A test paper for detecting hexestrol and a method for preparing the same

Abstract

The application discloses a test paper for detecting hexestrol and a preparation method thereof, and belongs to the technical field of small molecule analysis and detection. The test paper for detecting hexestrol comprises two parts of a test paper main body and a photothermal signal probe. The test paper strip main body comprises, in a horizontal direction, a sample pad, a nitrocellulose membrane (NC membrane) and an absorption pad which are sequentially and overlappedly pasted on a PVC base plate from top to bottom. The NC membrane comprises a detection zone (T zone) and a quality control zone (C zone). The T zone is fixed with hexestrol-BSA, and the C zone is fixed with a secondary antibody. The photothermal signal probe is an As2MoO6@NiS-mAb mixed solution. When the test paper is used for detecting 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 present application belongs to the technical field of small molecule analysis and detection, and particularly relates to a test paper for detecting hexestrol and a preparation method thereof. BACKGROUND

[0002] Lateral flow immunoassay (LFIA) has the characteristics of fast analysis performance, good selectivity, low cost, small sample requirement, easy mass production and stable long-term storage, and is widely used in the design of portable analysis and detection sensors. Compared with the colorimetric mode, the light-thermal LFIA using light-thermal signal probes has an increase of tens to hundreds of times in sensitivity. The signal of this detection method is caused by temperature change due to light-thermal effect, and the background signal is low, the sensing is sensitive, and it can be performed on various substrates of different colors, and has strong application potential. However, the light-thermal conversion efficiency of the existing light-thermal signal probe is low, especially the response to hexestrol is poor, which further affects the detection sensitivity of hexestrol. SUMMARY

[0003] [TECHNICAL PROBLEM]

[0004] The light-thermal conversion efficiency of the existing light-thermal signal probe is low, especially the response to hexestrol is poor, which leads to low detection sensitivity of hexestrol and poor quantitative effect of the hexestrol target.

[0005] [TECHNICAL SCHEME]

[0006] To solve the defects and deficiencies in the prior art, the purpose of the present application is to provide a test paper for detecting hexestrol and a preparation method thereof. The test paper uses As2MoO6@NiS composite material with high light-thermal conversion efficiency as a signal probe, which has a lower detection limit, a wider detection range, better specificity and higher sensitivity when used for detecting hexestrol.

[0007] NiS is a metal sulfide with certain potential in light-thermal conversion, but the light-thermal performance of NiS is usually affected by its own particle size, specific surface area and morphology, especially the material compounded with it. As2MoO6 is an inorganic compound with specific optical and thermal properties; the composite material obtained by compounding As2MoO6 with NiS is found to have high light-thermal conversion efficiency and can be used for specific detection of hexestrol.

[0008] To achieve the above purpose, the technical scheme provided is as follows:

[0009] The first purpose of the present application is to provide a test paper for detecting hexestrol, which comprises a test paper body and a light-thermal signal probe:

[0010] The test strip body comprises: a sample pad, a nitrocellulose membrane (NC membrane) and an absorbent pad which are overlapped and pasted in sequence from top to bottom along the horizontal direction on a PVC base plate; wherein the nitrocellulose membrane is used to realize the separation and detection of analytes and other substances in the sample, the sample pad is used for sample loading, and the absorbent pad is used to absorb excess liquid; the PVC base plate provides physical support for the test paper;

[0011] The nitrocellulose membrane comprises a test zone (T zone) and a quality control zone (C zone); wherein the T zone is fixed with hexestrol-BSA; and the C zone is fixed with a secondary antibody, which is a secondary antibody against the primary antibody, i.e. an anti-primary antibody;

[0012] The photothermal signal probe comprises a mixture of As2MoO6@NiS composite material, hexestrol antibody (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 body structure, or the photothermal signal probe is dried on a combination pad, and the combination pad is used by being inserted between the sample pad and the NC membrane.

[0014] In an embodiment, the source of the hexestrol antibody (primary antibody) includes mouse, rat and rabbit.

[0015] In an embodiment, 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.

[0016] In an embodiment, the preparation of hexestrol-BSA: (1) first prepare hexestrol carboxymethyl ether: dissolve 100-120 mg of hexestrol, 400-500 mg of KOH and 6-10 mL of DMSO by ultrasonic treatment at 100-400 W for 5 min, add 100 mg of bromoacetic acid (previously dissolved in 1 mL of DMSO) under magnetic stirring to perform nucleophilic substitution reaction, after reaction for 1.5-2 h, add about 50 mL of ice water to terminate the reaction; repeat extraction with 8-10 mL of ethyl acetate for 3 times to collect unreacted hexestrol, collect the water phase with a clean beaker, and add 2 mol / L HCl dropwise to the water phase under stirring conditions, followed by the appearance of white precipitate, centrifuge (9000 rpm, 15 min) the precipitate repeatedly with ultrapure water until the pH value of the supernatant is about 7.0; vacuum freeze-dry the centrifuged precipitate to obtain white powder as hexestrol-CME;

[0017] (2) Preparation of hexestrol-BSA: 3-4 mg of hexestrol-CME, 5-7 mg of NHS and 6-8 mg of EDC-HCl were weighed in a 10 mL centrifuge tube, 0.5-1.0 mL of DMSO was added, and the reaction was stirred gently for 10-20 h; 15-20 mg of BSA was dissolved in 4-6 mL of carbonate buffer (50 mmol / L, pH 9.6), and the activated hexestrol-CME solution was slowly added dropwise to the BSA solution for coupling reaction, and the reaction 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 liquid was 8-12 mmol / L PBS (pH 7.4), the dialysis temperature was 4°C, the dialysis liquid was replaced every 10-15 h, and continuous dialysis was performed for 3 days; after dialysis was completed, the solution in the dialysis bag was aliquoted and frozen for storage; the obtained solution was a hexestrol-BSA solution.

[0018] In an embodiment, the preparation of the As2MoO6@NiS composite material comprises: adding As2MoO6 nanocrystals into deionized water containing polyethylene glycol, ultrasonicating, stirring overnight to obtain a mixed solution; then adding the mixed solution into 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, stirring overnight; collecting a dark gray powder by centrifugation and drying to obtain the As2MoO6@NiS composite material.

[0019] In an 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 an embodiment, the polyethylene glycol has a MW of 5000-10000.

[0022] In an embodiment, the ultrasonicating conditions are: ultrasonicating power of 120-600 W, 20-30 min.

[0023] In an embodiment, the specific preparation method of the As2MoO6@NiS-mAb mixed solution is: 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, then adding a hexestrol antibody, oscillating and reacting at room temperature for 30-60 min, then adding bovine serum albumin BSA for blocking, oscillating and reacting at room temperature for 1-1.5 h, removing the supernatant after centrifugation, and then redissolving the remaining material in a buffer solution to obtain the As2MoO6@NiS-mAb mixed solution.

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

[0025] A second object of the present application is to provide a preparation method of the test paper for detecting hexestrol as described above, the preparation method of the test paper comprising the following steps:

[0026] (1) The NC film is pasted in the middle of the PVC base plate, and the sample pad and the water absorption pad are respectively overlapped on the left and right ends of the NC film, so that they cover the NC film by 2-3 mm. The assembled large card is cut into paper strips with a width of 3-5 mm, and a detection line (T zone) and a quality control line (C zone) are made on the NC film. The two lines are spaced 4-6 mm apart from each other;

[0027] (2) Hexestrol-BSA is added or sprayed in the T zone, and a second antibody against the first antibody, i.e. anti-first antibody, is added or sprayed in the C zone. After drying, the test paper strip is stored in a vacuum bag for standby;

[0028] (3) The photothermal signal probe, i.e. As2MoO6@NiS-mAb mixed solution, is stored in a sealed container in the form of a solution or a freeze-dried powder; or the photothermal signal probe, i.e. As2MoO6@NiS-mAb mixed solution, is dried on a binding pad, and the binding pad is inserted between the sample pad and the NC film for use.

[0029] In an embodiment, the T zone is added or sprayed with a 5-20 mmol / L PBS solution containing 0.5-10 mg / mL hexestrol-BSA; and the C zone is added or sprayed with a second antibody against the first antibody, i.e. anti-first antibody, specifically: the C zone is added or sprayed with a 5-20 mmol / L PBS solution containing 0.05-10 mg / mL second antibody.

[0030] A third object of the present application is to provide a method for qualitatively detecting hexestrol based on the test paper as described above, comprising:

[0031] The sample to be tested is mixed with the As2MoO6@NiS-mAb mixed solution in a running buffer for 3-10 min, and then placed in the main body of the test paper strip. After 15-30 min, it 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 is obtained using a thermal imaging or temperature measuring device for interpretation;

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

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

[0034] When the sample contains hexestrol, the temperature of the T zone decreases;

[0035] The quality control zone always shows purple as a reference for verifying the effectiveness of the test paper results.

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

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

[0038] In an embodiment, the test paper for quantitatively detecting hexestrol further comprises:

[0039] The photothermal signal probe, i.e. the As2MoO6@NiS-mAb mixed solution, is dried on the conjugate pad, the conjugate pad is inserted between the sample pad and the NC membrane, then the sample to be tested is mixed with the running buffer and placed in the test strip, after 15-30 min, the test strip is taken out and dried, placed under the near-infrared LED light source for excitation, and the temperature result is obtained using a thermal imaging or temperature measuring device, and quantitative analysis is performed according to the quantitative relationship model.

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

[0041] In an 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 an embodiment, the intelligent display end of the thermal imaging or temperature measuring device includes a computer or a smart phone.

[0043] In an embodiment, the near-infrared LED light source has a power of 1-50 W / cm 2 and irradiates for 1-10 min.

[0044] In an embodiment, the quantitative relationship model is constructed by preparing a series of standard samples with different concentrations of hexestrol, and constructing a quantitative relationship model according to the concentration of hexestrol and the temperature result obtained using a thermal imaging or temperature measuring device.

[0045] The test paper detection principle for detecting hexestrol of the application is as follows: the quality control area (C area) is used as a reference for verifying the effectiveness of the test paper result, and always shows purple. After the sample solution and As2MoO6@NiS-mAb are premixed, they move to the direction of the absorbent paper under the capillary action. When there is no hexestrol in the sample, the As2MoO6@NiS-mAb is captured by the T area hexestrol-BSA, and the T area shows purple, and under the excitation of the laser, the As2MoO6@NiS-mAb generates the LSPR effect, the temperature rises, and the excess As2MoO6@NiS-mAb is captured by the second antibody in the C area, so that the C area also shows purple. When the sample contains hexestrol, the hexestrol 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 decreases. With the increase of the concentration of hexestrol in the sample, the As2MoO6@NiS-mAb captured in the T area is less and less, and the temperature of the T area gradually decreases, which is inversely proportional to the concentration of the analyte.

[0046] [Advantages]

[0047] (1) The As2MoO6@NiS composite material provided by the application is applied in the preparation of test paper and the detection of target objects, without changing the conventional structure of the test paper, and is used as a photothermal signal probe, which can be used independently of the form of the test paper, is convenient to store, and can be fixed on the binding pad of the test paper for convenient carrying.

[0048] (2) The detection method comprising the test paper provided by the application, the test paper for detecting hexestrol is based on the plasmonic resonance effect, and the As2MoO6@NiS composite material with high photothermal conversion efficiency and small size is used as a T area fixed photothermal signal probe, near-infrared LED light sources are excited, and the collection of the photothermal signal is realized by combining an intelligent terminal and an infrared thermal imaging accessory, so that the sample liquid substrate color and the test paper fluorescence background interference are effectively removed, and the signal-to-noise ratio is improved.

[0049] (3) The detection method provided by the application has a lower detection limit, a wider detection range and better specificity, and has higher sensitivity than other photothermal detection test papers, and is suitable for rapid detection of hexestrol. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The flowchart of the test paper for detecting hexestrol of the application is shown in the figure.

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

[0052] Figure 3 This is a data graph showing the response of the test strip of the present invention to water samples of different concentrations of hexestrol under photothermal mode;

[0053] Figure 4 This is a graph showing the response of the test strip of the present invention to hexanestilbene and four other estrogens. Detailed Implementation

[0054] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate 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 of ethylene glycol (containing 10% hydrochloric acid by volume), then mixed thoroughly. 40 mL of ethanol was added, and the mixture was stirred for 1.0 h. The solution was then transferred to a 100 mL autoclave and heated at 180 °C for 15 h. After the reaction, the solution was removed and centrifuged. The precipitate was washed with deionized water and ethanol and dried at 100 °C for 12 h. Then, it was calcined at 450 °C (heating rate of 2 °C / min) for 2.5 h to obtain As2MoO6 nanocrystals.

[0059] (2) Preparation of As2MoO6@NiS composite material

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

[0061] Performance characterization

[0062] The transmission electron microscopy, photo-thermal conversion efficiency, and energy spectrum of Ni element and Mo element of the As2MoO6@NiS composite material were determined. The results are shown in Figure 2 The transmission electron microscopy showed that two types of nanoparticles were uniformly combined together, and the energy spectrum of Ni element and Mo element was simultaneously exhibited, indicating that the As2MoO6@NiS composite material was successfully prepared.

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

[0064] Example 2

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

[0066] 1 mL of As2MoO6@NiS was taken in a centrifuge tube, 4 μL of 0.2 mol / L K2CO3 solution was added to adjust the pH value of the system, and then 5 μL of 1 mg / mL hexestrol antibody was added. After mixing, the mixture was shaken at room temperature for 45 min. After the reaction was completed, 100 μL of BSA (m / m = 5%) was added for blocking for 1 h. After centrifugation at 12000 r / min for 30 min, the supernatant was removed, and the mixture was resuspended in 100 μL of buffer solution (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose). The As2MoO6@NiS-mAb mixture was obtained and stored at 4°C for standby use.

[0067] 2. Preparation of detection zone (T zone) solution

[0068] (1) 80 mg hexoestrol, 400 mg KOH and 6 mL DMSO were mixed, and dissolved by ultrasonic wave with 100 W for 5 min, and then 80 mg bromoacetic acid (previously dissolved in 1 mL DMSO) was added under magnetic stirring for nucleophilic substitution reaction, and about 50 mL ice water was added after 2 h reaction to terminate the reaction; 10 mL ethyl acetate was used for repeated extraction for 3 times to collect unreacted hexoestrol, and the water phase was collected in a clean beaker, and 2 mol / L HCl was added dropwise to the water phase under stirring, and then white precipitate appeared, and the precipitate was repeatedly centrifuged (9000 rpm, 15 min) with ultrapure water until the pH value of the supernatant was about 7.0, and the precipitate after centrifugation was vacuum freeze-dried to obtain white powder as hexoestrol-CME;

[0069] (2) 4 mg hexoestrol-CME, 6 mg NHS and 7 mg EDC·HCl were weighed in a 10 mL centrifuge tube, 1 mL DMSO was added, and the mixture was stirred and reacted for 10 h; 22 mg BSA was dissolved in 4 mL carbonate buffer (50 mmol / L, pH 9.6), and the activated hexoestrol-CME solution was slowly added dropwise to the BSA solution for coupling reaction, and the mixture was stirred and reacted for 10 h; after the reaction was completed, the reaction solution was transferred to a dialysis bag for dialysis to remove impurities, the dialysis liquid was 10 mmol / L PBS (pH 7.4), the dialysis temperature was 4°C, the dialysis liquid was replaced every 10 h, and the dialysis was continuously performed for 3 days; after the dialysis was completed, the solution in the dialysis bag was divided and stored in a freezer, and the obtained solution was hexoestrol-BSA solution.

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

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

[0072] The goat anti-mouse secondary antibody was diluted to 0.4 mg / mL with 10 mmol / L PBS solution with pH 7.4.

[0073] Example 3

[0074] Preparation of hexoestrol test strip, including the following:

[0075] The test strip was prepared according to Figure 1The combination mode is composed of a sample pad, a nitrocellulose membrane (NC), a combination pad and a PVC base plate. The NC membrane is pasted in the middle of the PVC base plate, and the sample pad and the water absorption pad are respectively overlapped on the left and right ends of the NC membrane, so as to cover the NC membrane by about 2 mm. The built large card is cut into a paper strip with a width of 3 mm, and a detection line (T zone) and a quality control line (C zone) are made on the NC membrane. The two lines are spaced 4 mm apart from each other. That is, a blank test paper strip is obtained. 0.5 μL of the T zone solution and 0.5 μL of the C zone solution are added to the T zone and the C zone, respectively. The test paper strip after spotting is placed in an oven and dried at 37°C for 60 min, and stored in a vacuum bag for standby.

[0076] Example 4

[0077] The preparation of the diethylstilbestrol test paper includes the following steps:

[0078] The test paper strip is prepared according to the combination mode Figure 1 The combination mode is composed of a sample pad, a nitrocellulose membrane (NC), a combination 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. The two lines are spaced 4 mm apart from each other. Then, the right end of the combination pad is overlapped on the left end of the NC membrane, so as to cover the NC membrane by about 2 mm. The left end of the sample pad is overlapped on the left end of the combination pad, so as to cover the combination pad by about 2 mm. The right end of the water absorption pad is overlapped on the right end of the NC membrane, so as to cover the NC membrane by about 2 mm. The built large card is cut into a paper strip with a width of 3 mm. Then, 10 μL of the As2MoO6@NiS-mAb mixed solution is fixed on the combination pad. 0.5 μL of the T zone solution and 0.5 μL of the C zone solution are added to the T zone and the C zone, respectively. The test paper strip after spotting is placed in an oven and dried at 37°C for 60 min, and stored in a vacuum bag for standby.

[0079] Example 5

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

[0081] (1) Quantitative relationship model construction

[0082] A 10 mL 1 mg / mL diethylstilbestrol standard solution is prepared by using acetonitrile. The solution is diluted to a concentration of 10 pg / mL, 10 pg / mL, 10 pg / mL, 10 pg / mL, 10 pg / mL, 10 pg / mL, 10 pg / mL, 10 pg / mL, 10 pg / mL and 10 pg / mL by using 10 mmol / L PBS, as a test solution for standby. - 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 a test solution for standby.

[0083] 80 μL of the sample solution was mixed with 10 μL of As2MoO6@NiS-mAb mixture 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 paper prepared in Example 3 was inserted into the centrifuge tube, after the liquid flowed through the NC membrane of the test paper, the near-infrared LED (power 1.36 W / cm 2 ) was irradiated for 3 min, the relationship between the change of temperature and the concentration of the sample solution was monitored by using a mobile phone and an infrared thermal imaging accessory, and a quantitative relationship model was constructed;

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

[0085] (2) The sample was determined as follows: 80 μL of the sample solution was mixed with 10 μL of As2MoO6@NiS-mAb mixture 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 paper strip was inserted into the centrifuge tube, after the test paper was dried, the near-infrared LED (power 1.36 W / cm 2 ) was irradiated for 3 min, the change of temperature was monitored by using a mobile phone and an infrared thermal imaging accessory, and the content of hexestrol in the sample solution was calculated according to the quantitative relationship model constructed in step (1).

[0086] Example 6 Specific detection

[0087] (1) 10 mL of 1 mg / mL hexestrol, estradiol, estrone, estriol, ethinyl estradiol standard solution was prepared with acetonitrile, and diluted to a concentration of 120 ng / mL with 10 mmol / L PBS as a sample solution for standby;

[0088] (2) The detection method of Example 5 was referred to, and each sample solution in step (1) was determined.

[0089] The results are shown in Figure 4 Compared with the blank, estradiol, estrone, estriol, and ethinyl estradiol had very weak effect on the temperature of the T zone of the test paper, only hexestrol had very significant effect on the temperature of the T zone of the test paper, indicating that the test paper had specific response to hexestrol.

[0090] In summary, the application provides an application of the As2MoO6@NiS composite material in preparation of test paper, without changing the conventional structure of the test paper, and the composite material is used as a photothermal signal probe, which can be used independently of the form of the test paper, is convenient to store, and can be fixed on a combination pad of the test paper and is convenient to carry; the test paper for detecting hexestrol provided by the application is based on a plasmonic resonance effect, the As2MoO6@NiS composite material with high photothermal conversion efficiency and small size is used as a T zone fixed photothermal signal probe, a near-infrared laser light source is excited, an intelligent terminal and an infrared thermal imaging accessory are combined to realize collection of the photothermal signal, and the color of a sample liquid substrate and the fluorescent background interference of the test paper are effectively removed, so that the signal-to-noise ratio is improved; the inspection method has a lower detection limit, a wider detection range and better specificity, and has higher sensitivity than other photothermal detection test papers.

[0091] The above provided examples are not used to limit the scope covered by the present application, and the described steps are not used to limit the execution order. The improvements of the present application made by those skilled in the art in combination with the existing common knowledge are also within the protection scope defined by the claims of the present application.

Claims

1. A test paper for detecting hexestrol, characterized by, The test paper comprises two parts of a photo-thermal signal probe and a test paper body: The photo-thermal signal probe comprises a mixture of As2MoO6@NiS composite material, hexestrol antibody, and bovine serum albumin, namely As2MoO6@NiS-mAb mixture; The test paper body comprises: a sample pad, a nitrocellulose membrane (NC membrane), and an absorbent pad which are sequentially overlapped and pasted on a PVC base plate in a horizontal direction from top to bottom; the nitrocellulose membrane is used to separate and detect analytes and other substances in a sample, the sample pad is used for sample loading, and the absorbent pad is used to absorb excess liquid; the PVC base plate provides physical support for the test paper; The photo-thermal signal probe is used in a form independent of the structure of the test paper body, or the photo-thermal signal probe is dried on a combination pad, and the combination pad is inserted between the sample pad and the NC membrane for use; The nitrocellulose membrane comprises a detection zone (T zone) and a quality control zone (C zone); the T zone is fixed with hexestrol-BSA, and the C zone is fixed with a secondary antibody which is a secondary antibody against the primary antibody, namely anti-primary antibody.

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

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

4. The test paper according to claim 1, characterized by The specific preparation method of the As2MoO6@NiS-mAb mixture is as follows: taking the As2MoO6@NiS composite material into a centrifuge tube, adding a weak alkaline solution to adjust the pH of the system to 6-8, oscillating and mixing, then adding hexestrol antibody, oscillating and reacting for 30-60 min at room temperature, adding bovine serum albumin (BSA) for blocking, oscillating and reacting for 1-1.5 h at room temperature, removing the supernatant after centrifugation, and then re-dissolving the remaining substance in a buffer solution, to obtain the As2MoO6@NiS-mAb mixture.

5. A method for producing the test paper for detecting hexestrol according to any one of claims 1 to 4, characterized by The preparation method comprises the following steps: (1) pasting the NC membrane in the middle of the PVC base plate, overlapping the sample pad and the water-absorbing pad on the left and right ends of the NC membrane respectively, and covering the NC membrane by 2-3 mm, cutting the built large card into paper strips with a width of 3-5 mm, and marking detection lines, T zone and quality control line, C zone on the NC membrane, and spacing the two lines from each other by 4-6 mm; (2) adding or spraying hexestrol-BSA on the T zone, and adding or spraying a secondary antibody against the primary antibody, namely anti-primary antibody, on the C zone, and then drying, and storing the test paper strip in a vacuum bag for standby; (3) storing the photo-thermal signal probe, namely the As2MoO6@NiS-mAb mixture, in a sealed container in the form of a solution or a freeze-dried powder; or drying the photo-thermal signal probe, namely the As2MoO6@NiS-mAb mixture, on a combination pad, and inserting the combination pad between the sample pad and the NC membrane for use.

6. A method for qualitatively detecting hexestrol 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 is mixed with the As2MoO6@NiS-mAb mixed solution in the running buffer for 3-10 min, and then placed in the test strip body. After 15-30 min, the test strip is taken out and dried. Then the dried test strip is placed under the near-infrared LED light source for excitation. The temperature is obtained by using a thermal imaging or temperature measuring device, and the result is interpreted. The photothermal temperature of the detection area is negatively correlated with the content of the diethylstilbestrol to be tested, and the color development intensity is negatively correlated with the content of diethylstilbestrol in the sample, specifically: When the sample does not contain diethylstilbestrol, the temperature of the T zone is high. When the sample contains diethylstilbestrol, the temperature of the T zone decreases. The quality control area serves as a reference for verifying the effectiveness of the test paper results and always shows purple color.

7. A method for quantitative detection of hexestrol 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 is mixed with the As2MoO6@NiS-mAb mixed solution in the running buffer for 3-10 min, and then placed in the test strip body. After 15-30 min, the test strip is taken out and dried. Then the dried test strip is placed under the near-infrared LED light source for excitation. The temperature is obtained by using a thermal imaging or temperature measuring device, and the result is interpreted according to the quantitative relationship model.

8. The method of claim 7, wherein, The method for quantitatively detecting diethylstilbestrol further comprises: 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. Then the sample to be tested is mixed with the running buffer and placed in the test paper. After 15-30 min, the test paper is taken out and dried, placed under the near-infrared LED light source for excitation, and the temperature result is obtained by using a thermal imaging or temperature measuring device. Quantitative analysis is performed according to 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 6.5-8.0 and a concentration of 10 mmol / L.

10. The method of any one of claims 6-8, wherein, 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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