Method, probe for quantitatively detecting sulfonylurea drugs and preparation method thereof

Abstract

The present invention discloses a method for quantitatively detecting sulfonylurea drugs, a probe and a preparation method thereof. In the quantitative detection method, the probe used comprises a combined zirconium-coordinated porphyrin metal-organic framework@polydopamine composite and an antibody, wherein the thickness of the dopamine shell layer in the zirconium-coordinated porphyrin metal-organic framework@polydopamine composite is 20-40 nm. The present invention effectively improves the dispersibility and stability of the material by uniformly dropping the ligand. Compared with the traditional one-pot method, the polydispersity coefficient of the synthesized material is reduced from 0.148 to 0.024. The quinone groups provided by the polydopamine outer shell coating are covalently coupled with the antibody without using a cross-linking agent, so that the antibody coupling rate is increased and exceeds 90% compared with the traditional colloidal gold probe. The detection limit of the immunochromatographic method based on this probe for sulfonylurea drugs is 0.22-4.27 μg / kg, and the sensitivity is 145 times that of the currently reported immunochromatographic methods. The present invention provides a detection means for simply operating, having high sensitivity, being reliable and capable of rapidly screening sulfonylurea drugs in health products.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly to a method for quantitatively detecting sulfonylurea drugs, a probe and a preparation method thereof. Background Art

[0002] Sulfonylureas (SUs) are a commonly used oral drug for the treatment of type 2 diabetes. The sulfonylurea drugs marketed in China are mainly glipizide, glibenclamide, glimepiride, gliquidone, tolbutamide, etc. Because of its rapid and complete absorption in the gastrointestinal tract, high binding rate to plasma proteins, and low price, it is widely used clinically. However, in order to pursue good effects and high profits, some illegal merchants often illegally add sulfonylurea drugs to health care products for assisting in reducing blood sugar. Long-term use of such health care products is likely to cause serious adverse reactions such as severe hypoglycemia and weight gain, posing a threat to the health of the public. Therefore, it is very important to establish a rapid, sensitive and accurate detection method for sulfonylurea drugs in health care products.

[0003] At present, an immunochromatography (Lateral flow immunoassay, LFIA) method for sulfonylurea drugs has been established based on colloidal gold, which has the advantages of simple operation, rapidity, low cost and no need for large-scale detection equipment. However, since colloidal gold binds antibodies by physical adsorption, the antibodies are easily detached from the surface of colloidal gold particles, resulting in unstable labeled probes. Therefore, the sensitivity and stability of the immunochromatography method established with colloidal gold as the labeling material are the bottlenecks of this technology, and it is urgent to invent a new labeling material to make up for the defects of colloidal gold. Summary of the Invention

[0004] In order to make up for the defects of the existing technology, the present invention provides a method for quantitatively detecting sulfonylurea drugs, a probe and a preparation method thereof. The present invention effectively controls the crystal growth rate by uniformly dropping ligands. Compared with the traditional one-pot method, the polydispersity coefficient of the synthesized material is reduced from 0.148 to 0.024, effectively improving the dispersibility and stability of the material. The quinone groups provided by the polydopamine coating on the material shell are covalently coupled with antibodies. During the coupling process, no cross-linking agent is required, and the antibody coupling rate is increased and exceeds 90% compared with the traditional colloidal gold probe. Based on the probe, an immunochromatography method has a detection limit for sulfonylurea drugs of 0.22 - 4.27 μg / kg, and the sensitivity is 145 times that of the currently reported immunochromatography methods. The present invention provides a detection means with simple operation, high sensitivity, reliability and capable of rapidly screening sulfonylurea drugs in health care products.

[0005] The technical problems to be solved by the present invention are realized through the following technical solutions:

[0006] First aspect, a method for quantitatively detecting sulfonylurea drugs, comprising the following steps:

[0007] (A) Sample pretreatment: Weigh the sample and place it in a centrifuge tube, add methanol solution, mix well, centrifuge to obtain the supernatant, and dilute the supernatant for testing.

[0008] (B) Standard curve preparation: According to the treatment method in step (A), prepare methanol solutions containing sulfonylurea drugs with different concentrations in a sample known to be free of sulfonylurea drugs, extract and dilute to obtain standard extraction solutions; Testing: Add the standard extraction solutions into enzyme-labeled wells, add the probe at the same time for incubation, and then insert the test strip for chromatographic reaction; Repeat each concentration several times, read the gray-scale values of the T and C line signals respectively, and add the ratio of the gray-scale value of the T line to the C line corresponding to a concentration of 0 μg / kg as the B0 value, and the ratio of the gray-scale value of the T line to the C line corresponding to other labeled concentrations as Bx. Take B X / B0 as the ordinate and the added concentration of sulfonylurea drugs as the abscissa to draw a standard curve.

[0009] (C) Testing the sample: Treat the sample to be tested according to step (A) to obtain the extract of the sample to be tested, and then perform detection according to the testing steps in step (B) to obtain the corresponding ratio of the gray-scale values of the T line to the C line, and then calculate the total content of sulfonylurea drugs in the sample according to the standard curve.

[0010] Wherein, the probe comprises a zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite and an antibody, and the thickness of the dopamine shell layer in the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite is 20-40 nm.

[0011] Second aspect, a probe for detecting sulfonylurea drugs, which comprises a zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite and an antibody, and the thickness of the dopamine shell layer in the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite is 20-40 nm.

[0012] Third aspect, a preparation method of the above-mentioned probe for detecting sulfonylurea drugs, which comprises the following steps:

[0013] (1) Synthesis of zirconium-coordinated porphyrin metal-organic framework: Weigh zirconium oxychloride octahydrate and benzoic acid and dissolve them in DMF, mix well to make a mixed solution; Weigh meso-tetra(4-carboxyphenyl)porphyrin and dissolve it in DMF and sonicate until there is no visible precipitate in the deep purple mixed solution to make a ligand solution; Drop the ligand solution into the mixed solution, place it in a constant-temperature magnetic stirrer for heating reaction; After the reaction is completed, wait for the reaction solution to cool naturally, centrifuge to collect spherical crystals, wash, and vacuum dry to obtain zirconium-coordinated porphyrin metal-organic framework (abbreviation: PCN-224).

[0014] (2) Synthesis of zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite: Weigh the zirconium-coordinated porphyrin metal-organic framework obtained in step (1), disperse it in an ethanol solution and sonicate it. Then add Tris-HCl buffer solution to adjust the pH of the solution, and add dopamine hydrochloride to the solution. Stir the reaction in the dark, then centrifuge to collect the solid, wash it, and finally redissolve it with deionized water and refrigerate it to obtain the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite (abbreviated as PCN-224@PDA);

[0015] (3) Synthesis of the probe: Centrifuge the solution redissolved in step (2), remove the supernatant, add borate buffer solution to redissolve it, sonicate and mix well. Then add an antibody to the liquid, mix well and incubate it on a shaker to allow the antibody to fully bind to the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite. Then add bovine serum albumin solution to block the unbound sites and incubate it on a shaker. Finally, centrifuge, discard the supernatant, resuspend the precipitate with phosphate buffer solution, sonicate and mix well to prepare the probe, and refrigerate it for later use.

[0016] Further, in step (2), the mass ratio of the zirconium-coordinated porphyrin metal-organic framework to dopamine hydrochloride is 2:(0.5 - 2.0), and the pH of the Tris-HCl solution is 7.0 - 8.5.

[0017] Further, in step (3), the volume ratio of the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite solution to the antibody is 1000:(0.5 - 2.0).

[0018] Further, in step (1), the heating reaction in the magnetic stirrer is carried out at 75 - 85 °C for 4.5 - 5.5 h.

[0019] Further, in step (1), the dropping of the ligand solution into the mixed solution means dropping the ligand solution into the mixed solution at a rate of 20 mL / h.

[0020] Fourthly, a preparation method of a zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite, which comprises the following steps:

[0021] (a) Synthesis of zirconium-coordinated porphyrin metal-organic framework: Weigh zirconium oxychloride octahydrate and benzoic acid in DMF, mix well to prepare a mixed solution; weigh meso-tetra(4-carboxyphenyl)porphyrin, dissolve it in DMF and sonicate it until there is no visible precipitate in the deep purple mixed solution to prepare a ligand solution; drop the ligand solution into the mixed solution, place it in a magnetic stirrer and heat it for reaction; after the reaction is completed, wait for the reaction solution to cool naturally, centrifuge to collect spherical crystals, wash them, and dry them under vacuum to obtain the zirconium-coordinated porphyrin metal-organic framework;

[0022] (b) Synthesis of zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite: Weigh the zirconium-coordinated porphyrin metal-organic framework obtained in step (a), disperse it in an ethanol solution and sonicate it. Then add Tris-HCl buffer solution to adjust the pH of the solution. Next, add dopamine hydrochloride to the solution, stir the reaction in the dark, then centrifuge to collect the solid, wash it, and finally redissolve it with deionized water and refrigerate it.

[0023] Furthermore, the mass ratio of zirconium-coordinated porphyrin metal-organic framework to dopamine hydrochloride is 2:(0.5 - 2.0), and the pH of the Tris-HCl solution is 7.0 - 8.5.

[0024] Furthermore, the addition of the ligand solution dropwise into the mixed solution means dropping the ligand solution into the mixed solution at a rate of 20 mL / h; the heating reaction in the magnetic stirrer is carried out at 75 - 85 °C for 4.5 - 5.5 h.

[0025] Furthermore, a preparation method of zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite specifically includes the following steps:

[0026] (a) Synthesis of zirconium-coordinated porphyrin metal-organic framework: Weigh 150 mg of zirconium oxychloride octahydrate and 1.4 g of benzoic acid in 30 mL of DMF, mix well for 10 min to prepare a mixed solution; then weigh 45 mg of meso-tetra(4-carboxyphenyl)porphyrin and dissolve it in 20 mL of DMF, sonicate for 30 min, and mix well for 30 s every 5 min until there is no visible precipitate in the deep purple mixed solution to prepare a ligand solution; the ligand solution is dropped into the mixed solution at a rate of 20 mL / h, and the reaction is carried out at 80 °C for 5 h in a heating magnetic stirrer; after the reaction solution naturally cools to room temperature, centrifuge to collect spherical crystals, then wash them 3 times with 48 mL of DMF and absolute ethanol respectively, and finally place the solid in a vacuum drying oven at 80 °C for vacuum drying for 5 h to obtain zirconium-coordinated porphyrin metal-organic framework;

[0027] (b) Synthesis of zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite: Weigh 24 mg of the dried zirconium-coordinated porphyrin metal-organic framework, disperse it in 24 mL of 75% ethanol solution, sonicate for 30 min, and mix well for 30 s every 5 min. Then add 24 mL of Tris-HCl buffer solution to adjust the pH of the solution. The concentration of the Tris-HCl buffer solution is 0.01 M and the pH is 7.0 - 8.5. Next, add 12 - 48 mg of dopamine hydrochloride to the solution, stir the reaction at 25 °C in the dark for 24 h; then centrifuge to collect the solid, wash it three times with 48 mL of absolute ethanol and deionized water respectively, and finally redissolve 12 mL with deionized water and place it in a 4 °C refrigerator for standby.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The detection method of the present invention not only has high sensitivity, but also improves the reproducibility of probe labeling and the coupling rate of antibodies. The immunochromatographic method established with zirconium-coordinated porphyrin metal-organic framework @ polydopamine as the signal amplification label in the present invention specifically uses a metal-organic framework polymer composite material as the labeling agent to establish an immunochromatographic method. Specifically, the quinone groups on the surface of the composite material can be covalently coupled with the amino groups of antibodies without activation, and no cross-linking agents such as EDC / NHS are required, which simplifies the labeling process and improves the reproducibility of probe labeling. Moreover, the direct coupling method overcomes the loss of antibodies caused by hydrolysis reactions that may occur when using cross-linking agents, resulting in a coupling rate of this material with antibodies > 90%, while the coupling rate of traditional colloidal gold with antibodies is < 90%. Further, by controlling the thickness of dopamine in the complex, the dispersibility and stability of the material are greatly improved, ultimately improving the detection sensitivity. The detection limit for sulfonylurea drugs is 0.22 - 4.27 μg / kg, and the sensitivity is 145 times that of the reported immunochromatographic methods currently.

[0030] (2) The dispersibility and stability of the labeling material are improved. In the present invention, by dripping the ligand at a constant speed, the crystal growth rate of zirconium-coordinated porphyrin metal-organic framework is effectively controlled. At the same time, the thickness of the polydopamine coating is effectively adjusted by controlling the amount of polydopamine used and the synthesis pH. Compared with the traditional one-pot synthesis method, the polydispersity coefficient of the material synthesized by this method is reduced from 0.148 to 0.024, thus effectively improving the dispersibility and stability of the labeling material. Description of the Drawings

[0031] Figure 1 A is a schematic diagram for the preparation of the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite probe of the present invention;

[0032] Figure 1 B and C are the detection principle diagrams for the chromatographic reaction of the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite probe of the present invention;

[0033] Figure 2A - K is the characterization of the preparation of the zirconium-coordinated porphyrin metal-organic framework @ polydopamine composite and probe in Example 1 of the present invention, Figure 2A 、 2D are the three-dimensional morphologies of PCN-224 and PCN-224@PDA characterized by scanning electron microscopy respectively; Figure 2B 、 2E are the particle sizes of the materials statistically measured by Nano Measurer through scanning electron microscopy, and finally the particle size distribution diagrams of PCN-224 and PCN-224@PDA are presented by Origin respectively; Figure 2C 、 2F are the material morphologies of PCN-224 and PCN-224@PDA characterized by transmission electron microscopy respectively and the elemental distribution in the materials analyzed by energy spectrum (EDS) in the transmission electron microscopy;Figure 2G Determining the crystal structure of the material by X-ray diffraction; Figure 2H Characterizing the types of surface elements of the material and semi-quantitatively determining the element content by X-ray photoelectron spectroscopy; Figure 2I Detecting the groups of the material by Fourier transform infrared spectrometer; Figure 2J Detecting the potential of the material by Zeta; Figure 2K Proving that the material has biocompatibility and can label proteins by polyacrylamide gel electrophoresis;

[0034] Figure 3a -e is the standard curve for detecting sulfonylurea drugs glipizide, glimepiride, glibenclamide, tolbutamide, and gliquidone in the immunochromatographic test strip prepared in the present invention;

[0035] Figure 4 It is a comparison of the performance of the immunochromatographic test strip after making a probe from the complex prepared under the condition of changing the pH of the Tris-HCl buffer solution (Tests 1-4) in the present invention;

[0036] Figure 5 It is a comparison of the Zeta potential of the complex prepared under the condition of changing the pH of the Tris-HCl buffer solution (Tests 1-4) in the present invention;

[0037] Figure 6 It is a comparison of the performance of the immunochromatographic test strip after making a probe from the complex prepared under the condition of changing the dosage of dopamine hydrochloride (Tests 5-8) in the present invention;

[0038] Figure 7 It is a comparison of the Zeta potential of the complex prepared under the condition of changing the dosage of dopamine hydrochloride (Tests 5-8) in the present invention;

[0039] Figure 8 It is to establish a standard curve for OD 450 and the concentration of sulfonylurea antibody by ELISA;

[0040] Figure 9 It is the antibody coupling rate diagram of colloidal gold and PCN-224@PDA at different sulfonylurea antibody concentrations; Detailed implementation manners

[0041] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0042] Unless otherwise specified, the meanings of the terms in this specification are the same as those generally understood by those skilled in the art, but in case of conflict, the definitions in this specification shall prevail.

[0043] In this text, "include", "comprise", "contain", "have" or other variants are intended to cover non-closed inclusion, and there is no distinction among these terms. The term "comprise" means that other steps and components can be added without affecting the final result. The term "comprise" also includes the terms "consist of" and "consist essentially of". The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional components, ingredients, steps, or limitations described herein.

[0044] All numerical values or expressions related to component amounts, process conditions, etc. used in the specification and claims should be understood to be modified by "about" in all cases. All ranges related to the same component or property include the endpoints, and these endpoints can be combined independently. Since these ranges are continuous, they include every numerical value between the minimum and maximum values. It should also be understood that any numerical range cited in this application is expected to include all sub-ranges within that range.

[0045] The embodiments of the present invention provide a method for quantitatively detecting sulfonylurea drugs, comprising the following steps:

[0046] (A) Sample pretreatment: Weigh the sample and place it in a centrifuge tube, add a methanol solution, mix well, and obtain the supernatant after centrifugation. Dilute the supernatant for subsequent measurement.

[0047] (B) Standard curve preparation: According to the treatment method in step (A), prepare methanol solutions containing sulfonylurea drugs with different concentrations in a sample known to be free of sulfonylurea drugs, and obtain standard extraction solutions after extraction and dilution; Testing: Add the standard extraction solutions into enzyme-labeled wells, and at the same time add a probe for incubation, then insert a test strip for chromatographic reaction. Repeat each concentration several times, and respectively read the gray scale values of the T and C lines. The ratio of the gray scale values of the T line to the C line corresponding to the added concentration of 0 μg / kg is the B0 value, and the ratio of the T / C gray scale values corresponding to other added concentrations is B X / B0 is used as the ordinate, and the added concentration of sulfonylurea drugs is used as the abscissa to plot a standard curve;

[0048] (C) Testing the sample: Treat the sample to be measured according to step (A) to obtain the test solution extraction solution, then perform detection according to the testing steps in step (B) to obtain the ratio of the gray scale values of the corresponding T and C lines, and then calculate the total content of sulfonylurea drugs in the sample according to the standard curve;

[0049] Wherein, the probe includes a zirconium-coordinated porphyrin metal-organic framework@polydopamine composite and an antibody, and the thickness of the dopamine shell layer in the zirconium-coordinated porphyrin metal-organic framework@polydopamine composite is 20 - 40 nm.

[0050] The immunochromatographic method established in the present invention uses zirconium-coordinated porphyrin metal-organic framework @ polydopamine as a signal method label. Specifically, a metal-organic framework polymer composite material is used as a marker to establish an immunochromatographic method. Specifically, the quinone groups on the surface of the composite material can be covalently coupled with the amino groups of antibodies without activation, simplifying the labeling process. Moreover, by controlling the thickness of dopamine in the complex, the dispersibility and stability of the material are greatly improved, and finally the detection sensitivity is enhanced. The detection limit for sulfonylurea drugs is 0.22 - 4.27 μg / kg, and the sensitivity is 145 times that of the currently reported immunochromatographic methods.

[0051] The embodiment of the present invention also provides a probe of zirconium-coordinated porphyrin metal-organic framework @ polydopamine, which comprises a combined zirconium-coordinated porphyrin metal-organic framework @ polydopamine complex and an antibody, wherein the thickness of the dopamine shell layer in the zirconium-coordinated porphyrin metal-organic framework @ polydopamine complex is 20 - 40 nm. The preparation method of the probe comprises the following steps:

[0052] (1) Synthesis of zirconium-coordinated porphyrin metal-organic framework: Weigh zirconium oxychloride octahydrate and benzoic acid and dissolve them in DMF, mix well to make a mixed solution; weigh meso-tetra(4-carboxyphenyl)porphyrin and dissolve it in DMF and sonicate until there is no visible precipitate in the deep purple mixed solution to make a ligand solution; drop the ligand solution into the mixed solution at a rate of 20 mL / h, place it in a constant-temperature magnetic stirrer and react at 75 - 85 °C for 4.5 - 5.5 h; after the reaction is completed, wait for the reaction solution to cool naturally, centrifuge to collect spherical crystals, wash, and dry in vacuum to obtain zirconium-coordinated porphyrin metal-organic framework;

[0053] (2) Synthesis of zirconium-coordinated porphyrin metal-organic framework @ polydopamine complex: Weigh the zirconium-coordinated porphyrin metal-organic framework obtained in step (1) and disperse it in 75% ethanol solution and sonicate, then add Tris-HCl buffer solution to adjust the pH of the solution, and then add dopamine hydrochloride to the solution, stir the reaction in the dark, then centrifuge to collect the solid, wash, and finally redissolve it with deionized water and refrigerate; the mass ratio of zirconium-coordinated porphyrin metal-organic framework to dopamine hydrochloride is 2:(0.5 - 2.0), and the pH of the Tris-HCl solution is 7.0 - 8.5;

[0054] (3) Synthesis of the probe: Centrifuge the reconstituted solution obtained in step (2), remove the supernatant, add borate buffer solution for reconstitution, sonicate and mix well. Then add an antibody to the liquid, mix well and incubate on a shaker to allow the antibody to fully bind to the zirconium-coordinated porphyrin metal-organic framework@polydopamine complex. The volume ratio of the zirconium-coordinated porphyrin metal-organic framework@polydopamine complex solution to the antibody is 1000:(0.5 - 2.0); then add bovine serum albumin solution to block the unbound sites and incubate on a shaker. Finally, centrifuge, discard the supernatant, resuspend the precipitate with phosphate buffer solution, sonicate and mix well to prepare the probe, and store it refrigerated for later use.

[0055] Taking the detection of sulfonylurea drugs as an example, the application method of the probe is further described. After adding the test detection solution to the probe, then insert the sulfonylurea drug test strip into the above-mentioned test detection solution for chromatographic detection.

[0056] The working principle of the test strip is as Figure 1 B and C. This test strip uses a competitive mode for detection. The test sample is treated with an extraction solution and diluted by a certain multiple to obtain a test solution. Take a certain amount of the test solution and put it into an enzyme immunoassay well, and at the same time add the probe for incubation. At this time, the probe binds to the drug in the test solution. Subsequently, insert the test strip. Under the action of capillary force, the liquid containing the probe will pass through the test line T and the quality control line C. The color development intensity of the test line depends on the accumulation of the probe. When the liquid migrates to the test line T, the probe specifically binds to the coating antigen and makes T turn brown. When the probe migrates to the quality control line C, it will bind to the goat anti-mouse IgG antibody and make the C line turn brown. When the concentration of sulfonylurea drugs in the reaction solution gradually increases, most of the probes have specifically bound to the sulfonylurea drugs, and the remaining small amount of probes bind to the coating antigen on the test line, so the test line gradually fades until it completely disappears. Regardless of the concentration of sulfonylurea drugs in the sample, the probe will bind to the quality control line C, making the quality control line always show brown. After the reaction, the test strip detects the ratio of the gray scale values of the T line and the C line through a reader and determines the concentration of sulfonylurea drugs according to the built-in standard curve.

[0057] The present invention will be described in detail below in conjunction with embodiments. The embodiments are only the preferred embodiments of the present invention and do not limit the present invention. Unless otherwise specified in this article, the solution concentrations are all volume concentrations.

[0058] Example 1

[0059] This example provides a preparation method of a zirconium-coordinated porphyrin metal-organic framework@polydopamine complex, which specifically includes the following steps:

[0060] (a) Synthesis of zirconium-coordinated porphyrin metal-organic framework (PCN-224): Weigh 150 mg of zirconium oxychloride octahydrate and 1.4 g of benzoic acid into 30 mL of DMF, mix well for 10 min to form a mixed solution. Then weigh 45 mg of meso-tetra(4-carboxyphenyl)porphyrin and dissolve it in 20 mL of DMF, sonicate for 30 min, and mix well for 30 s every 5 min until there is no visible precipitate in the dark purple mixed solution to form a ligand solution. The ligand solution is added dropwise into the mixed solution at a rate of 20 mL / h, and the reaction is carried out at 80 °C for 5 h in a constant-temperature magnetic stirrer. After the reaction solution is naturally cooled to room temperature, the spherical crystals are collected by centrifugation, then washed three times with 48 mL of DMF and absolute ethanol respectively, and finally the solid is placed in a vacuum drying oven at 80 °C for vacuum drying for 5 h to obtain zirconium-coordinated porphyrin metal-organic framework;

[0061] (b) Synthesis of zirconium-coordinated porphyrin metal-organic framework@polydopamine composite (PCN-224@PDA): Weigh 24 mg of dry zirconium-coordinated porphyrin metal-organic framework and disperse it in 24 mL of 75% ethanol solution, sonicate for 30 min, and mix well for 30 s every 5 min. Then add 24 mL of Tris-HCl buffer solution to adjust the pH. The concentration of Tris-HCl buffer solution is 0.01 M and the pH is 7.5. Then add 12 mg of dopamine hydrochloride to the solution, and stir and react in the dark at 25 °C for 24 h. Then centrifuge to collect the solid, wash it three times with 48 mL of absolute ethanol and deionized water respectively, and finally redissolve 12 mL with deionized water and store it in a refrigerator at 4 °C for later use.

[0062] The characterization of zirconium-coordinated porphyrin metal-organic framework@polydopamine composite is as follows:

[0063] (i) Scanning electron microscope and transmission electron microscope: As shown in Figure 2A 、D, PCN-224 presents a spherical morphology, and PCN-224@PDA shows a rough and irregular morphology. Figure 2B 、E, by Nano Measurer statistics, the size of PCN-224 is about 100 nm, and the size of PCN-224@PDA is about 120 - 140 nm. Figure 2C 、F, through transmission electron microscopy and EDX energy spectrum analysis, it shows that elements such as Zr (red), C (green), N (yellow) and O (blue) are evenly distributed in PCN-224 and PCN-224@PDA, indicating the successful synthesis of PCN-224@PDA composite material.

[0064] (ii) X-ray diffraction: As shown in the Figure 2G X-ray diffraction pattern, the crystal forms of PCN-224 and PCN-224@PDA are similar, proving that the dopamine coating layer does not affect the morphology (crystal crystallinity).

[0065] (iii) X-ray photoelectron spectroscopy: As Figure 2H shown, the peak of Zr3d at 182.9 eV in PCN-224 is weakened in the spectrum of PCN-224@PDA, which further proves the successful preparation of PCN-224@PDA with a dopamine-coated shell.

[0066] (iv) Infrared characterization: As Figure 2I shown, the characteristic bands of PCN-224 (1708, 1602, 1415, 1178, 869, 800, 769, and 721 cm -1 ) refer to the fingerprint peaks of C-N groups and Zr. In addition, in the infrared data of PCN-224@PDA, the peak at 1514 cm -1 is the N-H vibration peak of the indole ring in polydopamine, and the fingerprint peak of Zr is weakened.

[0067] (v) Potential characterization: As Figure 2J shown, the potential of PCN-224 is +34 mV, and it becomes -31 mV after coating with dopamine, indicating that the coating changes the surface charge distribution of the material.

[0068] Example 2

[0069] This example provides a method for preparing a probe of a zirconium-coordinated porphyrin metal-organic framework@polydopamine complex, which includes the following steps: Centrifuge 1 mL of the zirconium-coordinated porphyrin metal-organic framework@polydopamine complex (PCN-224@PDA) re-solution obtained in Example 1, remove the supernatant, add 1 mL of 0.05 M pH 8.4 borate buffer solution for re-dissolution, sonicate for 30 s, mix well for 30 s, then add 2 μL of 5 mg / mL sulfonylurea broad-spectrum antibody to the liquid. After that, place the mixture on a shaker (25 °C, 250 rpm / min) and incubate for 1 h to allow the sulfonylurea broad-spectrum antibody to fully bind to the PCN-224@PDA material. Then add 50 μL of 10% bovine serum albumin solution to block the unbound sites and incubate on a shaker for 1 h. Finally, centrifuge (4 °C, 10000 g, 5 min), discard the supernatant, resuspend the precipitate with 200 μL of 0.02 M pH 7.4 phosphate buffer solution, sonicate for 5 s, and mix well for 10 s to prepare the PCN-224@PDA-Abs probe, which is stored in a 4 °C refrigerator for later use.

[0070] Characterize the probe as follows:

[0071] (i) Potential characterization: As Figure 2J shown, the potential becomes -26 mV after labeling with the antibody, indicating that labeling with the antibody changes the surface charge of PCN-224@PDA.

[0072] (ii) Electrophoresis characterization: AsFigure 2K As shown, through polyacrylamide gel electrophoresis, for BSA, a colored band appears at a molecular weight of approximately 60 kDa. When BSA reacts with PCN-224@PDA, a corresponding colored band also appears at 60 kDa. However, under the same conditions, pure PCN-224@PDA does not show any bands, indicating that PCN-224@PDA can successfully label proteins.

[0073] Example 3

[0074] This example provides a method for preparing an immunochromatographic test strip for rapid detection of sulfonylurea drugs, including the following steps:

[0075] (1) Preparation of the nitrocellulose membrane: Dissolve the sulfonylurea drug-coated antigen in 0.01 M pH 9.6 carbonate buffer solution to prepare an antigen-coated solution with a concentration of 0.5 mg / mL. Dissolve the goat anti-mouse antibody in 0.02 M pH 7.4 phosphate buffer solution to prepare a secondary antibody-coated solution with a concentration of 0.2 mg / mL. Spray the antigen-coated solution and the secondary antibody-coated solution onto the nitrocellulose membrane CN 140 at a speed of 0.8 μL / cm to form a test line T and a control line C. The distance between the test line T and the control line C is 5 mm. Mark it and dry the membrane in an oven at 37°C for 8 h. Finally, place it in a self-sealing bag filled with desiccant and store it at room temperature for later use.

[0076] (2) Preparation of the sample pad and the absorbent pad: Immerse the cut sample pad SB08 in a solvent containing 0.75% Tween-20, 0.3% polyvinylpyrrolidone, 0.5% bovine serum albumin, and 0.03% Proclin 300 for 1 min until fully submerged. Finally, dry it in an oven at 37°C for 8 h. The solvent is phosphate buffer solution, which is obtained by adding 81 mL of 0.2 mol / L Na2HPO4 and 19 mL of 0.2 mol / L NaH2PO4 and diluting to 1000 mL with deionized water. Cut the absorbent pad into a size of 300 mm in length and 24 mm in width, dry it in an oven at 60°C for 2 h, place it in a self-sealing bag filled with desiccant, and store it at room temperature for later use.

[0077] (3) Assembly of the test strip: The test strip contains a PVP bottom plate, a nitrocellulose membrane, a sample pad, and an absorbent pad. First, stick the nitrocellulose membrane to the PVP bottom plate, then stick the treated sample pad near the test line end and the treated absorbent pad near the control line end. Both pads overlap the nitrocellulose membrane by 1 - 2 mm. Finally, cut the assembled test strip into a size of 60 mm in length and 3.05 mm in width using a strip cutter to obtain an immunochromatographic test strip for rapid detection of sulfonylurea drugs.

[0078] Example 4

[0079] This embodiment provides a method for on-site quantitative detection of sulfonylurea drugs (taking glipizide as an example) in hypoglycemic health care products (tea leaves), including the following steps:

[0080] (A) Sample pretreatment: Weigh 1 g of tea leaf sample and place it in a 10 mL centrifuge tube. Add 1 mL of methanol solution, shake for 5 min, then put it into a centrifuge (10000 g, 10 min). After centrifugation, obtain the supernatant. Dilute the supernatant 5 times with 0.02 M phosphate buffer solution at pH 7.4 for further testing.

[0081] (B) Standard curve preparation: According to the treatment method in step (A), prepare 1 mL of methanol solution containing sulfonylurea drug glipizide with different concentrations in 1 g of sample known to be free of sulfonylurea drugs. After extraction and dilution, obtain the standard extraction solution. Test: Add the standard extraction solution into the enzyme-linked immunosorbent assay (ELISA) wells, and at the same time add 1.7 μL of the probe prepared in Example 2 and let it stand for reaction for 3 min. Then insert the test strip prepared in Example 3 and chromatograph for 7 min. Repeat each concentration 3 times. The microplate reader (model: FIC-Q1, which can read visible light signals) reads the gray scale values of the T and C lines respectively. The ratio of the gray scale values of the T line to the C line corresponding to the added concentration of 0 μg / kg is the B0 value, and the ratio of the T / C gray scale values corresponding to other added concentrations is Bx. Taking B X / B0 as the ordinate and the added concentration of sulfonylurea drugs as the abscissa, draw the standard curve as Figure 3a , and finally obtain the standard curve of glipizide.

[0082] The method for preparing the standard curves of other sulfonylurea drugs such as glimepiride, glibenclamide, tolbutamide, and gliquidone is the same as that of glipizide, as shown in Figure 3b -e, which will not be elaborated here. Finally, the limits of detection (LOD) of glipizide, glimepiride, glibenclamide, tolbutamide, and gliquidone are 0.22 μg / kg, 0.58 μg / kg, 1.24 μg / kg, 3.72 μg / kg, and 4.27 μg / kg respectively, and the linear ranges are 0.75 - 44.78 μg / kg, 1.61 - 50.79 μg / kg, 3.08 - 68.25 μg / kg, 8.19 - 121.32 μg / kg, and 10.31 - 209.53 μg / kg respectively.

[0083] (C) Test sample: Treat the test sample according to step (A) to obtain the test solution extract, and then perform the test according to step (B) to obtain the corresponding signal intensity. According to the standard curve already entered in the instrument, the total content of sulfonylurea drugs in the hypoglycemic health care product (tea leaves) (converted with glipizide) can be calculated, realizing the rapid quantitative detection of the sample.

[0084] Comparative experiment

[0085] 1. Based on Example 1, only the pH of the Tris-HCl buffer in step b was changed. After corresponding probes were prepared, they were applied to Example 4 (detecting glipizide) for immunochromatographic testing. The thickness of the dopamine shell, the polymer dispersity index (PDI), and the inhibition rate affecting the performance of the test strip prepared at different pH values of the Tris-HCl buffer are shown in Table 1 below. The comparative graph showing the influence on the performance of the test strip is as shown in Figure 4 shown.

[0086] Table 1 Thickness of dopamine shell, PDI, and inhibition rate of immunochromatographic test at different pH values of Tris-HCl buffer

[0087]

[0088] From Table 1 and Figure 4 it can be seen that the thickness of the dopamine shell can be controlled by changing the pH of the Tris-HCl buffer. When the thickness of the dopamine shell is less than 20 nm (such as in Experiment 1), the color development of the test strip is light and it is difficult to determine the result. When it is higher than 40 nm (such as in Experiments 3 and 4), although the color development is deep, the inhibition rate is significantly reduced and the cut-off value also increases accordingly.

[0089] 2. Based on Example 1, only the amount of dopamine hydrochloride added was changed. After corresponding probes were prepared, they were applied to Example 4 (detecting glipizide) for immunochromatographic testing. The thickness of the dopamine shell, the PDI, and the inhibition rate affecting the performance of the test strip prepared with different amounts of dopamine hydrochloride added are shown in Table 2 below. The comparative graph showing the influence on the performance of the test strip is as shown in Figure 5 shown.

[0090] Table 2 Thickness of dopamine shell, PDI, and inhibition rate of immunochromatographic test at different amounts of dopamine hydrochloride added

[0091]

[0092] From Table 2 and Figure 5 it can be seen that the thickness of the dopamine shell can be controlled by changing the amount of dopamine hydrochloride added. When the thickness of the dopamine shell is less than 20 nm (such as in Experiment 5), the color development of the test strip is light and it is difficult to determine the result. When it is higher than 40 nm (such as in Experiments 7 and 8), although the color development is deep, the inhibition rate is significantly reduced and the cut-off value also increases accordingly.

[0093] III. Based on Example 1, under the condition of only changing the synthesis method of zirconium-coordinated porphyrin metal-organic framework (PCN-224), after being made into a probe, it was applied to Example 4 (detecting glipizide) for immunochromatographic testing. The particle size, PDI, yield of the complex, and the cut-off value of the immunochromatographic test prepared by different synthesis methods of zirconium-coordinated porphyrin metal-organic framework (PCN-224) are shown in Table 3.

[0094] Table 3 Particle size, PDI, and yield of the complex under different synthesis methods of zirconium-coordinated porphyrin metal-organic framework (PCN-224)

[0095]

[0096] Zr6(BA) 12 +1.5TCPP → Zr6(TCPP) 1.5 +12BA The yield is calculated according to the formula, m 实际 / m 理论 , where m is the dry weight of Zr6(TCPP). 1.5 Dry weight.

[0097] The hydrothermal method is specifically as follows: Weigh 150 mg of zirconium oxychloride octahydrate, 45 mg of meso-tetra(4-carboxyphenyl)porphyrin, and 1.4 g of benzoic acid in 30 mL of DMF, mix well and sonicate for 30 min, mix well for 30 s every 5 min of sonication, add the solution to a high-pressure reaction kettle, and prepare using the hydrothermal method. The reaction temperature of the hydrothermal method is 120 °C, and the reaction time is 24 h.

[0098] PCN-224 synthesized by two different methods was coated with dopamine and conjugated with antibodies to form a probe. The probe was used to detect glipizide drug diluted with phosphate buffer. The cut-off value obtained from the material formed in Example 1 of the present invention was 45 μg / kg, while the cut-off value obtained from the material formed by the hydrothermal method was 140 μg / kg. The probe based on Example 1 has higher performance and obvious advantages in terms of synthesis time and PDI.

[0099] IV. Based on Example 1, the coupling rate of colloidal gold and PCN-224@PDA antibody was compared. The specific steps were as follows: Prepare sulfonylurea antibody solutions with concentrations of 0.125, 0.25, 0.5, 1, and 2 μg / mL. Take 100 μL of each solution and add it to an enzyme-labeled well, and place it in a 37 °C water bath for 12 h. Then wash 2 times, pat dry, and add 120 μL of 6% fish gelatin and incubate at 37 °C for 3 h. After incubation, discard the liquid by centrifugation and dry it at 37 °C for 30 min. Then add 100 μL of enzyme-labeled secondary antibody with a concentration of 0.5 μg / mL and incubate at 37 °C for 30 min, and wash 5 times. Add 100 μL of TMB chromogenic solution and incubate at 37 °C for 10 min. Finally, add 50 μL of termination solution, OD 450Read the values and establish an antibody concentration calibration curve, such as Figure 8 .

[0100] Take 0.4 mg of colloidal gold or PCN-224@PDA label and add it to sulfonylurea antibodies with different concentrations of 0.75, 1, 1.25, 1.5, and 1.75 μg / mL for labeling. After reacting for 1 h, centrifuge to obtain the supernatant. Take 100 μL of the unbound antibody supernatant solution and add it to the enzyme-labeled wells. Repeat the above experiment. Finally, substitute the obtained OD 450 value into the antibody calibration curve to calculate the concentration of unbound antibody in the supernatant, and calculate the coupling rate as Figure 9 . The coupling rate calculation formula is (known added antibody concentration - unbound antibody concentration) / known added antibody concentration. It can be seen from the figure that the coupling rate of the PCN-224@PDA probe is always greater than that of the colloidal gold probe, which is attributed to the fact that the surface of PCN-224@PDA is rich in quinone groups and can bind to more antibodies, being superior to the colloidal gold labeling material.

[0101] The above-described embodiments only represent the implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. Any technical solutions obtained by means of equivalent substitution or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A method for quantitatively detecting sulfonylurea drugs, comprising the following steps: (A) Sample pretreatment; (B) Standard curve preparation: Add the standard extraction solution into the ELISA wells, add the probe at the same time for incubation, and then insert the test strip for chromatographic reaction; Use BX / B0 as the ordinate and the sulfonylurea drug addition concentration as the abscissa to plot the standard curve; (C) Test sample: Detect the ratio of the gray values of the corresponding T line and C line, and then calculate the total content of sulfonylurea drugs in the sample according to the standard curve; Among them, the probe includes a zirconium-coordinated porphyrin metal-organic framework@polydopamine composite and an antibody. The thickness of the dopamine shell layer in the composite is 25-35 nm; The size of the composite is 120-140 nm, and the preparation method of the composite is as follows: (a) Synthesis of zirconium-coordinated porphyrin metal-organic framework: Weigh 150 mg of zirconium oxychloride octahydrate and 1.4 g of benzoic acid in 30 mL of DMF, mix well for 10 min to make a mixed solution; Then weigh 45 mg of meso-tetrakis(4-carboxyphenyl)porphyrin and dissolve it in 20 mL of DMF, sonicate for 30 min, and mix well for 30 s every 5 min until there is no visible precipitate in the deep purple mixed solution to make a ligand solution; The ligand solution is added dropwise into the mixed solution at a rate of 20 mL / h, and the reaction is carried out at 80 °C for 5 h in a constant temperature magnetic stirrer; After the reaction solution naturally cools to room temperature, centrifuge to collect spherical crystals, and then wash 3 times with 48 mL of DMF and absolute ethanol respectively. Finally, place the solid in a vacuum drying oven at 80 °C for vacuum drying for 5 h to obtain the zirconium-coordinated porphyrin metal-organic framework; (b) Synthesis of zirconium-coordinated porphyrin metal-organic framework@polydopamine composite: Weigh 24 mg of the dried zirconium-coordinated porphyrin metal-organic framework and disperse it in 24 mL of 75% ethanol solution, sonicate for 30 min, and mix well for 30 s every 5 min. Then add 24 mL of Tris-HCl buffer solution to adjust the pH. The concentration of the Tris-HCl buffer solution is 0.01 M and the pH is 7.

5. Then add 12 mg of dopamine hydrochloride to the solution, and stir and react at 25 °C in the dark for 24 h; Then centrifuge to collect the solid, wash it three times with 48 mL of absolute ethanol and deionized water respectively, and finally redissolve 12 mL with deionized water and store it in a 4 °C refrigerator for standby.

2. A method for preparing an immunochromatographic probe for detecting sulfonylurea drugs, characterized in that It includes the following steps: (1) Synthesis of zirconium-coordinated porphyrin metal-organic framework: Weigh 150 mg of zirconium oxychloride octahydrate and 1.4 g of benzoic acid in 30 mL of DMF, mix well for 10 min to make a mixed solution; Then weigh 45 mg of meso-tetrakis(4-carboxyphenyl)porphyrin and dissolve it in 20 mL of DMF, sonicate for 30 min, and mix well for 30 s every 5 min until there is no visible precipitate in the deep purple mixed solution to make a ligand solution; The ligand solution is added dropwise into the mixed solution at a rate of 20 mL / h, and the reaction is carried out at 80 °C for 5 h in a constant temperature magnetic stirrer; After the reaction solution naturally cools to room temperature, centrifuge to collect spherical crystals, and then wash 3 times with 48 mL of DMF and absolute ethanol respectively. Finally, place the solid in a vacuum drying oven at 80 °C for vacuum drying for 5 h to obtain the zirconium-coordinated porphyrin metal-organic framework; (2) Synthesis of zirconium-coordinated porphyrin metal-organic framework@polydopamine composite: Weigh 24 mg of dry zirconium-coordinated porphyrin metal-organic framework and disperse it in 24 mL of 75% ethanol solution. Sonicate for 30 min, mix well for 30 s every 5 min of sonication, then add 24 mL of Tris-HCl buffer solution to adjust the pH. The concentration of Tris-HCl buffer solution is 0.01 M and the pH is 7.

5. Then add 12 mg of dopamine hydrochloride to the solution and stir and react at 25 °C in the dark for 24 h; then centrifuge to collect the solid, wash it three times with 48 mL of absolute ethanol and deionized water respectively, and finally redissolve it in 12 mL of deionized water and store it in a refrigerator at 4 °C for later use; the thickness of the dopamine shell in the composite is 25 - 35 nm; the size of the composite is 120 - 140 nm; (3) Synthesis of the probe: Centrifuge the solution obtained by redissolving in step (2), remove the supernatant, add borate buffer solution to redissolve, sonicate and mix well, then add an antibody to the liquid and incubate it on a shaker to allow the antibody to fully bind to the zirconium-coordinated porphyrin metal-organic framework@polydopamine composite; then add bovine serum albumin solution to block the excess active sites in the polydopamine coating and incubate it on a shaker. Finally, centrifuge, discard the supernatant, resuspend the precipitate with phosphate buffer solution, sonicate and mix well to prepare the probe, and store it refrigerated for later use.

3. The preparation method according to claim 2, characterized in that In the step (3), the volume ratio of the zirconium-coordinated porphyrin metal-organic framework@polydopamine composite solution to the antibody is 1000:(0.5 - 2.0).

4. An immunochromatographic probe for detecting sulfonylurea drugs, characterized in that Prepared according to the method described in claim 2 or 3.

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