Polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe and its detection test strip and preparation method

Abstract

The invention discloses a polydopamine-wrapped ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, a detection test strip and a preparation method thereof; the technical scheme comprises polydopamine-wrapped ZIF-8 nanoparticles and anti-folate polyclonal antibodies coupled to the polydopamine-wrapped ZIF-8 nanoparticles; the invention aims to provide a probe which is stable and not prone to coagulation in a high-salt environment, has good temperature and chemical stability, and the nanoparticles are black-gray in shape and have obvious labeled color; and a test strip containing the polydopamine-wrapped ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, wherein the detection test strip has high specificity and good sensitivity; the invention belongs to the technical field of immunoassay.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and specifically relates to a polydopamine-wrapped ZIF-8 nanoparticle-anti-folate polyclonal antibody probe. The invention also relates to a test strip containing the probe, and a preparation method of the test strip. Background Art

[0002] Folic acid (FA) is a necessary substance for the human body to utilize sugar and amino acids, and is a substance necessary for the growth and reproduction of body cells. Folic acid acts in the body in the form of tetrahydrofolic acid, which participates in the synthesis and conversion of purine nucleic acids and pyrimidine nucleotides in the body. Folic acid plays an important role in the production of nucleic acids (RNA, DNA). Folic acid helps the metabolism of proteins, and together with vitamin B12, promotes the production and maturation of red blood cells, and is an indispensable substance for the production of red blood cells. Folic acid plays an important role in the division and growth of cells and the synthesis of nucleic acids, amino acids, and proteins. Folic acid deficiency in the human body can lead to abnormalities in red blood cells, an increase in immature cells, anemia, and a decrease in white blood cells. Folic acid is an indispensable nutrient for fetal growth and development. Folic acid deficiency in pregnant women may cause low birth weight, cleft lip and palate, heart defects, etc. If folic acid is deficient in the first three months of pregnancy, it can cause neural tube defects in the fetus, leading to deformities. Therefore, women who are preparing to become pregnant can start taking 100 to 300 micrograms of folic acid every day before pregnancy. Common methods for detecting folic acid include colorimetry, thin layer chromatography, high performance liquid chromatography, microbiological method and isotope radioimmunoassay. Although these methods have their own advantages, their application scope is limited, and they are accompanied by disadvantages such as instruments or radioactivity. Therefore, it is very meaningful to develop a rapid and sensitive detection method for the rapid detection of folic acid.

[0003] Immunochromatographic test strips are currently a widely used on-site rapid detection technology. The more mature immunochromatographic test strip technology uses colloidal gold nanoparticles to label antibodies to capture the target to achieve the purpose of detection. The synthesis method of colloidal gold nanoparticles is simple, the synthesis conditions are easy to achieve, and the molar absorption coefficient is high. Therefore, it is the most widely used signal marker in the current immunochromatographic method. However, colloidal gold is easy to aggregate under high salt concentrations, and the "dead gold" phenomenon often occurs. Summary of the invention

[0004] In view of the shortcomings and deficiencies of the above prior art, the first object of the present invention is to provide a probe which is stable and not easy to precipitate in a high-salt environment, has good temperature and chemical stability, and has black-gray nanoparticles with obvious labeled colors.

[0005] The second object of the present invention is to provide a test strip of ZIF-8 nanoparticles-anti-folate polyclonal antibody probes wrapped with polydopamine, which has high specificity and good sensitivity.

[0006] To this end, the first technical solution provided by the present invention is as follows:

[0007] A polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe comprises polydopamine-coated ZIF-8 nanoparticles (ZIF-8@PDA) and anti-folate polyclonal antibodies coupled to the polydopamine-coated ZIF-8 nanoparticles.

[0008] Furthermore, the polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, the anti-folate polyclonal antibody is prepared according to the following steps:

[0009] 1) Preparation of complete folic acid antigen

[0010] The amino group on folic acid was activated by glutaraldehyde method, and then carrier proteins bovine serum albumin BSA and ovalbumin OVA were added for coupling, respectively, to obtain the complete immunofolate antigen FA-BSA and the complete folate coating antigen FA-OVA;

[0011] 2) Mixing and emulsifying the prepared complete immune folate antigen FA-BSA and immune adjuvant;

[0012] 3) The emulsified mixture was injected subcutaneously on the neck and back of New Zealand white rabbits using a syringe. After 4-5 immunization cycles, blood was collected from the ear vein to obtain anti-folate polyclonal antibodies.

[0013] Wherein: the folic acid hapten is a compound represented by formula (I):

[0014]

[0015] Formula (I).

[0016] Complete folic acid antigen, that is, the compound represented by formula (II):

[0017]

[0018] Formula (II).

[0019] Furthermore, the polydopamine-coated ZIF-8 nanoparticles-anti-folate polyclonal antibody probe is prepared by the following steps:

[0020] Step 1: Mix a methanol solution of zinc nitrate hexahydrate Zn(NO3)2·6H2O and 2-methylimidazole for 30 minutes, centrifuge and dry the precipitate to obtain ZIF-8 nanoparticles;

[0021] Step 2: Mix and stir the ZIF-8 nanoparticles, polydopamine hydrochloride, pH 8.5 Tris-HCl solution, and ethanol-water mixed solution for 24 hours, centrifuge and dry the precipitate to obtain polydopamine-coated ZIF-8 nanoparticles.

[0022] The second technical solution provided by the present invention is a method for preparing the above-mentioned polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, which comprises mixing an aqueous solution of polydopamine-coated ZIF-8 nanoparticles, an alkaline solution, and rabbit serum containing anti-folate polyclonal antibodies, reacting for 1 hour, then adding a blocking solution for blocking, and finally centrifuging the obtained precipitate to obtain the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe.

[0023] Furthermore, in the preparation method of the above-mentioned polydopamine-coated ZIF-8 nanoparticles-anti-folate polyclonal antibody probe, the mass ratio of the polydopamine-coated ZIF-8 nanoparticles to the anti-folate polyclonal antibody is 100:(1-20).

[0024] Furthermore, in the preparation method of the above-mentioned polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, the volume ratio of the aqueous solution of the ZIF-8@PDA nanoparticles, the alkaline solution, the rabbit serum containing anti-folate polyclonal antibodies and the blocking solution is 1000: (0-8): (1-20): 100.

[0025] Furthermore, in the preparation method of the above-mentioned polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, the aqueous solution concentration of the ZIF-8@PDA nanoparticles is 0.625~10 mg / mL, the alkaline solution is 0.2M potassium carbonate solution, the concentration of anti-folate polyclonal antibodies in the rabbit serum containing anti-folate polyclonal antibodies is 50 mg / mL, and the blocking solution is 0.1 mg / mL bovine serum albumin solution.

[0026] The last technical solution provided by the present invention is the use of the above-mentioned polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe in the preparation of an immunochromatographic test strip for detecting folic acid.

[0027] An immunochromatographic test strip for detecting folic acid, which is composed of a sample pad, a nitrocellulose membrane, a binding pad and a water-absorbing pad which are sequentially attached to a bottom plate;

[0028] The conjugate pad is coated with the polydopamine-wrapped ZIF-8 nanoparticle-anti-folate polyclonal antibody probe according to claim 1, and the nitrocellulose membrane is sequentially marked with a detection line of folate-coated antigen FA-OVA and a quality control line of goat anti-rabbit IgG antibody.

[0029] Furthermore, the concentration of the polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe is 5-30 mg / mL, and the spraying amount is 7.5-10.0 μL / cm; the concentration of the coated antigen FA-OVA is 5-10 mg / mL, and the spraying amount is 1 μL / cm; the concentration of the goat anti-rabbit IgG antibody is 0.1-1.0 mg / mL, and the spraying amount is 1 μL / cm; the distance between the quality control line and the detection line is 5 mm.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides a ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe, in which the ZIF-8@PDA nanoparticles have a distinct gray-black color, and can replace the tracing effect of colloidal gold in traditional immunoassay strips, and the preparation cost is also much lower than that of colloidal gold. The complex is applied to the test strip and combined with the folic acid-coated antigen to form a competitive immunoassay test strip, which has high specificity for folic acid, good sensitivity, and low detection limit (0.38ng / mL), and can quickly and accurately detect folic acid in milk powder samples.

[0032] 2. The technical solution provided by the present invention uses the glutaraldehyde method to prepare folic acid immune antigens to obtain better anti-folate polyclonal antibodies. At the same time, the immunochromatographic test strip method using ZIF-8@PDA nanoparticles to label anti-folate polyclonal antibodies has reliable and accurate results, which can be used for rapid on-site detection of folic acid content in food, medicine and medical industries. At the same time, the materials used are easy to prepare and the cost is relatively low, and it has good application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the technical roadmap described in the present invention.

[0034] Figure 2 It is the SDS-PAGE electrophoresis diagram of the antigen prepared in Example 1 of the present invention.

[0035] Figure 3 It is a transmission electron microscope image of ZIF-8 nanoparticles and ZIF-8@PDA nanoparticles prepared in Example 3 of the present invention.

[0036] Figure 4 It is a schematic diagram of the results of pH optimization of the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe prepared in Example 4 of the present invention.

[0037] Figure 5 It is a schematic diagram of the optimization results of the dosage of ZIF-8@PDA nanoparticles of the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe prepared in Example 4 of the present invention.

[0038] Figure 6 It is a schematic diagram of the optimization results of the dosage of the anti-folate polyclonal antibody of the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe prepared in Example 4 of the present invention.

[0039] Figure 7 The figure is a graph of the folic acid standard detected by the test strip prepared in Example 5 of the present invention and the corresponding standard curve. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0041] In the examples of the present invention, folic acid, potassium carbonate, and zinc nitrate hexahydrate were purchased from Shanghai McLean Biochemical Technology Co., Ltd., ovalbumin and dopamine hydrochloride were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., bovine serum albumin was purchased from Guangzhou Ruishu Biotechnology Co., Ltd., and the SDS-PAGE detection kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.

[0042] Example 1 Conjugation of folic acid and carrier protein

[0043] 1. The method for coupling folic acid with carrier bovine serum albumin is as follows: take 8 mg of folic acid and dissolve it in 4 mL of pH 7.4, 0.01 M phosphate buffer as solution A; then take 9 mg of bovine serum albumin (BSA), dissolve it in 1 mL of PBS solution, and slowly stir to dissolve it as solution B; slowly add solution A dropwise into solution B, and slowly stir to mix at 4°C in the dark; add 30 μL of 50% glutaraldehyde, and slowly stir to react for 2 hours at 4°C in the dark; add 1.5 mL of 1 mol / L glycine solution to the above solution to terminate the reaction; centrifuge to obtain the supernatant, put it into a dialysis bag, dialyze it in pH 7.4, 0.01 M phosphate buffer for 72 hours (change the water 2-3 times a day), divide it into portions and store it at -20°C as an immunogen.

[0044] 2. The method for coupling folic acid with carrier ovalbumin is as follows: take 8 mg of folic acid and dissolve it in 4 mL of pH 7.4, 0.01 M phosphate buffer as solution A; then take 6 mg of ovalbumin (OVA), dissolve it in 1 mL of PBS solution, and slowly stir to dissolve it as solution B; slowly add solution A dropwise into solution B, and slowly stir to mix at 4°C in the dark; add 30 μL of 50% glutaraldehyde, and slowly stir to react for 2 hours at 4°C in the dark; add 1.5 mL of 1 mol / L glycine solution to the above solution to terminate the reaction; use an ultrafiltration tube for ultrafiltration to obtain an immunogen or coating source solution, which is divided and stored at -20°C as a coating source.

[0045] 3. Preliminary characterization of antigens: The obtained complete antigen FA-BSA and coated antigen FA-OVA were characterized by SDS-PAGE electrophoresis. Figure 2 As shown, lane 1 is the immune antigen FA-BSA after coupling, lane 2 is bovine serum albumin BSA, M is the electrophoresis marker band, lane 3 is ovalbumin OVA, and lane 4 is the coated antigen FA-OVA after coupling. It was found that the bands of the coupled proteins were higher than those of the carrier protein, indicating that the molecular weight of the protein increased after coupling, the target molecule was successfully coupled to the carrier protein, and the two antigens were successfully prepared.

[0046] Example 2 Preparation of anti-folate polyclonal antibodies

[0047] 1. Animal immunization. Take 1mL of the obtained complete antigen FA-BSA (1mg / mL) and mix it with an equal volume of immune adjuvant (mix with Freund's complete adjuvant for the first immunization, and use Freund's incomplete adjuvant for the booster immunization), and emulsify it with an emulsifier. If the solution becomes a paste after 10-15 minutes of emulsification, it can be dropped into deionized water to observe whether the droplets spread in the water. The water surface can be gently shaken. If it spreads, it is necessary to continue emulsification, otherwise it proves that the emulsification has been successful. Select a 1.5-2kg New Zealand white rabbit (female) and inject the emulsified mixed liquid into the subcutaneous part of the neck and back of the New Zealand white rabbit.

[0048] 63 days after immunization of New Zealand white rabbits, rabbit anti-folate polyclonal antibodies were obtained by collecting blood from the ear vein. The collected rabbit blood was placed at 4°C overnight, and the blood clots were completely shrunk. The supernatant was collected and stored at -20°C for 10 minutes at 4°C, and recorded as anti-folate polyclonal antibodies.

[0049] The specific immunization experimental scheme for New Zealand white rabbits is shown in Table 1:

[0050] Table 1 Immunization scheme for New Zealand white rabbits

[0051] Immunization time operate Location Immunogen dose Adjuvant Day 1 First immunization Subcutaneous neck and back 1000μg / each Freund's complete adjuvant Day 14 Second immunization Subcutaneous neck and back 1000μg / each Freund's incomplete adjuvant Day 28 The third immunization Subcutaneous neck and back 1000μg / each Freund's incomplete adjuvant Day 42 Fourth immunization Subcutaneous neck and back 1000μg / each Freund's incomplete adjuvant Day 56 Fifth immunization Subcutaneous neck and back 1000μg / each Freund's incomplete adjuvant Day 63 Blood collection Auricular vein —— —— .

[0052] Example 3 Preparation of ZIF-8 Nanoparticles and ZIF-8@PDA Nanoparticles

[0053] 1. The synthesis of ZIF-8 nanoparticles is as follows: add 1.4665 g of zinc nitrate hexahydrate Zn(NO3)2·6H2O in 100 mL of methanol and fully dissolve it, quickly pour in 3.2445 g of 2-methylimidazole, stir at room temperature for 30 minutes to see a milky turbid solution, centrifuge at 10000 rpm for 20 minutes at room temperature, leave the precipitate and put it in an oven at 50°C for overnight drying, and weigh it.

[0054] 2. The steps of using polydopamine (PDA) to encapsulate ZIF-8 nanoparticles are as follows: take 40 mg of ZIF-8 nanoparticles and disperse them in the prepared ethanol-water (v / v=3:4) mixed solution, and ultrasonically disperse them for 10 minutes until there are no obvious particles in the solution; add 8 mg of dopamine hydrochloride to the solution and stir vigorously; then add 25 mL of 10 mM, pH 8.5 Tris-HCl solution, change to gentle stirring, and react for 24 hours in the dark; after centrifugation at 11000 rpm for 15 minutes, wash the precipitate with water and ethanol respectively, dry it in an oven at 50°C overnight, weigh it, and prepare it into a 10 mg / mL solution for storage in the dark.

[0055] 3. Characterization of ZIF-8@PDA nanoparticles: The two nanoparticles were characterized by transmission electron microscopy for morphology and particle size statistics. Figure 3 As shown, the left picture is a transmission electron microscope image of ZIF-8 nanoparticles, and the right picture is a transmission electron microscope image of ZIF-8@PDA nanoparticles. From the transmission electron microscope image, it can be clearly seen that the ZIF-8 nanoparticles are in a hexagonal nanoparticle structure. From the transmission electron microscope image of ZIF-8@PDA nanoparticles, it can be seen that there is an obvious black film layer of polydopamine successfully wrapped on the surface of ZIF-8 nanoparticles. From the actual solution color comparison, it can also be seen that the ZIF-8 nanoparticle solution before wrapping is milky white, and the ZIF-8@PDA nanoparticle solution after wrapping is gray-black, and the color becomes more obvious and easy to identify. According to observation and statistics, the particle size of ZIF-8 nanoparticles and ZIF-8@PDA nanoparticles is about 50nm.

[0056] Example 4 Preparation of ZIF-8@PDA nanoparticles-anti-folate polyclonal antibody probe

[0057] The preparation of ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe is specifically carried out as follows: 1 mL of ZIF-8@PDA nanoparticle solution is added, 0.2 M K2CO3 is added, and then rabbit serum (containing anti-folate polyclonal antibody) is added, and the mixture is reacted at room temperature for 1 hour, and then 100 μL of 0.1 mg / mL BSA solution is added to block the unbound sites. After reacting at room temperature for another 1 hour, the precipitate is centrifuged to obtain the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe complex. The precipitate is dissolved in 100 μL of the complex solution, wherein the formula of the complex solution is: PBS buffer containing 5% sucrose, 2% fucose, 1% PEG 20000, 1% BSA and 0.25% Tween-20.

[0058] To demonstrate the advantages of the technical solution of Example 4, the development process of the technical solution provided by this application is given below:

[0059] 1. Explore the pH of the coupling system

[0060] Take 1mL of 5mg / mL ZIF-8@PDA nanoparticle aqueous solution, add 0, 2, 4, 6, 8μL 0.2MK2CO3 to change the pH of the solution, then add 10μL rabbit serum, react at room temperature for one hour, then add 100μL 0.1mg / mL BSA solution to block unbound sites, react for 1 hour, centrifuge to get the precipitate. Dissolve the conjugate in the complex solution. Then spray the conjugate on the conjugate pad and observe the color development at different pH.

[0061] like Figure 4 As shown, with the increase of pH value of ZIF-8@PDA nanoparticle aqueous solution, the quality control line and the detection line become darker. In order to balance the visible signal output and economic cost, it is preferred to add 2 μL 0.2 M K2CO3 to the ZIF-8@PDA nanoparticle aqueous solution for reaction.

[0062] 2. Explore the dosage of ZIF-8@PDA nanoparticles

[0063] Dilute 1 mL of 10 mg / mL ZIF-8@PDA nanoparticle aqueous solution at a volume ratio of 1:1, 1:2, 1:4, 1:8, and 1:16, add 2 μL of 0.2M K2CO3 to change the pH of the solution, then add 10 μL of rabbit serum, react at room temperature for 1 hour, and then add 100 μL of 0.1 mg / mL bovine serum albumin solution to block unbound sites. After 1 hour of reaction, centrifuge to obtain the precipitate. Dissolve the conjugate in the complex solution. Then spray the conjugate on the conjugate pad and observe the color development of ZIF-8@PDA nanoparticle solutions of different concentrations.

[0064] like Figure 5 As shown in Figure 2, as the amount of ZIF-8@PDA nanoparticles decreased, the intensity of the quality control line and the detection line gradually weakened. In order to balance the visible signal output and economic cost considerations, ZIF-8@PDA nanoparticles with a dilution ratio of 1:2 were preferred.

[0065] 3. Explore the dosage of anti-folate polyclonal antibodies

[0066] Take 1mL of ZIF-8@PDA nanoparticle aqueous solution (10mg / mL) with a dilution ratio of 1:2, add 2μL of 0.2M K2CO3 to change the pH of the solution, then add 1, 5, 10, 15, 20μL of rabbit serum (containing anti-folate polyclonal antibody), react at room temperature for 1 hour, and then add 100μL of 0.1mg / mL bovine serum albumin solution to block unbound sites. After 1 hour of reaction, centrifuge to obtain the precipitate. Dissolve the conjugate in the complex solution. Then spray the conjugate on the conjugate pad and observe the color development of anti-ferritin polyclonal antibody at different concentrations.

[0067] like Figure 6 As shown, within the range of 1-10 μL of anti-folate polyclonal antibody, the color intensity of the control line and the test line gradually increased with the increase of the anti-folate polyclonal antibody content. By adding anti-folate polyclonal antibody in the range of 10-20 μL, the intensity of the test line and the control line was almost the same. Considering the economic cost and sensitivity, 10 μL was preferred as the optimal amount of anti-folate polyclonal antibody.

[0068] Example 5 Preparation of immunochromatographic test strips based on ZIF-8@PDA

[0069] 0.5, 1, 2, 5, 10 mg / mL of coated antigen FA-OVA and 1.0 mg / mL of goat anti-rabbit antibody were drawn on the nitrocellulose membrane at a sample loading of 1 μL / cm as the test line and quality control line, respectively. At the same time, 10 mg / mL ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe was sprayed on the conjugate pad at a sample loading of 8.5 μL / cm. The nitrocellulose membrane was attached to the bottom plate, the absorbent pad (close to the end of the quality control line) was attached on the top of the bottom plate, and the conjugate pad and sample pad were attached under the bottom plate. The distance between the quality control line and the test line was 5 mm, and the pads and the pads and the bottom plate were overlapped at a distance of 1 mm. After drying at room temperature for 10 minutes, 100 μL of deionized water was added to the sample pad, and the results were observed after waiting for 15 minutes.

[0070] The results showed that at 0.5-2 mg / mL, the band on the test line gradually became obvious, but the color of the band was still significantly lighter than the quality control line; as the coating antigen concentration increased to a gradient of 2-10 mg / mL, the color of the test line became as dark as the quality control line, indicating that the antibodies binding to folic acid on the test line had reached saturation at this time. Therefore, 2 mg / mL of antibody was preferably used as the marking concentration of the test line.

[0071] To demonstrate the advantages of the technical solution of Example 5, the development process of the technical solution provided by this application is given below:

[0072] Optimization of gold spraying dosage

[0073] 5mg / mL coated antigen FA-OVA and 1mg / mL goat anti-rabbit antibody were drawn on the nitrocellulose membrane at a loading volume of 1μL / cm as the test line and quality control line, respectively. At the same time, ZIF-8@PDA nanoparticles-anti-folate polyclonal antibody probe was sprayed on the conjugate pad at a loading volume of 7.5-10.0μL / cm. The nitrocellulose membrane was attached to the bottom plate, the absorbent pad (close to the end of the quality control line) was attached to the top of the bottom plate, and the conjugate pad and sample pad were attached to the bottom of the bottom plate. The spacing between the quality control line and the test line was 5mm, and the pads and the pads and the bottom plate were overlapped at a distance of 1mm. After drying at room temperature for 10min, 100μL of deionized water was added to the sample pad, and the results were observed after waiting for 15min.

[0074] The results showed that at 7.5-8.5 μL / cm, as the spraying amount increased, the color of the test line became darker, but at a gradient of 8.5-10.0 μL / cm, there was no obvious difference in the test line. It can be seen that when the sample loading amount was 8.5 μL / cm, the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe could form a clearly discernible signal with the coated antigen on the test line. Therefore, a sample loading amount of 8.5 μL / cm was preferred.

[0075] Example 6: Testing of standard samples

[0076] 5 mg / mL coated antigen FA-OVA and 1 mg / mL goat anti-rabbit antibody were drawn on the nitrocellulose membrane at a loading volume of 1 μL / cm as the test line and quality control line, respectively. At the same time, ZIF-8@PDA nanoparticles-anti-folate polyclonal antibody probe was sprayed on the conjugate pad at a loading volume of 8.5 μL / cm. The nitrocellulose membrane was attached to the base plate, the absorbent pad (the end close to the quality control line) was attached on the top of the base plate, and the conjugate pad and sample pad were attached under the base plate. The spacing between the quality control line and the test line was 5 mm, and the pads and the base plate were overlapped at a distance of 1 mm. After drying at room temperature for 10 minutes, 100 μL of different concentrations of folic acid standard solution (0, 0.1, 1, 10, 100, 1000, 10000, 100000 ng / mL) were added. After 15 minutes, it can be observed with the naked eye, and the image is taken with a smartphone for grayscale calculation. A four-parameter fitting curve was established with folic acid content as the horizontal axis and gray value as the vertical axis.

[0077] When the ferritin concentration exceeded 100 ng / mL, it was observed that the color intensity of the ZIF-8@PDA nanoparticle-based immunochromatographic test strip reached saturation, and the test line of the test strip gradually disappeared. The lowest visually detectable amount was 1 ng / mL. The color intensity difference was plotted on the Y axis and the folic acid concentration was plotted on the X axis, and the four-parameter logarithmic equation defined below was used for fitting: Y = (AD) / [1+(X / C)^B]+D, where A = the response at the high asymptote, B = the slope factor, and C = the value corresponding to 50% specific binding (IC 50） The four-parameter logarithmic equation is y = 18013 / [1+(x / 50.48)^0.45]+1763.09, R 2 =0.999.

[0078] The principle of the folic acid rapid test strip of the present invention is that when the pretreated milk powder serum sample is dripped into the test strip sample pad, the sample solution diffuses to the other end due to the capillary action of the nitrocellulose membrane carrier. During the movement, the corresponding antigen-antibody reaction occurs and is displayed by the color of the ZIF-8@PDA nanoparticles. If the sample solution to be tested contains folic acid, the folic acid first reacts with the binding pad, which is the ZIF-8@PDA nanoparticle-anti-folic acid polyclonal antibody. When the ZIF-8@PDA nanoparticles diffuse to the detection line with the sample solution, the anti-folate polyclonal antibody has bound to the folic acid molecules and does not bind to the folic acid-coated antigen on the test line, so no detection mark is displayed. However, the goat anti-rabbit IgG antibody can bind to the antibody again, and only the quality control line is colored. On the contrary, when the sample solution does not contain folic acid, the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody and the coated antigen on the test line undergo an antigen-antibody reaction, and the detection line is colored. The goat anti-rabbit IgG antibody on the quality control line can also bind to the antibody, and the quality control line is also colored. If neither the quality control line nor the detection line on the cellulose membrane is colored, the test strip is invalid.

[0079] In order to better use the technical solution provided by this application, the following is a method for using the technical solution provided by this application: The detection method comprises the following steps:

[0080] Drop 80-150 μL of pre-treated milk powder clear solution to be tested onto the sample pad in the folic acid rapid test strip. If both the test line and the quality control line are colored, the sample to be tested does not contain folic acid. If the test line does not color but the quality control line is colored, the sample to be tested contains folic acid.

[0081] The milk powder supernatant sample to be tested is pre-treated to remove fat and impurity proteins, and the obtained supernatant solution is subjected to sample processing.

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe, characterized in that: The invention comprises a nanoparticle of ZIF-8 wrapped with polydopamine, and an anti-folate polyclonal antibody coupled to the nanoparticle of ZIF-8 wrapped with polydopamine; The anti-folate polyclonal antibody is prepared according to the following steps: 1) Preparation of complete folic acid antigen The amino group on folic acid was activated by glutaraldehyde method, and then carrier proteins bovine serum albumin BSA and ovalbumin OVA were added for coupling, respectively, to obtain the complete immunofolate antigen FA-BSA and the complete folate coating antigen FA-OVA; 2) Mixing and emulsifying the prepared complete folate antigen FA-BSA and the immune adjuvant; 3) The emulsified mixture is injected into the subcutaneous part of the neck and back of New Zealand white rabbits using a syringe. After 4-5 immunization cycles, blood is collected from the ear vein to obtain anti-folate polyclonal antibodies; The polydopamine-wrapped ZIF-8 nanoparticles are prepared according to the following steps: Step 1: Mix a methanol solution of zinc nitrate hexahydrate Zn(NO3)2·6H2O and 2-methylimidazole for 30 minutes, centrifuge and dry the precipitate to obtain ZIF-8 nanoparticles; Step 2: ZIF-8 nanoparticles, polydopamine hydrochloride, Tris-HCl solution at pH 8.5, and ethanol-water mixed solution were mixed and stirred for 24 hours, and the precipitate was centrifuged and dried to obtain polydopamine-coated ZIF-8 nanoparticles; Step 3: The aqueous solution of polydopamine-coated ZIF-8 nanoparticles, the alkaline solution, and the rabbit serum containing anti-folate polyclonal antibodies were mixed and reacted for 1 hour, and then the blocking solution was added for blocking. Finally, the precipitate was centrifuged to obtain the ZIF-8@PDA nanoparticle-anti-folate polyclonal antibody probe.

2. The polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe according to claim 1, characterized in that: The mass ratio of the polydopamine-coated ZIF-8 nanoparticles to the anti-folate polyclonal antibodies is 100:(1-20).

3. The polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe according to claim 1, wherein the volume ratio of the aqueous solution of the ZIF-8@PDA nanoparticles, the alkaline solution, the rabbit serum containing anti-folate polyclonal antibodies and the blocking solution is 1000:(0-8):(1-20):

100.

4. The polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe according to claim 1, characterized in that: The aqueous solution concentration of the ZIF-8@PDA nanoparticles is 0.625-10 mg / mL, the alkaline solution is a 0.2 M potassium carbonate solution, the concentration of the anti-folate polyclonal antibody in the rabbit serum containing the anti-folate polyclonal antibody is 50 mg / mL, and the blocking solution is a 0.1 mg / mL bovine serum albumin solution.

5. Use of the polydopamine-coated ZIF-8 nanoparticle-anti-folate polyclonal antibody probe according to claim 1 in preparing an immunochromatographic test strip for detecting folic acid.

6. An immunochromatographic test strip for detecting folic acid, characterized in that: It is composed of a sample pad, a nitrocellulose membrane, a binding pad and a water-absorbing pad which are sequentially attached to a bottom plate; The conjugate pad is coated with the polydopamine-wrapped ZIF-8 nanoparticle-anti-folate polyclonal antibody probe according to claim 1, and the nitrocellulose membrane is sequentially marked with a detection line of folate-coated antigen FA-OVA and a quality control line of goat anti-rabbit IgG antibody.

7. The immunochromatographic test strip for detecting folic acid according to claim 6, characterized in that: The concentration of the polydopamine-wrapped ZIF-8 nanoparticle-anti-folate polyclonal antibody probe is 5-30 mg / mL, and the spraying amount is 7.5-10.0 μL / cm; the concentration of the coated antigen FA-OVA is 5-10 mg / mL, and the spraying amount is 1 μL / cm; the concentration of the goat anti-rabbit IgG antibody is 0.1-1.0 mg / mL, and the spraying amount is 1 μL / cm; the distance between the quality control line and the detection line is 5 mm.

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