrol using the test paper further comprises:

[0039] 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 then mixed with the running buffer and placed in a test strip. After 15 to 30 minutes, the test strip is taken out and dried, and then placed under a near-infrared LED light source for excitation. The temperature result is obtained using thermal imaging or temperature measurement equipment, and quantitative analysis is performed based on the quantitative relationship model.

[0040] In one embodiment, the running buffer is a PBS solution with a pH of 6.5 to 8.0 and a total concentration of 10 mmol / L; the PBS solution contains the following components: the content is in mass percentage, including: 5 to 15% sucrose, 1 to 10% BSA, and 0.15 to 1% Tween-20.

[0041] In one embodiment, the thermal imaging or temperature measuring device includes any one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, or a temperature measuring gun.

[0042] In one embodiment, the intelligent display terminal of the thermal imaging or temperature measuring device includes a computer or a smart phone.

[0043] In one embodiment, the near-infrared LED light source power is 1-50 W / cm 2 , irradiate for 1-10 minutes.

[0044] In one embodiment, the quantitative relationship model is: a series of concentrations of standard substances are prepared using hexestrol, and the quantitative relationship model is constructed based on the hexestrol concentrations and the corresponding temperature results obtained using thermal imaging or temperature measurement equipment.

[0045] The test paper detection principle of the present invention for detecting hexane estradiol is as follows: the quality control area (area C) is used as a reference for verifying the validity of the test paper result, and always shows 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 hexane estradiol in the sample, As2MoO6@NiS-mAb is captured by hexane estradiol-BSA in the T area, and the T area is visible to the naked eye as purple. Under the excitation of laser, As2MoO6@NiS-mAb produces LSPR effect, the temperature rises, and the excess As2MoO6@NiS-mAb is captured by the secondary antibody in the C area, so that the C area also shows purple; when hexane estradiol is contained in the sample, hexane estradiol specifically binds to 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 hexaneestrol 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.

[0046] [Beneficial Effects]

[0047] (1) The As2MoO6@NiS composite material provided by the present invention is used in the preparation of test paper and target object detection, without changing the conventional structure of the test paper, and can be used as a photothermal signal probe. It can be used independently of the test paper form for convenient storage, and can also be fixed on the binding pad of the test paper for easy carrying;

[0048] (2) The detection method provided by the present invention includes a test paper, which is used to detect hexane estradiol. The test paper is based on the plasma resonance effect, uses a high photothermal conversion efficiency and a micro-sized As2MoO6@NiS composite material as a T-zone fixed photothermal signal probe, and is excited by a near-infrared LED light source. It 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 paper, thereby improving the signal-to-noise ratio;

[0049] (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 hexaneestrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of the process of detecting hexaneestrol by the test paper of the present invention;

[0051] Figure 2The performance diagram of the test paper photothermal signal probe of the present invention; (A) is a transmission electron microscope image of the As2MoO6@NiS composite material; (B) is a data diagram of the photothermal conversion efficiency of the As2MoO6@NiS composite material; (C) Ni element energy spectrum diagram; (D) Mo element energy spectrum diagram;

[0052] Figure 3 This is a data diagram of the response of the test paper of the present invention to water samples with different concentrations of hexaneestrol in the photothermal mode;

[0053] Figure 4 The figure is a response diagram of the test paper of the present invention to hexane estradiol and four other estrogens. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0055] Example 1

[0056] The preparation of As2MoO6@NiS composite material includes the following steps:

[0057] (1) Preparation of As2MoO6 nanocrystals

[0058] 0.7 g Na2MoO4·2H2O and 3.0 g AsCl3 were dissolved in 8 mL ethylene glycol (containing 10% hydrochloric acid by volume), and then mixed evenly. 40 mL ethanol was added, and after stirring for 1.0 h, the solution was transferred to a 100 mL autoclave and heated at 180°C for 15 h. After the reaction, the solution was centrifuged, and the precipitate was washed with deionized water and ethanol, and dried at 100°C for 12 h. Then, the precipitate was calcined at 450°C (2°C / min heating rate) for 2.5 h to obtain As2MoO6 nanocrystals.

[0059] (2) Preparation of As2MoO6@NiS composites

[0060] 2 mg of As2MoO6 nanocrystals prepared in step (1) were added to 10 mL of deionized water containing 20 mg of polyethylene glycol (MW = 7000), ultrasonicated for 30 minutes, and stirred overnight; then 1 mL of the above mixed solution was added to 1 mL of a mixed aqueous solution with a mass fraction of 0.1% (containing 5 wt% nitric acid) and a molar ratio of 2:1 of nickel chloride and sodium sulfide, and stirred overnight; refrigerated storage was performed to obtain an As2MoO6@NiS composite material solution, which was used directly; or dark gray precipitate was collected by centrifugation at 10000 rpm and dried at 80°C to obtain an As2MoO6@NiS composite material powder.

[0061] Performance Characterization

[0062] The transmission electron microscopy, photothermal conversion efficiency and Ni and Mo element energy spectra of As2MoO6@NiS composite materials were measured; the results are as follows Figure 2 , transmission electron microscopy shows that the two types of nanoparticles are evenly combined together, and the element energy spectrum simultaneously shows the Ni element and Mo element energy spectrum, indicating that the As2MoO6@NiS composite material was successfully prepared.

[0063] Figure 2 B is the photothermal conversion efficiency data of As2MoO6@NiS composite material and As2MoO6 material. The results show that As2MoO6@NiS composite material has a photothermal conversion efficiency of 75%, which is significantly better than As2MoO6 material (40%).

[0064] Example 2

[0065] 1. Preparation of the signal probe As2MoO6@NiS-mAb mixed solution, including the following:

[0066] 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 hexane 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 (20mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain As2MoO6@NiS-mAb mixed solution, which was stored at 4℃ for later use.

[0067] 2. Preparation of the test area (T zone) solution

[0068] (1) 80 mg of hexestrol, 400 mg of KOH and 6 mL of DMSO were mixed and dissolved by ultrasonic treatment at 100 W for 5 min. 80 mg of bromoacetic acid (pre-dissolved in 1 mL of DMSO) was added under magnetic stirring to carry out nucleophilic substitution reaction. After reacting for 2 h, about 50 mL of ice water was added to terminate the reaction. Unreacted hexestrol was extracted three times with 10 mL of ethyl acetate. The aqueous phase was collected in a clean beaker. 2 mol / L HCl was added dropwise to the aqueous phase under stirring, and a white precipitate appeared. The precipitate was washed by repeated centrifugation with ultrapure water (9000 rpm, 15 min) until the pH value of the supernatant was about 7.0. The precipitate after centrifugation was vacuum freeze-dried to obtain a white powder, which was hexestrol-CME.

[0069] (2) Weigh 4 mg of estradiol-CME, 6 mg of NHS and 7 mg of EDC·HCl in a 10 mL centrifuge tube, add 1 mL of DMSO, and gently stir for 10 h. Weigh 22 mg of BSA and dissolve it in 4 mL of carbonate buffer (50 mmol / L, pH 9.6). Slowly add the activated estradiol-CME solution dropwise into the BSA solution for coupling reaction. Stir for 10 h. After the reaction is completed, transfer the reaction solution to a dialysis bag for dialysis to remove impurities. The dialysis solution is 10 mmol / L PBS (pH 7.4) at a dialysis temperature of 4°C. The dialysis solution is replaced every 10 h for 3 consecutive days. After the dialysis is completed, the solution in the dialysis bag is divided and frozen for storage. The resulting solution is the estradiol-BSA solution.

[0070] Hexestrol-BSA was diluted to 0.6 mg / mL with 10 mmol / L PBS solution at pH 7.4.

[0071] 3. Preparation of solution in quality control area (area C)

[0072] Dilute the goat anti-mouse secondary antibody to 0.4 mg / mL with 10 mmol / L PBS solution at pH 7.4.

[0073] Example 3

[0074] The preparation of the hexane estradiol test strips includes the following:

[0075] Test strips according to Figure 1Combination mode: paste the NC membrane in the middle of the PVC bottom plate, overlap the sample pad and the absorbent pad on the left and right ends of the NC membrane respectively, so that it covers the NC membrane by about 2mm, cut the assembled large card into 3mm wide strips, make the detection line (T zone) and the quality control line (C zone) on the NC membrane respectively, and the two lines are separated by 4mm; that is, a blank test strip is obtained; add 0.5μL T zone solution and 0.5μL C zone solution to the T zone and C zone, put the test strip after spotting in an oven, dry it at 37℃ for 60min, and store it in a vacuum bag for later use.

[0076] Example 4

[0077] The preparation of hexane estradiol test paper includes the following:

[0078] Test strips according to Figure 1 The 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 strips; then 10 μL As2MoO6@NiS-mAb mixed solution 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, and 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.

[0079] Example 5

[0080] A method for detecting hexaneestrol based on the test paper prepared in Example 3, the method comprising the following steps:

[0081] (1) Construction of quantitative relationship model

[0082] Prepare 10 mL of 1 mg / mL hexaneestrol standard solution in acetonitrile and dilute to a concentration of 10 with 10 mmol / L 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, used as test solution;

[0083] 80 μL of the test solution, 10 μL of As2MoO6@NiS-mAb mixed solution and 10 μL of running buffer (10 mmmol / L PBS solution, containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) were mixed in a centrifuge tube for 10 min. Then, the test paper prepared in Example 3 was inserted into the centrifuge tube. After all the liquid had flowed through the NC membrane of the test paper, a near-infrared LED (power of 1.36 W / cm 2 ) for 3 minutes, using a mobile phone and infrared thermal imaging accessories to monitor the relationship between temperature changes and the concentration of the test liquid, and construct a quantitative relationship model;

[0084] The results are as follows Figure 3 ; The quantitative relationship model constructed is: y = 4.654x + 0.457, R 2 =0.9897, the detection limit can reach 10 -1 pg / mL;

[0085] (2) Determination of hexaneestrol in samples 80 μL of sample solution was mixed with 10 μL of As2MoO6@NiS-mAb mixed solution and 10 μL of running buffer (10 mmol / L 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, monitor the temperature change using a mobile phone and infrared thermal imaging accessories, and calculate the content of hexaneestrol in the sample solution based on the quantitative relationship model constructed in step (1).

[0086] Example 6 Specificity Detection

[0087] (1) Prepare 10 mL of 1 mg / mL standard solutions of hexaneestradiol, estradiol, estrone, estriol, and ethinylestradiol with acetonitrile, dilute to a concentration of 120 ng / mL with 10 mmol / L PBS, and use them as test solutions;

[0088] (2) Referring to the detection method of Example 5, each test solution in step (1) is measured.

[0089] The results are as follows Figure 4 As shown, compared with the blank, estradiol, estrone, estriol, and ethinyl estradiol have very weak effects on the T zone temperature of the test paper, and only hexane estradiol has a very significant effect on the T zone temperature of the test paper, indicating that the test paper has a specific response to hexane estradiol.

[0090] In summary, the As2MoO6@NiS composite material provided in the present application does not need to change the conventional structure of the test paper in the application of preparing test paper. It can be used as a photothermal signal probe, and can be used independently of the test paper form for convenient storage, or fixed on the binding pad of the test paper for use, which is convenient to carry; the test paper for detecting hexane estradiol of the present invention 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 laser light source excitation, 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 paper, 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 papers.

[0091] 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 hexaneestrol, characterized in that: The test paper comprises two parts: a photothermal signal probe and a test paper body: The photothermal signal probe comprises a mixture of As2MoO6@NiS composite material, hexane estradiol antibody (i.e., primary antibody), and bovine serum albumin (i.e., As2MoO6@NiS-mAb mixed solution); The photothermal signal probe is used in a form independent of the test paper main structure, 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; The test paper 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, and the absorption pad is used to absorb excess liquid, and the PVC bottom plate provides physical support for the test paper; The nitrocellulose membrane includes a detection area, namely, a T area, and a quality control area, namely, a C area; wherein hexane estradiol-BSA 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.

2. The test paper according to claim 1, characterized in that The secondary antibody includes any one of goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody and donkey anti-rabbit secondary antibody.

3. The test paper according to claim 1, characterized in that The preparation of the As2MoO6@NiS composite material includes: adding As2MoO6 nanocrystals into deionized water containing polyethylene glycol, ultrasonicating, stirring at room temperature overnight to obtain a mixed solution; then adding the mixed solution into a mixed aqueous solution of nickel chloride and sodium sulfide in a molar ratio of 2:1-1:2 with a concentration of 0.1%-1% (containing 5wt% nitric acid) and stirring at room temperature overnight; collecting dark gray powder by centrifugation and drying to obtain the obtained product.

4. The test paper according to claim 1, characterized in that The specific preparation method of the As2MoO6@NiS-mAb mixed solution is: take the As2MoO6@NiS composite material into a centrifuge tube, add a weak alkaline solution to adjust the pH of the system to 6-8, oscillate and mix, then add hexane estradiol antibody, oscillate and react at room temperature for 30-60 minutes, add bovine serum albumin BSA for blocking, oscillate and react at room temperature for 1-1.5 hours, remove the supernatant after centrifugation, and then dissolve the remaining substance in a buffer solution to obtain the As2MoO6@NiS-mAb mixed solution.

5. A method for preparing a test paper for detecting hexaneestrol according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) Paste the NC membrane in the middle of the PVC base plate, overlap the sample pad and the absorbent pad on the left and right ends of the NC membrane, and cover the NC membrane by 2-3 mm. Cut the assembled large card into 3-5 mm wide strips, and make the test line, T zone, and the quality control line, C zone, on the NC membrane, respectively. The interval between the two lines is 4-6 mm. (2) dripping or spraying hexane estradiol-BSA on the T area, and dripping or spraying the secondary antibody against the source of the primary antibody, i.e., the anti-primary antibody, on the C area. After drying, the test strip is stored in a vacuum bag for later use; (3) 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.

6. A method for qualitatively detecting hexaneestrol based on the test paper according to any one of claims 1 to 4, characterized in that: The method comprises: Place the sample to be tested and the As2MoO6@NiS-mAb mixed solution in the running buffer and mix for 3-10 minutes, then put it into the test strip body. After 15-30 minutes, take it out and dry it. Then, place the dried test strip under a near-infrared LED light source for excitation, take it out and use thermal imaging or temperature measurement equipment to obtain the temperature for interpretation; The photothermal temperature of the detection zone is negatively correlated with the hexane estradiol content to be measured, and the color intensity is negatively correlated with the hexane estradiol content in the sample, specifically: When the sample does not contain hexaneestrol, the temperature of the T zone is high; When the sample contained hexaneestrol, the temperature of the T zone decreased; The quality control area serves as a reference for verifying the validity of the test paper results and always appears purple.

7. A method for quantitatively detecting hexaneestrol based on the test paper according to any one of claims 1 to 4, characterized in that: The method comprises: The sample to be tested and the As2MoO6@NiS-mAb mixed solution are placed in the running buffer and mixed for 3-10 minutes, then placed in the test paper body. After 15-30 minutes, the test paper strip is taken out and dried, and then the dried test paper strip is placed under a near-infrared LED light source for excitation. The temperature result is obtained using thermal imaging or temperature measurement equipment, and quantitative analysis is performed based on the quantitative relationship model.

8. The method according to claim 7, characterized in that The method for quantitatively detecting hexaneestrol also includes: 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 then mixed with the running buffer and placed in the test paper. After 15 to 30 minutes, the test paper is taken out and dried, and then placed under a near-infrared LED light source for excitation. The temperature results are obtained using thermal imaging or temperature measurement equipment, and quantitative analysis is performed based on the quantitative relationship model.

9. The method according to any one of claims 6 to 8, characterized in that: The running buffer is a PBS solution with a pH of 6.5 to 8.0 and a 10 mmol / L concentration.

10. The method according to any one of claims 6 to 8, characterized in that: The thermal imaging or temperature measuring device includes any one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager or a temperature measuring gun.

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