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

Abstract

The application discloses a test paper for detecting diethylstilbestrol and a preparation method thereof, and belongs to the technical field of small molecule analysis and detection. The test paper for detecting diethylstilbestrol comprises two parts of a test paper main body and a photothermal signal probe. The test paper 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 diethylstilbestrol-BSA, and the C zone is fixed with a secondary antibody. The photothermal signal probe is a Bi2MoO6@NiS-mAb mixed solution. When the test paper is used for detecting diethylstilbestrol, the detection mode is flexible, the linear range is wide, the sensitivity is high, and the specificity is good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of small molecule analysis and detection, and particularly relates to a test paper for detecting diethylstilbestrol 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 a sensitivity improved by tens to hundreds of times. The signal of this detection method is caused by temperature change due to light-thermal effect, the background signal is low, the sensing sensitivity is high, and the detection can be performed on various substrates of different colors, which has strong application potential. However, the light-thermal conversion efficiency of the existing light-thermal signal probe is low, especially the light-thermal signal response to diethylstilbestrol is poor, which still affects the detection sensitivity of diethylstilbestrol. SUMMARY

[0003] [TECHNICAL PROBLEM]

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

[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 diethylstilbestrol and a preparation method thereof. The test paper uses a Bi2MoO6@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 diethylstilbestrol.

[0007] Bi2MoO6 is a semiconductor photocatalyst with visible light response. Its unique double-layer structure makes it have a more ideal solar energy utilization rate and higher visible light catalytic activity than single-layer structure photocatalysts. Its energy band structure is beneficial to the separation of photo-generated electrons and holes, thereby improving the photocatalytic efficiency. NiS is a sulfide that usually has good light absorption capacity and can absorb most of the visible light and part of the ultraviolet light in sunlight. NiS can convert the absorbed light energy into heat energy, which makes it have potential application value in the field of light-thermal conversion. The combination of Bi2MoO6 and NiS may produce a synergistic effect, that is, the two materials promote each other in the process of photocatalysis or light-thermal conversion, thereby improving the overall light-thermal conversion performance.

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

[0009] A first object of the present application is to provide a test paper for detecting diethylstilbestrol, which comprises two parts of a test paper body and a photothermal signal probe:

[0010] The test paper body comprises: a sample pad, a nitrocellulose membrane (NC membrane) and an absorbent pad which are sequentially overlapped and pasted in horizontal direction from top to bottom on a PVC base plate; wherein the nitrocellulose membrane is used to realize 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 for absorbing excess liquid, and the PVC base plate provides physical support for the test paper;

[0011] The nitrocellulose membrane comprises a test zone (T zone) and a control zone (C zone); wherein the T zone is fixed with diethylstilbestrol-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 Bi2MoO6@NiS composite material, diethylstilbestrol antibody (primary antibody) and bovine serum albumin, i.e. Bi2MoO6@NiS-mAb mixed solution;

[0013] The photothermal signal probe is used in a form independent of the structure of the test paper body, 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 diethylstilbestrol antibody (primary antibody) source 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 diethylstilbestrol-BSA:

[0017] (1) First, prepare diethylstilbestrol carboxymethyl ether: dissolve 100-120 mg of diethylstilbestrol, 400-500 mg of KOH and 6-10 mL of DMSO by ultrasonicating 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 1.5-2 h of reaction, add about 50 mL of ice water to terminate the reaction; collect the unreacted diethylstilbestrol by repeatedly extracting 3 times with 8-10 mL of ethyl acetate, 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, repeatedly centrifuge (9000 rpm, 15 min) and wash the precipitate with ultrapure water until the pH value of the supernatant is about 7.0, vacuum freeze-dry the centrifuged precipitate to obtain white powder of diethylstilbestrol-CME;

[0018] (2) Preparation of dienoestrol-BSA: 3-4 mg of dienoestrol-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 dienoestrol-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 dienoestrol-BSA solution.

[0019] In an embodiment, the preparation of the Bi2MoO6@NiS composite material comprises: adding Bi2MoO6 nanoparticles into deionized water containing ethylene glycol, ultrasonicating, stirring overnight to obtain a mixed solution; then adding the mixed solution into an equal volume of NiS aqueous solution and stirring overnight; collecting the dark gray powder by centrifugation and drying to obtain the Bi2MoO6@NiS composite material.

[0020] In an embodiment, the mass-volume ratio of the Bi2MoO6 nanoparticles, polyethylene glycol and water is 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 condition is: ultrasonicating power is 120-600 W, and the ultrasonicating time is 20-30 min.

[0023] In an embodiment, the mass fraction of the NiS aqueous solution is 0.1%-1% (containing 5 wt% nitric acid).

[0024] In an embodiment, the specific preparation method of the Bi2MoO6@NiS-mAb mixed solution is: taking the Bi2MoO6@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 dienoestrol 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 substance in a buffer solution to obtain the Bi2MoO6@NiS-mAb mixed solution.

[0025] In an embodiment, the buffer includes one or more of a phosphate buffer, a borate buffer, or a carbonate buffer.

[0026] A second object of the present application is to provide a preparation method of the test paper for detecting diethylstilbestrol as described above, the preparation method of the test paper comprising the following steps:

[0027] (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, covering 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;

[0028] (2) Diethylstilbestrol-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;

[0029] (3) The photothermal signal probe, i.e., Bi2MoO6@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., Bi2MoO6@NiS-mAb mixed solution, is dried on a combination pad, and the combination pad is inserted between the sample pad and the NC film for use.

[0030] In an embodiment, the T zone is added or sprayed with a 5-20 mmol / L PBS solution containing 0.5-10 mg / mL diethylstilbestrol-BSA; and the C zone is added or sprayed with a 5-20 mmol / L PBS solution containing 0.05-10 mg / mL second antibody against the first antibody, i.e., anti-first antibody.

[0031] A third object of the present application is to provide a method for qualitatively detecting diethylstilbestrol based on the test paper as described above, comprising:

[0032] The sample to be tested and the Bi2MoO6@NiS-mAb mixed solution are mixed in the running buffer for 3-10 min, and then placed in the test paper main body. After 15-30 min, the test paper is taken out and dried. Then the dried test paper strip is placed under a near-infrared LED light source for excitation, and the temperature is obtained using a thermal imaging or temperature measuring device for interpretation.

[0033] The photothermal temperature of the detection zone is negatively correlated with the content of the diethylstilbestrol to be detected, and the color development intensity is negatively correlated with the content of diethylstilbestrol in the sample, specifically:

[0034] When the sample does not contain diethylstilbestrol, the temperature of the T zone is high;

[0035] When the sample contains diethylstilbestrol, the temperature of the T zone decreases.

[0036] The quality control area serves as a reference for verifying the effectiveness of the test paper results, and always shows a purple color.

[0037] A fourth object of the present application is to provide a method for quantitatively detecting diethylstilbestrol based on the above-mentioned test paper, comprising:

[0038] The sample to be tested and the Bi2MoO6@NiS-mAb mixed solution are mixed in the running buffer for 3-10 min, placed in the test paper strip body, and after 15-30 min, the test paper strip is taken out and dried, and then the dried test paper 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.

[0039] In one embodiment, the test paper for quantitatively detecting diethylstilbestrol further comprises:

[0040] The photothermal signal probe, i.e. the Bi2MoO6@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 strip, and after 15-30 min, the test paper 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.

[0041] In one embodiment, the running buffer is a PBS solution with a total concentration of 10 mmol / L and a pH of 6.5-8.0; the PBS solution contains the following components: 5-15% sucrose, 1-10% BSA, and 0.15-1% Tween-20.

[0042] 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.

[0043] In one embodiment, the intelligent display end of the thermal imaging or temperature measuring device includes a computer or a smart phone.

[0044] In one embodiment, the near-infrared LED light source has a power of 1-50 W / cm 2 and irradiates for 1-10 min.

[0045] In one embodiment, the quantitative relationship model is constructed by preparing a series of standard samples of diethylstilbestrol at different concentrations, and constructing a quantitative relationship model according to the concentration of diethylstilbestrol and the temperature result obtained using a thermal imaging or temperature measuring device.

[0046] The test paper detection principle for detecting diethylstilbestrol 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 color. After the sample solution and Bi2MoO6@NiS-mAb are premixed, they move to the direction of the water absorption paper under the capillary action. When there is no diethylstilbestrol in the sample, the Bi2MoO6@NiS-mAb is captured by the T area diethylstilbestrol-BSA, and the T area shows purple color, and under the excitation of the laser, the Bi2MoO6@NiS-mAb generates LSPR effect, the temperature rises, and the excess Bi2MoO6@NiS-mAb is captured by the second antibody in the C area, so that the C area also shows purple color. When the sample contains diethylstilbestrol, the diethylstilbestrol specifically binds to part of the Bi2MoO6@NiS-mAb, thereby reducing the total amount of Bi2MoO6@NiS-mAb captured in the T area, and the LSPR effect of the Bi2MoO6@NiS-mAb in the T area is reduced or even disappears, and the temperature decreases. With the increase of the concentration of diethylstilbestrol in the sample, the Bi2MoO6@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.

[0047] [Advantages]

[0048] (1) The Bi2MoO6@NiS composite material provided by the application is applied in the preparation of test paper and the detection of target substances, without the need to change the conventional structure of the test paper strip, and is used as a photothermal signal probe, which can be used independently of the test paper form, is convenient to store, and can be fixed on the conjugate pad of the test paper for convenient carrying.

[0049] (2) The detection method comprising the test paper provided by the application, the test paper for detecting diethylstilbestrol is based on the plasmonic resonance effect, and the Bi2MoO6@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 source excitation, combination of intelligent terminal and infrared thermal imaging accessories to realize the collection of photothermal signals, effectively remove the sample liquid substrate color and test paper fluorescence background interference and thus improve the signal-to-noise ratio.

[0050] (3) The detection method provided by the application has a lower detection limit, a wider detection range and better specificity, and is more sensitive than other photothermal detection test papers, and is suitable for rapid detection of diethylstilbestrol. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 It is a flowchart for detecting diethylstilbestrol by the test paper of the application;

[0052] Figure 2 It is a scanning electron microscope image, an X-ray energy spectrum image and a spectrum fitting curve image of Bi elements and Mo elements of the Bi2MoO6@NiS composite material in the photothermal signal probe of the test paper of the application;

[0053] Figure 3 The photothermal conversion efficiency fitting curve of Bi2MoO6@NiS composite material and Bi2MoO6 in the test paper photothermal signal probe of the application;

[0054] Figure 4 The response condition of the test paper of the application to different concentrations of diethylstilbestrol water sample in the photothermal mode and the working curve graph;

[0055] Figure 5 The response condition graph of the test paper of the application to diethylstilbestrol and another four kinds of estrogens. DETAILED DESCRIPTION

[0056] The technical solutions in the embodiments of the application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application. The following specific embodiments further describe the application.

[0057] Embodiment 1

[0058] Preparation of Bi2MoO6@NiS composite material, including the following steps:

[0059] (1) Preparation of Bi2MoO6 nanoparticles

[0060] 1.0g of Na2MoO4·2H2O and 3.0g of Bi(NO3)3·5H2O were respectively dissolved in 10mL of ethylene glycol, then they were mixed uniformly, 50mL of ethanol was added, after stirring for 1.0h, the solution was transferred to a 100mL autoclave, heated at 180℃ for 15h, after reaction, centrifuged, the precipitate was washed with deionized water and ethanol, and dried at 100℃ for 12h, then calcined at 450℃ (2℃ / min heating rate) for 2.5h, to obtain Bi2MoO6 nanocrystals;

[0061] (2) Preparation of Bi2MoO6@NiS composite material

[0062] Bi2MoO6@NiS composite material powder was obtained by adding 1 mL of the Bi2MoO6@NiS suspension prepared in Example 1 to 1 mL of a NiS aqueous solution (mass fraction of 0.1%, containing 5 wt% nitric acid) and stirring overnight, and then storing in a refrigerator. The Bi2MoO6@NiS composite material powder can be directly used. Alternatively, the Bi2MoO6@NiS composite material powder can be obtained by centrifuging the Bi2MoO6@NiS suspension at 10,000 rpm for 20 min at room temperature, collecting the dark gray powder, and drying at 50°C.

[0063] Performance characterization

[0064] The scanning electron microscope and energy spectrum of the Bi2MoO6@NiS composite material were determined, and the results are shown in FIG. 2. Figure 2 As can be seen from the spectrum, the needle-shaped Bi2MoO6@NiS composite material containing Bi, Mo and Ni was successfully prepared.

[0065] Figure 3 FIG. 3 is a temperature rising curve of the Bi2MoO6@NiS composite material and Bi2MoO6; the Bi2MoO6@NiS composite material has a photo-thermal conversion efficiency of 74%, which is significantly better than that of Bi2MoO6 (51%).

[0066] Example 2

[0067] 1. Preparation of signal probe Bi2MoO6@NiS-mAb mixed solution, including the following steps:

[0068] 1 mL of the Bi2MoO6@NiS suspension prepared in Example 1 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 dienestrol antibody was added and mixed. After mixing, the mixture was oscillated 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. The supernatant was removed by centrifugation at 12,000 r / min for 30 min, and then the Bi2MoO6@NiS-mAb mixed solution was redissolved in 100 μL of buffer solution (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose), and stored at 4°C for standby.

[0069] 2. Preparation of detection zone (T zone) solution

[0070] Preparation of dienoestrol-BSA: (1) 100 mg dienoestrol, 500 mg KOH and 6 mL DMSO were mixed, and dienoestrol was dissolved by ultrasonic treatment at 100 W for 5 min. 100 mg bromoacetic acid (previously dissolved in 1 mL DMSO) was added under magnetic stirring to carry out nucleophilic substitution reaction. After 2 h of reaction, 50 mL ice water was added to terminate the reaction. 10 mL ethyl acetate was used to repeat extraction for 3 times to collect unreacted dienoestrol. The water phase was collected in a clean beaker, and 2 mol / L HCl was added dropwise to the water phase under stirring, and white precipitate appeared. The precipitate was repeatedly centrifuged (9000 rpm, 15 min) with ultrapure water until the pH value of the supernatant was about 7.0. The precipitate after centrifugation was vacuum freeze-dried to obtain white powder, which was dienoestrol-CME;

[0071] (2) 3.3 mg dienoestrol-CME, 6.2 mg NHS and 7.1 mg EDC·HCl were weighed in a 10 mL centrifuge tube, 0.5 mL DMSO was added, and the mixture was gently stirred for 12 h of reaction. 20 mg BSA was dissolved in 4 mL carbonate buffer (50 mmol / L, pH 9.6), and the activated dienoestrol-CME solution was slowly added dropwise to the BSA solution to carry out coupling reaction. After 12 h of stirring reaction, 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), and the dialysis temperature was 4°C. The dialysis liquid was replaced every 12 h, and the dialysis was continuously carried out for 3 days. After dialysis was completed, the solution in the dialysis bag was aliquoted and stored in a freezer. The obtained solution was dienoestrol-BSA solution.

[0072] The dienoestrol-BSA was diluted to 0.6 mg / mL with 10 mmol / L PBS solution at pH 7.0.

[0073] 3. Preparation of quality control area (C area) solution

[0074] The goat anti-mouse secondary antibody was diluted to 0.4 mg / mL with 10 mmol / L PBS solution at pH 7.0.

[0075] Example 3

[0076] Preparation of dienoestrol test strip, including the following:

[0077] 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 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 strip after spotting is placed in an oven and dried at 37°C for 60 min, and stored in a vacuum bag for standby.

[0078] Example 4

[0079] The preparation of the dienoestrol test strip includes the following steps:

[0080] The test 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 Bi2MoO6@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 strip after spotting is placed in an oven and dried at 37°C for 60 min, and stored in a vacuum bag for standby.

[0081] Example 5

[0082] A method for detecting dienoestrol based on the paper strip prepared in Example 3, the method comprising the following steps:

[0083] (1) Quantitative relationship model construction

[0084] A 10 mL 1 mg / mL dienoestrol standard solution is prepared with acetonitrile, and diluted to a concentration of 10 - 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;

[0085] 80 μL of the sample solution was mixed with 10 μL of Bi2MoO6@NiS-mAb 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, and then the test strip prepared in Example 3 was inserted into the centrifuge tube. After the test strip was dried at 50-80°C for 10 min, it was irradiated with a near-infrared LED (power 1.36 W / cm 2 ) for 3 min, and the relationship between the temperature change and the concentration of the sample solution was monitored using a mobile phone and an infrared thermal imaging accessory to construct a quantitative relationship model.

[0086] The results are shown in Figure 4 ; the constructed quantitative relationship model is y = 5.146x + 0.279, R 2 = 0.9917, and the detection limit can reach 10 -1 pg / mL.

[0087] (2) Determination of diethylstilbestrol in samples

[0088] 80 μL of the sample solution was mixed with 10 μL of Bi2MoO6@NiS-mAb 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, and then the test strip was inserted into the centrifuge tube. After the test strip was dried, it was irradiated with a near-infrared LED (power 1.36 W / cm 2 ) for 3 min, and the temperature change was monitored using a mobile phone and an infrared thermal imaging accessory. The content of diethylstilbestrol in the sample solution was calculated according to the quantitative relationship model constructed in step (1).

[0089] Example 6 Specific detection

[0090] (1) 10 mL of 1 mg / mL diethylstilbestrol, estradiol, estrone, estriol, and ethinyl estradiol standard solutions were prepared with acetonitrile, respectively, and diluted to a concentration of 120 ng / mL with 10 mmol / L PBS as the sample solution for standby.

[0091] (2) The detection method of Example 5 was referred to for determination of each sample solution in step (1).

[0092] The results are shown in Figure 5 Compared with the blank, estradiol, estrone, estriol, and ethinyl estradiol had very weak effects on the temperature of the T zone of the test strip, and only diethylstilbestrol had a very significant effect on the temperature of the T zone of the test strip, indicating that the test strip had a specific response to diethylstilbestrol.

[0093] In summary, the Bi2MoO6@NiS composite material provided by the application in the application of preparing test paper does not need to change the conventional structure of the test paper strip, is used as a photothermal signal probe, 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 diethylstilbestrol is based on a plasmonic resonance effect, a Bi2MoO6@NiS composite material with high photothermal conversion efficiency and small size is used as a T area 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, sample liquid substrate color and test paper fluorescence background interference are effectively removed, and 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.

[0094] The above provided examples are not used to limit the scope covered by the application, and the described steps are not used to limit the execution order. The person skilled in the art makes obvious improvements to the application combined with the existing common knowledge, which also falls within the protection scope defined by the claims of the application.

Claims

1. A test paper for detecting diethylstilbestrol, characterized by, The test paper comprises a photo-thermal signal probe and a test paper strip body: The photo-thermal signal probe comprises a mixture of Bi2MoO6@NiS composite material, dienestrol antibody, and bovine serum albumin, namely Bi2MoO6@NiS-mAb mixture; The test paper strip 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 strip 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 dienestrol-BSA, and the C zone is fixed with a secondary antibody which is a secondary antibody against the primary antibody, namely anti-primary antibody; The preparation of the Bi2MoO6@NiS composite material comprises: adding Bi2MoO6 nanoparticles into deionized water containing polyethylene glycol, ultrasonicating, stirring overnight to obtain a mixture; then adding the mixture into an equal volume of NiS aqueous solution and stirring overnight; collecting deep gray powder by centrifugation and drying to obtain the Bi2MoO6@NiS composite material.

2. The test paper according to claim 1, characterized in that 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.

3. The test paper according to claim 1, characterized by The specific preparation method of the Bi2MoO6@NiS-mAb mixture is as follows: taking the Bi2MoO6@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 dienestrol antibody, oscillating and reacting at room temperature for 30-60 min, 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 re-dissolving the remaining substance in a buffer solution to obtain the Bi2MoO6@NiS-mAb mixture.

4. A method for producing the test paper for detecting dienoestrol according to any one of claims 1 to 3, 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-in 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 by 4-6 mm; (2) adding or spraying dienestrol-BSA on the T zone and adding or spraying the 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 Bi2MoO6@NiS-mAb mixture, in a sealed container in the form of a solution or a freeze-dried powder.

5. A method for the qualitative detection of diethylstilbestrol based on the test strip according to any one of claims 1 to 3, characterized in that, The method comprises: The mixture of the sample to be tested and the Bi2MoO6@NiS-mAb mixed solution is mixed in the running buffer for 3-10 min, and then placed in the test paper main body. After 15-30 min, the test paper is taken out and dried. Then the dried test paper 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 zone is negatively correlated with the content of the dienestrol to be detected, and the color development intensity is negatively correlated with the content of dienestrol in the sample, specifically: When the sample does not contain dienestrol, the temperature of zone T is high. When the sample contains dienestrol, the temperature of zone T decreases. The quality control zone serves as a reference for verifying the effectiveness of the test paper and always shows purple color.

6. A method for the quantitative determination of diethylstilbestrol based on the test strip according to any one of claims 1 to 3, characterized in that, The method comprises: The mixture of the sample to be tested and the Bi2MoO6@NiS-mAb mixed solution is mixed in the running buffer for 3-10 min, and then placed in the test paper main body. After 15-30 min, the test paper is taken out and dried. Then the dried test paper is placed under the near-infrared LED light source for excitation. The temperature result is obtained by using a thermal imaging or temperature measuring device, and the result is interpreted according to the quantitative relationship model.

7. The method of claim 6, wherein, The method for quantitatively detecting dienestrol further comprises: The photothermal signal probe, i.e. the Bi2MoO6@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 strip. After 15-30 min, the test paper is taken out and dried. Then the dried test paper is placed under the near-infrared LED light source for excitation. The temperature result 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 according to any one of claims 5 to 7, characterized in that, The running buffer is a PBS solution with pH 6.5-8.0 and 10 mmol / L.

9. The method according to any one of claims 5 to 7, characterized in that, The thermal imaging or temperature measuring device comprises any one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, or a temperature measuring gun.

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