An immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers, and preparation method and application thereof

Abstract

The present invention discloses an immunochromatographic test strip with metal polyphenol network fluorescent microspheres as a marker, and a preparation method and application thereof. The method uses MPNFM as a marker material, not only has a fast labeling speed, but also has a high protein coupling rate, which is very beneficial to improving the detection sensitivity of the immunochromatographic method. By overlapping and pasting a sample pad, a glass fiber pad sprayed with an MPNFM test object antibody probe, a nitrocellulose membrane sprayed with an artificial coupled antigen of the test object and sprayed with anti-mouse antibody or anti-rabbit antibody as a quality control line, and absorbent paper on a bottom plate in sequence, and then cutting it into a 4 mm wide test strip with a strip cutter, it can be used for actual sample detection. Compared with the traditional immunochromatographic detection method using colloidal gold as a marker, this method has higher qualitative and quantitative sensitivity and is more stable.

Description

Technical Field

[0001] The invention relates to the field of medical testing, and in particular to an immunochromatographic test strip using metal polyphenol network fluorescent microspheres MPNFM as a marker, and a preparation method and application thereof. Background Art

[0002] Fluorescence immunochromatography is a new rapid detection method that combines fluorescent nanomaterials with immunochromatography technology. It is widely used in medical diagnosis, drug detection, food safety, environmental monitoring and other fields. The biocompatibility of fluorescent nanomaterials is the key to the sensitivity of fluorescence immunochromatography. At present, the preparation methods of fluorescent microspheres mainly include microemulsion method, swelling method, etc., but the biocompatibility of fluorescent microspheres prepared by these synthetic methods is still unsatisfactory and cannot achieve efficient antibody coupling.

[0003] In order to improve the biocompatibility of fluorescent microspheres, the surface of fluorescent microspheres is usually modified by functionalization, such as modifying the carboxyl group to facilitate covalent conjugation of antibodies using the EDC method, or adding intermediates, such as polymer chains or cell surface proteins (protein G), to facilitate directional conjugation of antibodies; however, this chemical conjugation process may cause the antibody to reduce its activity or even lose its ability to bind to the antigen, and the adsorption of the protein corona during closure may cover the chemically conjugated antibody target recognition site.

[0004] Compared with the widely used chemical coupling, antibodies can achieve stable and strong coverage through electrostatic interaction, which is an easier, more flexible and more efficient probe targeting process. Therefore, some studies have added a coating layer with strong adhesion, such as polydopamine, to the surface of fluorescent microspheres to facilitate direct coupling of antibodies through electrostatic adsorption. However, the additional pretreatment operation of this method is cumbersome and complicated, the stability is poor, and the fluorescence is quenched, resulting in poor sensitivity. Summary of the invention

[0005] Based on this, the purpose of the present invention is to propose an immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers and a preparation method and application thereof, so as to solve the problem of poor biocompatibility of fluorescent microspheres prepared by traditional methods and effectively improve the sensitivity and stability of immunochromatographic detection.

[0006] According to the present invention, an immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers comprises a bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper sequentially overlapped on the bottom plate;

[0007] The glass fiber mat is sprayed with a probe prepared by MPNFM-labeled antibodies to be tested, and the nitrocellulose membrane is sequentially provided with a detection T line sprayed with artificially coupled antigens or antibodies to be tested and a quality control C line sprayed with anti-mouse antibodies or anti-rabbit antibodies.

[0008] Furthermore, the MPNFM is prepared by mixing and coordinating metal ions with a polyphenol aqueous solution, adding the mixture to a fluorescent dye solution dissolved in an organic solvent, stirring the mixture for reaction, adding a Tris buffer solution to adjust the pH to 8-10, centrifuging the mixture to discard the supernatant, and washing the precipitate on both sides with ultrapure water before redissolving the mixture.

[0009] Furthermore, the probe is prepared by adding the antibody to be tested to the MPNFM, mixing and reacting at room temperature for 0.4-0.6 h, adding a blocking agent, reacting at room temperature for 0.8-1.2 h, centrifuging and precipitating, and re-dissolving.

[0010] Furthermore, the metal ion is one of Fe(III), Fe(II), Co(II), Ni(II), Cu(II), Zn(IIII), Zr(II), Mg(II), Mn(II), and Ca(II).

[0011] Furthermore, the polyphenol is one of tannic acid, gallic acid, anthocyanin and epicatechin.

[0012] Furthermore, the organic solvent is one of tetrahydrofuran, N,N-dimethylformamide, pyridine, 1,4-dioxane, dimethyl sulfoxide, and acetone.

[0013] Furthermore, the final concentration of the antibody to be detected after adding the antibody to be detected is 1-5 μg / mL, the final concentration of the blocking agent after adding the blocking agent is 1-20%, and the blocking agent is any one of bovine serum albumin, casein, and ovalbumin.

[0014] Furthermore, the centrifugation speed is 10000-15000 r / min, the time is 15-30 min, and the precipitate obtained after centrifugation is redissolved using 0.01 M, pH=7.4 phosphate buffer.

[0015] According to the second aspect of the present invention, there is also provided a method for preparing an immunochromatographic test strip using metal polyphenol network fluorescent microspheres as a marker, which is used to prepare the above-mentioned immunochromatographic test strip, and the preparation method comprises:

[0016] S01. Preparation of nitrocellulose membrane:

[0017] (1) Use 0.01 M, pH = 7.4 phosphate buffer solution to adjust the concentration of the artificial conjugated antigen and secondary antibody (anti-mouse antibody or anti-rabbit antibody) to 0.01-1.0 mg / mL respectively;

[0018] (2) Spray the artificially coupled antigens of different analytes after adjusting the concentration onto the upper part of the nitrocellulose membrane as the test line, and spray the anti-mouse antibody or anti-rabbit antibody onto the lower part of the nitrocellulose membrane as the quality control line; the test line and the quality control line are separated by a certain distance, and the spraying volume of both is 0.3-0.8 μL / cm;

[0019] (3) Dry the nitrocellulose membrane sprayed with the test line and quality control line at 37°C for 4 h and store it in a drying oven at room temperature for later use.

[0020] S02, preparation of glass fiber mat for MPNFM antibody probe;

[0021] S03, assembling test strips;

[0022] S04, cutting, bagging, drying, sealing and storage.

[0023] According to the third aspect of the present invention, there is also provided the use of the above-mentioned immunochromatographic test strip in immunochromatographic detection.

[0024] In summary, the immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers and the preparation method and application thereof proposed in the present invention have the following advantages:

[0025] (1) As the fluorescent dye begins to disperse and dissolve in the organic solvent, it self-aggregates into nuclei due to hydrophobic effects after the addition of the aqueous phase. The metal ions and polyphenols coordinate in the aqueous phase to form a 3D cross-linked metal polyphenol network, which is deposited on the surface of the formed fluorescent dye nanoaggregates, thereby forming a core-shell MPNFM. The metal polyphenol network on the surface of MPNFM has a strong adsorption effect and excellent biocompatibility, which significantly improves its antibody coupling efficiency.

[0026] (2) The immunochromatographic method using MPNFM as a marker prepared by the present invention is the first to use MPNFM as a marker. Compared with fluorescent microspheres prepared by traditional methods, MPNFM has a higher antibody coupling efficiency, can improve its detection sensitivity, and has good biocompatibility. The prepared immunochromatographic method using MPNFM as a marker has strong stability. At present, there is no related report on the establishment of an immunochromatographic method using MPNFM as a marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the structure of the immunochromatographic test strip using MPNFM as a marker;

[0028] Figure 2 The flowchart of the method for preparing the immunochromatographic test strip using MPNFM as a marker;

[0029] Figure 3 This is the transmission electron microscopy characterization image of MPNFM;

[0030] Figure 4 The results are the comparison of antibody coupling rates of MPNFM, microemulsion fluorescent microspheres, and swelling fluorescent microspheres;

[0031] Figure 5 Standard curves for the detection of Escherichia coli O157:H7 based on MPNFM (A) and AuNP (B).

[0032] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] Example 1 Preparation of MPNFM

[0036] Synthesis of MPNFM: 2 mL of 0.25 mM Zr (IIII) and 2 mL of 1 mM tannic acid aqueous solution were mixed and coordinated, added to a fluorescent dye (1 mg / mL) dissolved in tetrahydrofuran, and 1 mL of 0.05 M Tris buffer solution (pH=9) was added. The mixture was stirred for 2 h, and the mixture was centrifuged to discard the supernatant. The precipitate was washed twice with ultrapure water and then redissolved in 1 mL of ultrapure water to obtain MPNFM.

[0037] It should be noted that since the fluorescent dye is initially dispersed and dissolved in the organic solvent, it self-nucleates due to the hydrophobic effect after the addition of the aqueous phase, and the metal ions and polyphenols coordinate in the aqueous phase to form a 3D cross-linked metal polyphenol network, which is deposited on the surface of the formed fluorescent dye nanoaggregates, thereby forming a core-shell MPNFM. The metal polyphenol network on the surface of MPNFM has a strong adsorption effect and excellent biocompatibility, which significantly improves its antibody coupling efficiency. Using MPNFM as a marker in the construction of a fluorescent immunochromatography method can improve its detection sensitivity. At present, there is no relevant report on the establishment of an immunochromatography method using MPNFM as a marker.

[0038] Furthermore, the transmission electron microscopy characterization of MPNFM is shown in Figure 3 shown by Figure 3 It can be seen that the core-shell MPNFM has good uniformity, the average particle size is about 100 nm, and the thickness of the metal polyphenol network shell is about 10 nm.

[0039] Furthermore, the antibody coupling efficiency of MPNFM, microemulsion fluorescent microspheres, and swelling fluorescent microspheres was compared. Figure 4 shown by Figure 4 It can be seen that MPNFM can reach an antibody coupling efficiency of 93.4% in 10 minutes, while the antibody coupling efficiencies of the microemulsion method fluorescent microspheres and the swelling method fluorescent microspheres after 240 minutes are 81.2% and 78.7%, respectively. MPNFM not only has a short antibody coupling time, but also a higher antibody coupling rate. It is a fluorescent material that can efficiently couple antibodies.

[0040] Example 2

[0041] See also Figure 2 , which is a flow chart of a method for preparing a test strip for an immunochromatographic detection method using MPNFM as a marker according to a second embodiment of the present invention, the method comprises steps S01 to S04, wherein:

[0042] Step S01, preparation of nitrocellulose membrane:

[0043] Escherichia coli O157:H7 antibody and anti-mouse antibody were diluted with 0.01 M PBS, pH = 7.4 to a concentration of 0.5 mg / mL and 0.2 mg / mL, respectively, and sprayed on the nitrocellulose membrane as the detection T line and quality control C line, respectively. The spray volume of the two lines was 0.74 μL / cm. The detection 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 4 h and store in a drying cabinet for later use.

[0044] Step S02, preparation of MPNFM antibody probe glass fiber mat:

[0045] 0.5 mg MPNFM was ultrasonicated for 3 min, and the concentration of MPNFM was adjusted to 0.1 mg / mL with 0.1 M borate buffer (pH=6.0). After oscillation and mixing, 20 μg of Escherichia coli O157:H7 monoclonal antibody was added to 5 mL MPNFM. After thorough mixing, the mixture was stirred at 4 °C for 0.5 h, and 500 μL of 1% casein was added. The mixture was blocked at room temperature for 1 h, and centrifuged at 12000 r / min for 15 min. The precipitate was re-dissolved with 500 μL PBS (0.01 M pH 7.4), sprayed onto a glass fiber mat at a volume of 3 μL / cm, and dried in vacuo for 4 h.

[0046] It should be noted that before adding the antibody to be tested to MPNFM, the MPNFM is pretreated, and the pretreatment process is: ultrasonicating the MPNFM for 3 min, and then adjusting the concentration of the MPNFM to 0.01-0.05 mg / mL with 0.02-0.2 M, pH=5-9 borate buffer.

[0047] Step S03, assembling the test strips:

[0048] (1) Sample pad, size 0.8 × 30 cm;

[0049] (2) Glass fiber mat, size 0.7 × 30 cm;

[0050] (3) Nitrocellulose membrane sprayed with detection T line and quality control C line, with a specification of 2.5×30 cm;

[0051] (4) Absorbent paper, size: 1.5 × 30 cm;

[0052] (5) PVC baseboard, size: 5.5×30 cm.

[0053] Discard the above materials as follows Figure 1 The components in the test strip structure diagram are pasted in sequence and assembled.

[0054] Step S04: cutting, bagging, drying, sealing and storage:

[0055] After assembly, use a cutter to cut into 4×55 mm test strips, put them into a ziplock bag, add desiccant, seal and store, the shelf life at room temperature is 6 months.

[0056] Example 3 Detection of Escherichia coli O157:H7 in milk using an immunochromatographic test strip based on MPNFM

[0057] The method for detecting Escherichia coli O157:H7 in a milk sample using the above-mentioned immunochromatographic test strip with MPNFM as a marker comprises the following steps:

[0058] 1. Dilute the milk sample 10 times with PBS (0.01 M pH 7.4) and use it as the sample solution for testing;

[0059] 2. Add 100 μL of the above sample solution to the sample well of the test strip and react for 15 min;

[0060] 3. Use a fluorescence reader to read the grayscale value of the T line of the test strip. According to the established standard curve, the content of E. coli O157:H7 in the sample can be calculated to achieve quantitative detection of positive samples.

[0061] 4. Establish a standard curve: add standard to the negative matrix, the concentration of E. coli O157:H7 is: 0, 1×10 2 , 2.5×10 2 , 5×10 2 , 1×10 3 , 2.5×10 3 , 5×10 3 , 1×10 4 , 2.5×10 4 , 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 1×10 6 , 1×10 7 CFU / mL, such as Figure 5 As shown: The standard curve was established by immunochromatographic test strips with MPNFM as the marker, and the linear regression equation was: y = -1527.9ln(x)-12086 (R²=0.9932), and the minimum detection limit was 3.14×10 3 CFU / mL.

[0062] 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 using metal polyphenol network fluorescent microspheres as markers, characterized in that: The immunochromatographic test strip comprises a bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped on the bottom plate; Wherein, the glass fiber mat is sprayed with a probe prepared by MPNFM-labeled antibodies to be detected, and the nitrocellulose membrane is sequentially provided with a detection T line sprayed with artificially coupled antigens or antibodies to be detected and a quality control C line sprayed with anti-mouse antibodies or anti-rabbit antibodies; The MPNFM is prepared by mixing metal ions with a polyphenol aqueous solution for coordination, adding the mixture to a fluorescent dye solution dissolved in an organic solvent for stirring and reacting, then adding a Tris buffer solution to adjust the pH to 8-10, centrifuging the mixture to discard the supernatant, and washing the precipitate on both sides with ultrapure water before redissolving it; The metal ion is one of Fe(III), Fe(II), Co(II), Ni(II), Cu(II), Zn(IIII), Zr(II), Mg(II), Mn(II), and Ca(II); The polyphenol is one of tannic acid, gallic acid, anthocyanin and epicatechin.

2. The immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers according to claim 1, characterized in that: The probe is prepared by adding the antibody to be tested to the MPNFM, mixing and reacting at room temperature for 0.4-0.6 h, adding a blocking agent, reacting at room temperature for 0.8-1.2 h, centrifuging to obtain a precipitate, and re-dissolving.

3. The immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers according to claim 1, characterized in that: The organic solvent is one of tetrahydrofuran, N,N-dimethylformamide, pyridine, 1,4-dioxane, dimethyl sulfoxide and acetone.

4. The immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers according to claim 2, characterized in that: The final concentration of the antibody to be detected after adding the antibody to be detected is 1-5 μg / mL, and the final concentration of the blocking agent after adding the blocking agent is 1-20%. The blocking agent is any one of bovine serum albumin, casein, and ovalbumin.

5. The immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers according to claim 1, characterized in that: The centrifugation speed is 10000-15000 r / min, the time is 15-30 min, and the precipitate obtained after centrifugation is redissolved with 0.01 M phosphate buffer with pH=7.

4.

6. A method for preparing an immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers, for preparing the immunochromatographic test strip according to any one of claims 1 to 5, characterized in that: The preparation method comprises: S01, preparation of nitrocellulose membrane; S02, preparation of glass fiber mat for MPNFM antibody probe; S03, assembling test strips; S04, cutting, bagging, drying, sealing and storage.

7. Use of an immunochromatographic test strip using metal polyphenol network fluorescent microspheres as markers according to any one of claims 1 to 5 in immunochromatographic detection for non-diagnostic purposes.

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