Immunochromatographic test strip and preparation method and detection method thereof

Abstract

The present invention discloses an immunochromatographic test strip and a preparation method and a detection method thereof. The immunochromatographic test strip uses UiO‑66@PDA as an immune marker. The filter paper, the sample pad, the glass fiber pad sprayed with UiO‑66@PDA@Ab, the nitrocellulose membrane sprayed with an artificial coupled antigen of the test object or the antibody of the test object as the detection line and sprayed with anti-mouse antibody or anti-rabbit antibody as the secondary antibody as the quality control line, and the absorbent paper are overlapped and pasted on the bottom plate in sequence for assembly. During the detection, quantitative detection and qualitative judgment are achieved by reading the signal data of the detection line and the quality control line on the immunochromatographic test strip or by direct observation. The immunochromatographic test strip prepared by the present invention uses UiO‑66@PDA as an immune marker for the first time, and its light absorption performance is excellent. Compared with the conventional colloidal gold as an immune marker, the coupled antibody effect has a higher detection sensitivity of the obtained immunochromatographic test strip.

Description

Technical Field

[0001] The invention belongs to the field of food safety detection and medical inspection, and specifically relates to an immunochromatographic test strip and a preparation method and a detection method thereof. Background Art

[0002] Immunochromatography is a detection method based on antigen-antibody specific reaction. It has the advantages of fast detection speed, good specificity, simple operation and low cost. Compared with other methods, it can better meet the needs of large-scale on-site detection. Therefore, it has developed rapidly in recent years and is widely used in food safety, medical testing and environmental pollutant monitoring. Among them, colloidal gold immunochromatography test strips are the most widely used immunochromatography products, but they have poor sensitivity and are easily interfered by the matrix.

[0003] Metal-organic framework materials (MOF) are a class of multifunctional porous materials formed by the interaction of various metal ions and organic ligands. As a new type of material, the composition and structure of metal-organic framework materials can be adjusted by adjusting the designed organic and inorganic units or changing the synthesis conditions, showing ultra-high specific surface area and porosity as well as high strength and ideal chemical activity. According to the different structural characteristics of MOF, the most representative ones are ZIF, UiO, MIL and PCN. Among them, the typical UiO series MOFs (UiO-66, UiO-67 and UiO-68) materials were first reported and synthesized by Lillerud et al. in 2008. UiO-66 is a porous material composed of clusters (Zr6O4(OH)4) coordinated and self-assembled with 12 bridging ligands to form a three-dimensional structure in which each octahedral central hole cage is surrounded by eight tetrahedral corner cages in its network structure.

[0004] Dopamine (DA) is an important neurotransmitter. In alkaline solution, it can self-polymerize to form polydopamine (PDA) under the oxidation of oxygen. Due to the rich surface functional groups (such as amine, hydroxide, catechol, etc.) on PDA, PDA has good biocompatibility and adhesion to proteins, making it suitable as an excellent surface coating material. At present, there are no reports on the application of UiO-66@PDA material on immunochromatographic test strips. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide an immunochromatographic test strip, which specifically adopts the following technical solutions:

[0006] An immunochromatographic test strip, wherein UiO-66@PDA is used as an immune marker.

[0007] The preparation process of UiO-66@PDA is as follows: UiO-66-NH2 is prepared by hydrothermal method, and then UiO-66@PDA is prepared by self-polymerization and oxidation of dopamine under alkaline conditions. The inventors used UiO-66-NH2 as the base material and coated a layer of polydopamine on UiO-66-NH2, which not only improved the light absorption capacity of UiO-66-NH2, but also improved the efficiency of UiO-66-NH2 coupling antibodies, so that the UiO-66@PDA labeled material showed a higher sensitivity on the immunochromatographic test strip.

[0008] Preferably, the above-mentioned immunochromatographic test strip includes a base plate, and filter paper, a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are overlapped and pasted in sequence on the base plate; the glass fiber pad is sprayed with UiO-66@PDA@Ab, and the UiO-66@PDA@Ab is prepared from the UiO-66@PDA and the antibody to be tested.

[0009] Preferably, the preparation process of the UiO-66@PDA@Ab is as follows: adding the antibody to be tested to the UiO-66@PDA, mixing and incubating for 1 hour, adding a blocking agent, incubating for 1 hour, centrifuging to obtain a precipitate, and re-dissolving to obtain the UiO-66@PDA@Ab.

[0010] The final concentration of the antibody to be detected after adding the antibody to be detected is 1-100 μg / mL, the final concentration of the blocking agent after adding the blocking agent is 0.1-1%, and the blocking agent is any one of casein, bovine serum albumin, ovalbumin, polyethylene glycol, and skim milk. The precipitate obtained after centrifugation is re-dissolved in a phosphate buffer of 0.01-0.1M, pH=6-9.

[0011] Preferably, the antibody to be detected is a monoclonal antibody, a polyclonal antibody, a nanobody or a phage-expressed antibody.

[0012] Preferably, the nitrocellulose membrane is coated with an artificially coupled antigen of the object to be detected or an antibody of the object to be detected as a detection line, and is coated with an anti-mouse antibody or an anti-rabbit antibody as a secondary antibody as a quality control line.

[0013] Among them, the artificially coupled antigen of the analyte refers to a complete antigen with immunogenicity and reactogenicity prepared by a chemical coupling method of a small molecule analyte and a macromolecular protein; wherein the small molecule analyte covers all small molecules that need to be detected in the field of food safety testing and medical testing; the coupling methods include diazo method, carbodiimide method, glutaraldehyde method, mixed acid anhydride method, and succinic anhydride method; the coupled macromolecular proteins include bovine serum albumin, casein, ovalbumin, and keyhole limpet hemocyanin; the coupling ratio is 1:5-1:200, and the coupling is followed by dialysis purification to obtain the desired artificially coupled antigen.

[0014] The preparation method of the nitrocellulose membrane is as follows:

[0015] (1) using 0.01-0.5M, pH=6.0-8.0 PBS (phosphate buffer solution) solution to adjust the coating (artificially coupled antigen to be detected or antibody to be detected) and the secondary antibody (anti-mouse antibody or anti-rabbit antibody) to a concentration of 0.01-10.0 mg / mL; wherein the antibody to be detected is a monoclonal antibody, a polyclonal antibody, a nanobody or a phage-expressed antibody;

[0016] (2) Spraying the artificially coupled antigen or antibody of the test object after adjusting the concentration onto the upper part of the nitrocellulose membrane as the test line, and spraying the anti-mouse antibody or anti-rabbit antibody onto the lower part of the nitrocellulose membrane as the quality control line; wherein a certain distance is set between the test line and the quality control line, and the spraying amount of both is 0.25-0.74 μL / cm;

[0017] (3) The nitrocellulose membrane sprayed with the test line and the quality control line is dried at 37°C overnight and then stored in a dry environment at room temperature for later use.

[0018] The present invention also provides a method for preparing the above-mentioned immunochromatographic test strip, comprising the following steps: overlapping and pasting filter paper, a sample pad, a glass fiber pad sprayed with UiO-66@PDA@Ab, a nitrocellulose membrane sprayed with an artificially coupled antigen of a test object or an antibody of a test object as a detection line and sprayed with an anti-mouse antibody or an anti-rabbit antibody as a secondary antibody as a quality control line, and absorbent paper on a bottom plate in sequence, and the assembly is completed.

[0019] The assembled test strip plate can be cut into required widths by a cutter. The test strip can be used directly or put into a plastic card case for use.

[0020] The present invention also provides a detection method for the above-mentioned immunochromatographic test strip, comprising the following steps: adding a sample to be tested to the immunochromatographic test strip, with a sample volume of 50-200 μL and a reaction time of 3-30 min; reading the signal data of the detection line and the quality control line on the immunochromatographic test strip, and calculating the concentration of the sample to be tested with a built-in standard curve to achieve quantitative detection; and observing whether the detection line and the quality control line are dark brown by using the immunochromatographic test strip to achieve qualitative judgment.

[0021] The beneficial effects of the present invention are as follows: the immunochromatographic test strip prepared by the present invention uses UiO-66@PDA as an immune marker for the first time, has excellent light absorption performance, and has a good coupling antibody effect. Compared with the traditional colloidal gold test strip (5ng / mL), its sensitivity is increased by 5 times (1ng / mL). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Shown is a schematic diagram of the structure of an immunochromatographic test strip;

[0023] Figure 2 Shown is the principle diagram of the competitive UiO-66@PDA immunochromatographic test strip detection;

[0024] Figure 3 Shown are the UV absorption graphs and physical images of UiO-66-NH2 and UiO-66@PDA;

[0025] Figure 4 The results of the UiO-66@PDA immunochromatographic test strip test are shown;

[0026] Figure 5 Shown is the result of Au NPs immunochromatographic test strip detection. DETAILED DESCRIPTION

[0027] The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0028] Example 1: Preparation of UiO-66@PDA

[0029] In a 100mL single-necked flask, 80mg ZrCl4, 420mg benzoic acid and 60mg 2-aminoterephthalic acid were added, followed by 20mL DMF, ultrasonication until completely dissolved, and then a reflux device was set up to allow the reaction solution to be magnetically stirred for 24h at 120℃. After the reaction was complete, DMF and ultrapure water were centrifuged and washed three times, and the precipitate was re-dissolved with ultrapure water; the preparation of UiO-66@PDA was to modify a layer of polydopamine on the basis of UiO-66-NH2. The specific process was as follows: 0.5mL of UiO-66-NH2 was added to 35mL of water: ethanol system (water: ethanol = 4:3, volume ratio), magnetic stirring was performed evenly, and then 8mg DA·HCl and 25mL 0.01M Tris-HCl (pH = 8.5) were added, and the reaction was left to react overnight in the dark for 12h. After the reaction is complete, wash the mixture three times with ultrapure water by centrifugation, re-dissolve the precipitate with ultrapure water to 5 mL, and store it at 4°C for later use.

[0030] Among them, the UV absorption graphs and physical pictures of UiO-66-NH2 and UiO-66@PDA are shown in Figure 3 shown.

[0031] Example 2: Preparation of a competitive immunochromatographic test strip for detecting enrofloxacin using UiO-66@PDA as an immune marker

[0032] 1. Preparation process of immunochromatographic test strips

[0033] 1. Preparation of Nitrocellulose Membrane

[0034] (1) Preparation of Enrofloxacin Artificial Antigen (ENR-BSA):

[0035] The coupling method is the carbodiimide method, the coupling protein is bovine serum albumin (BSA), the coupling ratio is 1:60, and after coupling, it is dialyzed and purified to obtain ENR-BSA.

[0036] (2) Preparation of test lines and quality control lines

[0037] ENR-BSA conjugate and anti-mouse antibody were coated on nitrocellulose membrane: ENR-BSA was diluted to a concentration of 1 mg / mL with 0.01 M PBS (phosphate buffer) at pH 7.5, and the resulting solution was sprayed on the membrane as a test line; the anti-mouse antibody was diluted to a concentration of 0.5 mg / mL, and the resulting solution was sprayed on the membrane as a quality control line. The spray volume of both lines was 0.74 μL / cm, the test line was 10 mm away from the top edge of the membrane, and the interval between the two lines was 5 mm. Dry at 37°C for 12 h and store in a drying cabinet for later use.

[0038] 2. Preparation of UiO-66@PDA@Ab glass fiber mat

[0039] (1) Preparation of UiO-66@PDA labeled ENR monoclonal antibody: 5 μg ENR monoclonal antibody was added to the prepared UiO-66@PDA, mixed and incubated for 1 h, then 10% BSA blocking agent was added and incubated at room temperature for 1 h. The precipitate was centrifuged and the obtained precipitate was re-dissolved in 0.01 M PBS, pH = 7.0 to 1 / 10 of the initial volume to prepare UiO-66@PDA@Ab, which was sprayed onto a glass fiber mat at 3 μL / cm and vacuum dried for 2 h.

[0040] (2) Adjust the standard curve: Add the negative matrix to the standard curve with ENR concentrations of 0, 0.05, 0.1, 0.25, 0.5, and 1 ppb. The results are as follows: Figure 4 As shown by Figure 4 It can be seen that as the concentration of the substance to be detected increases, the T line signal value gradually decreases. Calculate R 2 The linear regression equation is: y = -1444.91 log x + 1120.89. Its sensitivity is 1 ng / mL.

[0041] 3. Assemble the test strips

[0042] (1) The sample pad size is 1×30cm;

[0043] (2) Glass fiber mat sprayed with UiO-66@PDA@Ab, with a size of 0.8 × 30 cm;

[0044] (3) Nitrocellulose membrane sprayed with test lines and quality control lines, with specifications of 2.5×30cm;

[0045] (4) Absorbent paper, size: 1.5 × 30 cm;

[0046] (5) PVC baseboard, specification: 5.5×30cm.

[0047] Discard the above materials as follows Figure 1 The components in the test strip structure diagram shown are pasted in sequence (including 1 sample pad, 2 glass fiber pad, 3 detection line, 4 quality control line, 5 nitrocellulose membrane, 6 absorbent paper and 7 PVC bottom plate). After assembly, cut into 4×55mm test strips with a cutter, put them into a plastic card shell, press them into an aluminum foil bag, add desiccant, and seal them for storage. The shelf life at room temperature is 12 months.

[0048] 2. Quantitative detection of ENR in samples

[0049] like Figure 2 As shown, the detection principle is as follows: after the sample to be tested is added to the sample well of the test strip and the reaction is completed, if the sample is negative, the sample is chromatographed along the chromatographic direction, and the UiO-66@PDA@Ab on the binding pad surges to the detection line position, and an immunological reaction based on antigen-antibody binding occurs with the artificially coupled antigen on the detection line to form an antigen-antibody complex, and the UiO-66@PDA@Ab gathers on the detection line and shows a dark brown color, and some UiO-66@PDA@Ab that is not bound to the artificially coupled antigen surges to the quality control line position, and binds to the corresponding anti-antibody so that the quality control line also shows a dark brown color. If the sample is positive, the analyte in the sample first binds to the UiO-66@PDA@Ab, and the UiO-66@PDA@Ab that binds to the analyte can no longer bind to the artificially coupled antigen of the analyte on the detection line, so the color of the detection line becomes lighter or even disappears, and the principle of the quality control line is the same as that of the negative sample.

[0050] The method for detecting ENR in a sample using the above-mentioned immunochromatographic test strip comprises the following steps:

[0051] 1. Add 100 μL of milk sample to the sample well of the test strip and react for 15 minutes;

[0052] 2. Insert the test strip into the detection window of the reader. The strength of the test line and quality control line signals will be displayed on the display as high and low values. According to the standard curve already entered in the instrument, the ENR content in the sample can be calculated to achieve quantitative detection of ENR in the sample.

[0053] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. An immunochromatographic test strip, characterized in that: The immunochromatographic test strip uses UiO-66@PDA as an immune marker; It comprises a bottom plate, and filter paper, a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are overlapped and pasted on the bottom plate in sequence; the glass fiber pad is sprayed with UiO-66@PDA@Ab, and the UiO-66@PDA@Ab is prepared from the UiO-66@PDA and an antibody to be detected; The preparation process of the UiO-66@PDA@Ab is as follows: adding the antibody to be tested to the UiO-66@PDA, mixing and incubating for 1 h, adding a blocking agent, incubating for 1 h, centrifuging to obtain a precipitate, and re-dissolving to obtain the UiO-66@PDA@Ab; The preparation process of UiO-66@PDA is as follows: UiO-66-NH2 is prepared by a hydrothermal method, and then UiO-66@PDA is prepared by self-polymerization and oxidation of dopamine under alkaline conditions; Among them, the preparation method of UiO-66-NH2 includes: adding 80 mg ZrCl4, 420 mg benzoic acid and 60 mg 2-aminoterephthalic acid into the reaction container respectively, and then adding 20 mL DMF, ultrasonicating until completely dissolved, and then setting up a reflux device so that the reaction solution is magnetically stirred at 120°C for 24 hours.

2. The immunochromatographic test strip according to claim 1, characterized in that: The final concentration of the antibody to be detected after adding the antibody to be detected is 1-100 μg / mL, and the final concentration of the blocking agent after adding the blocking agent is 0.1-1%. The blocking agent is any one of casein, bovine serum albumin, ovalbumin, polyethylene glycol, and skim milk.

3. The immunochromatographic test strip according to claim 1, characterized in that: The precipitate obtained after centrifugation was reconstituted with 0.01-0.1 M phosphate buffer, pH = 6-9.

4. The immunochromatographic test strip according to claim 1, characterized in that: The antibody to be detected is a monoclonal antibody, a polyclonal antibody, a nanobody or a phage-expressed antibody.

5. The immunochromatographic test strip according to claim 1, characterized in that: The nitrocellulose membrane is coated with an artificially coupled antigen of the object to be detected or an antibody of the object to be detected as a detection line, and is coated with an anti-mouse antibody or an anti-rabbit antibody as a secondary antibody as a quality control line.

6. A method for preparing the immunochromatographic test strip according to any one of claims 1 to 5, characterized in that: The following steps are involved: The filter paper, sample pad, glass fiber pad sprayed with UiO-66@PDA@Ab, nitrocellulose membrane sprayed with artificial conjugated antigen of the test object or antibody of the test object as the detection line and sprayed with anti-mouse antibody or anti-rabbit antibody as the secondary antibody as the quality control line, and absorbent paper are overlapped and pasted on the bottom plate in sequence, and the assembly is completed.

7. A detection method of the immunochromatographic test strip according to any one of claims 1 to 5, characterized in that: The following steps are involved: The sample to be tested is added to the immunochromatographic test strip, the sample volume is 50-200 μL, the reaction time is 3-30 min, and the signal data of the detection line and the quality control line on the immunochromatographic test strip are read, and the concentration of the sample to be tested is calculated by the built-in standard curve to achieve quantitative detection. The qualitative judgment is achieved by observing whether the detection line and the quality control line are dark brown through the immunochromatographic test strip.

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