Color developing test paper for aflatoxin detection and preparation method thereof

Abstract

The present invention discloses a color test paper for aflatoxin detection and a preparation method thereof. 71‑x Y x Nb6B 23 Nanocrystalline / amorphous composite magnetic iron-based nanoparticles are mixed with colloidal gold particles, labeled with corresponding antibodies, and then sprayed on a nitrocellulose membrane. Finally, a polyvinyl chloride base plate, a sample pad, a nitrocellulose membrane, and absorbent paper are assembled to form a color test paper. 71‑x Y x Nb6B 23 The mixed particles of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold have a specific adsorption effect on aflatoxin, forming color blocks under the action of particle agglomeration, thereby realizing the visual detection of aflatoxin.

Description

Technical Field

[0001] The invention belongs to the technical field of aflatoxin detection and relates to a color developing test paper for aflatoxin detection and a preparation method thereof. Background Art

[0002] The ingredients of common agricultural products and their products are complex and diverse, and the aflatoxin content is extremely low. Therefore, it is particularly urgent to explore a highly sensitive aflatoxin content determination method that is suitable for complex agricultural product systems.

[0003] Currently, the method for rapid detection of aflatoxins on the market is the gold-labeled test paper method, which is a solid-phase immunoassay. This method utilizes the reaction of aflatoxins in the sample with quantitative specific antibodies. The excess free antibodies bind to the coated antigen in the enzyme-labeled plate (detection line T line, T coated with antigen-coupled aflatoxin B1 coupled with bovine serum albumin) to develop color, and the content is determined by comparison with the standard. Therefore, this method requires the liquid to be tested to flow through the C line (quality control line, coated with sheep anti-mouse immunoglobulin G) and the T line in sequence for detection (such as Chinese patent CN201410208109.4). Magnetic nanoparticles are usually used as adsorbents for magnetic solid-phase extraction. After functional modification, they can achieve selective extraction of aflatoxins. The detection of aflatoxins can then be achieved by combining them with methods such as high-performance liquid chromatography-fluorescence detection. Therefore, these two methods are still cumbersome for daily aflatoxin detection. The detection of aflatoxins by magnetic nanoparticles requires the support of instruments and equipment, and the detection cost is high. Summary of the Invention

[0004] The present invention provides a colorimetric test paper for aflatoxin detection and a preparation method thereof. The test paper comprises a mixture of magnetic iron-based nanoparticles and colloidal gold particles labeled with an aflatoxin monoclonal antibody. When aflatoxin is present in the test sample, the nanoparticles are enriched and aggregated to develop color, enabling visual detection of aflatoxin. The magnetic iron-based nanoparticles are amorphous and metastable materials with enhanced surface activity and high adsorption properties. Upon encountering aflatoxin, they promote aggregation of the mixed particles, enabling detection of aflatoxin and producing a colorimetric effect.

[0005] The technical solution adopted in the present invention is as follows:

[0006] The method for preparing a color test paper for aflatoxin detection comprises the following steps:

[0007] Step 1, preparation of nanocrystalline / amorphous composite magnetic iron-based nanoparticles: using the laser inertial condensation method to 71-x Y x Nb6B 23 (x=0-7) amorphous alloy as target material to produce nanocrystalline / amorphous composite magnetic iron-based nanoparticles;

[0008] Step 2: In an argon-protected glove box, nanocrystalline / amorphous composite magnetic iron-based nanoparticles are added to the colloidal gold solution, the pH value thereof is adjusted to 7.5-8.5 with potassium carbonate solution, and ultrasonic vibration is performed to fully mix;

[0009] Step 3: Add the corresponding aflatoxin monoclonal antibody to the mixed solution obtained in step 2, stir evenly, let it stand until fully combined, then add sterile bovine serum albumin with a mass concentration of 10%, stir until fully uniform, let it stand at 4°C to 10°C overnight, then centrifuge at 12000-14000 r / min for 30-50 minutes, discard the supernatant, add 0.01-0.02 mol / L phosphate buffer to the precipitate and resuspend it to obtain a mixed solution of nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold;

[0010] Step 4: evenly spraying the mixed solution of nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold onto the nitrocellulose membrane, and drying the mixture at 37° C. to 45° C. with exhaust air until the mixture is fully dry, thereby obtaining a nitrocellulose membrane to which the nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold are attached;

[0011] Step 5: Assemble the polyvinyl chloride base plate, the nitrocellulose membrane obtained in step 4, and the absorbent paper, and cut them into strips to obtain a colorimetric test paper for aflatoxin detection.

[0012] In the present invention, the aflatoxins are common aflatoxins, including but not limited to aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, etc.

[0013] In the present invention, in step 3, the aflatoxin monoclonal antibody is an antibody corresponding to the aflatoxin to be detected, including but not limited to aflatoxin B1 monoclonal antibody, aflatoxin B2 monoclonal antibody, aflatoxin G1 monoclonal antibody, aflatoxin G2 monoclonal antibody, aflatoxin M1 monoclonal antibody, etc.

[0014] Preferably, in step 1, the Fe 71-x Y x Nb6B 23 Amorphous alloy, x=3 to 5. In the specific embodiment of the present invention, the Fe 71-x Y x Nb6B 23 Amorphous alloy, x = 3 or 5.

[0015] Preferably, in step 2, the particle size of the colloidal gold is 5 to 20 nm.

[0016] Preferably, in step 2, the mass ratio of the nanocrystalline / amorphous composite magnetic iron-based nanoparticles to colloidal gold is 1:5 to 1:8.

[0017] In step 2 of the present invention, the pH value is adjusted to 7.5-8.5 because when the pH value is equal to or slightly alkaline to the isoelectric point of the protein, the protein is electrically neutral. At this time, the electrostatic interaction between the protein molecules and the nanoparticles is small, but the surface tension of the protein molecules is the largest. The protein molecules are in a weak hydrated state and are more easily adsorbed on the surface of the nanoparticles, thereby forming a protein layer, which prevents the nanoparticles from contacting each other and keeps them in a stable state.

[0018] In step 2 of the present invention, sterile bovine serum albumin with a mass concentration of 10% is added to play a blocking role and is used as a stabilizer to prevent aggregation and precipitation of proteins and nanoparticles.

[0019] Preferably, in step 3, the time for standing until the mixture is fully combined is 0.5 to 5 hours, more preferably 2 to 3 hours.

[0020] Preferably, in step 4, the spray volume of the mixed solution of nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold is 2 to 10 μL / cm 2 .

[0021] The present invention also provides a color developing test paper for aflatoxin detection prepared by the above preparation method.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention first discovered that mixing nanocrystalline / amorphous composite magnetic iron-based nanoparticles with colloidal gold particles, after labeling with aflatoxin monoclonal antibodies, can achieve adsorption and aggregation of aflatoxin, thereby developing color and allowing direct visual observation. Compared with conventional gold-labeled test paper, the preparation process is simpler, and there is no need to set up detection T lines and quality control C lines on the nitrocellulose membrane, making detection more convenient, greatly reducing the difficulty of aflatoxin detection, and realizing simple detection of aflatoxin.

[0024] (2) The method of the present invention can be further extended to other monoclonal antibody-labeled test strips, and then to the detection of other toxins, thereby realizing the visual detection of aflatoxins and other toxins, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Fe with a mass ratio of 1:5 66 Y5Nb6 23 The color development effect of the test paper made of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold mixed particles on aflatoxin. The yellow dotted circle indicates the agglomeration effect of the colored blocks after color development.

[0026] Figure 2 Fe 68 Y3Nb6 23 The color development effect of the test paper composed of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold mixed particles on aflatoxin. The yellow circles and rectangles are the red blocks after aggregation.

[0027] Figure 3 Fe with a mass ratio of 1:2 66 Y5Nb6 23 Comparison of the color development of aflatoxin by test paper made of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold.

[0028] Figure 4 To add only Fe 66 Y5Nb6 23 Comparison of the color development of aflatoxin by test paper made of nanocrystalline / amorphous composite magnetic iron-based nanoparticles.

[0029] Figure 5 This is a comparison chart of the color development of aflatoxin by test paper made by adding only purchased commercial colloidal gold particles.

[0030] Table 1 shows the adsorption test results of aflatoxin B1 by the test paper made of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold mixed particles with a mass ratio of 1:5. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0032] In the following examples, the colloidal gold used was purchased from Shanghai Jieyi Biotechnology Co., Ltd., with a particle size of 15 nm. 71-x Y x Nb6B 23 (x=0-7) Nanocrystalline / amorphous composite magnetic iron-based nanoparticles can be purchased commercially or prepared by referring to conventional methods. 66 Y5Nb6 23 For example, Fe 66 Y5Nb6 23 Nanocrystalline / amorphous composite magnetic iron-based nanoparticles, the specific preparation method is as follows:

[0033] Step 1: Fe 70 B 30 , Fe, Nb, rare earth element Y raw materials are weighed, and the master alloy ingot of the composition is prepared by arc melting method. 66 Y5Nb6 23, polishing to remove the oxide film on the surface of the ingot to prevent crystallization due to heterogeneous nucleation in the second step;

[0034] Step 2: preparing an amorphous alloy target with a thickness of 1 mm and a length of 20 to 40 mm by arc melting-absorption method, and verifying its amorphous nature by X-ray diffraction analysis;

[0035] Step 3: Pre-treat the surface of the target material, grind and polish it, clean it ultrasonically with alcohol, and then fix it in the corresponding position in the high vacuum powder making chamber;

[0036] Step 4: Seal the chamber and pump the air pressure in the powder making chamber to ultra-high vacuum state (less than 10 -6 Pa), and after the vacuum is stable, slowly input 800Pa of high-purity helium into the powder making chamber;

[0037] Step 5: Add liquid nitrogen to the liquid nitrogen cooling tank until the tank is full of liquid nitrogen.

[0038] In step 6, aim the external laser at the metal target in the cavity. Adjust the laser angle so that the emitted laser light is perpendicular to the target. Also, adjust the distance between the laser lens and the target to within 75 cm. This ensures that the laser reaches the target at maximum power. The metal vapor or atoms evaporating from the metal target surface collide with the inert helium gas in the chamber, rapidly cooling the metal. This forms a new solid-phase nanopowder. Due to the ultra-low temperature of the liquid nitrogen in the tank, the powder is adsorbed and deposited on the copper roller outside the tank.

[0039] Step 7: Rotate the scraper next to the copper roller to scrape the nanocrystalline / amorphous composite magnetic iron-based nanoparticles on the copper roller into the square column powder collecting tank below to collect Fe 66 Y5Nb6 23 Nanocrystalline / amorphous composite magnetic iron-based nanoparticles.

[0040] Example 1

[0041] Step 1: Take 50 ml of commercial colloidal gold solution (particle size: 15 nm) (colloidal gold content is about 1%) and add Fe 66 Y5Nb6 23 1 mg of nanocrystalline / amorphous composite magnetic iron-based nanoparticles (the mass ratio of nanocrystalline / amorphous composite magnetic iron-based nanoparticles to colloidal gold particles is about 1:5) was added, and then the pH value was adjusted to 8.0 with 0.2 mol / L potassium carbonate solution, and ultrasonically shaken for 0.5 h to fully mix.

[0042] Step 2: adding 0.2 ml of purchased aflatoxin B1 monoclonal antibody (5 mg / ml) to the mixed solution, stirring evenly, and letting it stand for 2 hours; adding 10 ml of sterile bovine serum albumin with a mass concentration of 10% dropwise, stirring for 1 hour, and letting it stand at 4°C overnight; centrifuging the labeled mixed solution at 12000 r / min for 30 min, discarding the supernatant, adding 5 ml of 0.02 mol / L phosphate buffer to the precipitate and resuspending it to obtain a mixed solution of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold labeled with aflatoxin B1 monoclonal antibody;

[0043] Step 3: a mixture of nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin B1 monoclonal antibody and colloidal gold was added at 10 μL / cm 2 The solution was sprayed on a nitrocellulose membrane at a spray rate of 1000 nm and dried at 39°C with ventilation for 5 hours to obtain a nitrocellulose membrane with nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin B1 monoclonal antibody and attached with colloidal gold.

[0044] Step 4: Assemble the polyvinyl chloride base plate, the sample pad, the nitrocellulose membrane obtained in step 3, and the absorbent paper, and cut them into strips to obtain aflatoxin B1 detection colorimetric test paper: using polyvinyl chloride as the bottom support, the coated nitrocellulose membrane and the absorbent paper are attached to the polyvinyl chloride base plate in a sequentially connected manner.

[0045] Characterization experiments and results:

[0046] (1) The aflatoxin solution extracted from moldy peanuts was tested by a professional organization and the concentration was 1.33 μg / ml. After taking 2 ml of the aflatoxin solution and adding 0.05 ml of the mixed solution of the nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold labeled with the above-mentioned aflatoxin B1 monoclonal antibody, the supernatant was centrifuged and the aflatoxin concentration was detected to be 0, which proved that the nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold labeled with the aflatoxin B1 monoclonal antibody had a significant adsorption effect on aflatoxin, as shown in Table 1.

[0047] Table 1 Adsorption test results of mixed solutions of nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold

[0048]

[0049] (2) The aflatoxin solution extracted from moldy peanuts was dropped onto the above-mentioned aflatoxin B1 detection color test paper. After standing for 10 minutes, it can be seen that the sample with the aflatoxin solution added has particles that aggregate and develop color, as shown in Figure 2. Figure 1 As shown in the yellow dotted circle, there is a clear red-purple agglomerate color block, which was not seen before the addition of aflatoxin. As a control, the addition of water or other liquids did not have this effect. Figure 1As shown, the test paper of the present invention can be used for simple detection and visual identification of aflatoxins.

[0050] Example 2

[0051] Fe was prepared by inert gas condensation method. 68 Y3Nb6 23 Nanocrystalline / amorphous composite magnetic iron-based nanoparticles. Then, the same steps as in Example 1 were followed to complete aflatoxin B1 monoclonal antibody labeling, spraying, and test paper assembly.

[0052] Drop the aflatoxin solution extracted from moldy peanuts onto the above test paper and let it stand for 10 minutes. You can also see a purple-red color block after adding the aflatoxin solution. Figure 2 As shown by the yellow dotted circle and rectangle, there was no aflatoxin before the addition of aflatoxin.

[0053] Comparative Example 1

[0054] This comparative example is substantially the same as Example 1, with the only difference being that the mass ratio of the nanocrystalline / amorphous composite magnetic iron-based nanoparticles to the colloidal gold particles is approximately 1:2.

[0055] Aflatoxin solution extracted from moldy peanuts was dropped onto the prepared test paper. After standing for 10 minutes, there was no obvious change in the pattern on the test paper. Figure 3 As shown, the visual detection effect of the test paper prepared with this mass ratio is not ideal.

[0056] Comparative Example 2

[0057] This comparative example is substantially the same as Example 1, except that no colloidal gold is added.

[0058] Aflatoxin solution extracted from moldy peanuts is dropped onto the prepared test paper. After standing for 10 minutes, the pattern on the test paper has no obvious change and the color is black. Figure 4 As shown, it shows that the test paper prepared without adding colloidal gold cannot effectively realize the visual detection of aflatoxin.

[0059] Comparative Example 3

[0060] This comparative example is substantially the same as Example 1, except that no nanocrystalline / amorphous composite magnetic iron-based nanoparticles are added.

[0061] Aflatoxin solution extracted from moldy peanuts was dropped onto the prepared test paper. After standing for 10 minutes, there was no obvious change in the pattern on the test paper. Figure 5 As shown, it is shown that the test paper prepared without adding nanocrystalline / amorphous composite magnetic iron-based nanoparticles cannot effectively realize the visual detection of aflatoxin.

Claims

1. A method for preparing a color test paper for aflatoxin detection, characterized in that: The following steps are involved: Step 1, preparation of nanocrystalline / amorphous composite magnetic iron-based nanoparticles: using the laser inertial condensation method to 71- x Y x Nb6B 23 Amorphous alloy was used as the target material to prepare nanocrystalline / amorphous composite magnetic iron-based nanoparticles, where x = 0~7; Step 2: In an argon-protected glove box, nanocrystalline / amorphous composite magnetic iron-based nanoparticles are added to a colloidal gold solution, the pH value thereof is adjusted to 7.5-8.5 with a potassium carbonate solution, and ultrasonic vibration is performed until the solution is fully mixed. The particle size of the colloidal gold is 5-20 nm, and the mass ratio of the nanocrystalline / amorphous composite magnetic iron-based nanoparticles to the colloidal gold is 1:5-1:

8. Step 3, adding the corresponding aflatoxin monoclonal antibody to the mixed solution obtained in step 2, stirring evenly, standing until fully combined, then adding sterile bovine serum albumin with a mass concentration of 10%, stirring until fully uniform, standing at 4°C to 10°C overnight, then centrifuging at 12000-14000 r / min for 30-50 minutes, discarding the supernatant, adding 0.01-0.02 mol / L phosphate buffer to the precipitate and resuspending it to obtain a mixed solution of aflatoxin monoclonal antibody-labeled nanocrystalline / amorphous composite magnetic iron-based nanoparticles and colloidal gold; Step 4: evenly spray the mixed solution of nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold onto the nitrocellulose membrane, and dry it at 37°C to 45°C with exhaust air until it is fully dry, thereby obtaining a nitrocellulose membrane with nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold attached thereto; Step 5: Assemble the polyvinyl chloride base plate, the nitrocellulose membrane obtained in step 4, and the absorbent paper, and cut them into strips to obtain a colorimetric test paper for aflatoxin detection.

2. The preparation method according to claim 1, characterized in that The aflatoxins are aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 or aflatoxin M1.

3. The preparation method according to claim 1, characterized in that In step 3, the aflatoxin monoclonal antibody is aflatoxin B1 monoclonal antibody, aflatoxin B2 monoclonal antibody, aflatoxin G1 monoclonal antibody, aflatoxin G2 monoclonal antibody or aflatoxin M1 monoclonal antibody.

4. The preparation method according to claim 1, characterized in that In step 1, the Fe 71-x Y x Nb6B 23 Amorphous alloy, x=3~5.

5. The preparation method according to claim 1, characterized in that In step 3, let it stand for 0.5 to 5 hours until it is fully combined.

6. The preparation method according to claim 1, characterized in that In step 3, let it sit for 2 to 3 hours until it is fully combined.

7. The preparation method according to claim 1, characterized in that In step 4, the spray volume of the mixed solution of nanocrystalline / amorphous composite magnetic iron-based nanoparticles labeled with aflatoxin monoclonal antibody and colloidal gold is 2-10 μL / cm 2 .

8. A colorimetric test paper for aflatoxin detection prepared according to the preparation method according to any one of claims 1 to 7.

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