Aflatoxin detection chromogenic test paper and preparation method thereof

Abstract

The application discloses a chromogenic test paper for detecting aflatoxin and a preparation method thereof. The method adds montmorillonite to Fe(OH)3 colloid and mixes, and drops the mixture on a nitrocellulose membrane and waits for it to dry. The mixed solution of the Fe(OH)3 colloid and the montmorillonite in the application has an adsorption effect on the aflatoxin, forms a color block under the particle agglomeration effect, and realizes visual detection of the aflatoxin.

Description

Technical Field

[0001] This invention belongs to the field of aflatoxin detection technology, and relates to a colorimetric test strip for aflatoxin detection and its preparation method. Background Technology

[0002] Aflatoxin is a class of naturally occurring toxins produced by fungi of the genus *Aspergillus*. Widely distributed in nature, especially in tropical and subtropical regions, it poses a serious threat to food safety and human health. Aflatoxin comprises several subtypes, including aflatoxin B1, B2, G1, G2, M1, and M2, with B1 being the most common and toxic subtype. Aflatoxin is a potentially potent carcinogen. Long-term exposure to aflatoxin can lead to various health problems, the most serious being liver cancer. Furthermore, aflatoxin is also associated with liver damage, immune system suppression, reproductive problems, and growth retardation. Therefore, various countries have established regulatory standards and limits for aflatoxin to ensure that aflatoxin levels in food do not exceed safe levels. These standards and limits vary depending on the type of food and national regulations. Producers and farmers also take a series of measures to reduce the risk of aflatoxin exposure, such as implementing anti-mold measures in farmland and maintaining dry and hygienic storage conditions for food.

[0003] Aflatoxin mainly forms under high temperature and humidity conditions and is commonly found in crops susceptible to mold contamination, such as corn, peanuts, cottonseed, rice, wheat, and nuts. These molds multiply on these crops, producing aflatoxin. Therefore, monitoring and controlling aflatoxin is crucial in the field of food safety. Currently, the most common method for rapid detection of aflatoxin on the market is the gold nanoparticle test strip method. This method utilizes the specific binding phenomenon of antigen and antibody; if aflatoxin is present in the sample, they will bind together to form an antibody-antigen complex. The test strip typically includes a test line and a control line. The test line is coated with an antibody, while the control line is coated with a control antibody unrelated to aflatoxin. Based on the color change on the test strip, users can determine the presence of aflatoxin in the sample. Generally, the degree of color change of the test line is directly proportional to the concentration of aflatoxin. However, this method is usually not very sensitive, and the biological materials are greatly affected by environmental interference, which may lead to measurement errors. Summary of the Invention

[0004] This invention provides a colorimetric test strip for aflatoxin detection and its preparation method. The test strip contains a mixed solution of ferric hydroxide colloid and montmorillonite. When the sample contains aflatoxin, the ferric hydroxide colloid has a strong adsorption effect on aflatoxin, causing it to aggregate and precipitate, resulting in a noticeable color change reaction.

[0005] The technical solution adopted in this invention is as follows: a colorimetric test paper for aflatoxin detection, wherein the test paper is coated with a mixture of Fe(OH)3 colloid and montmorillonite.

[0006] Preferably, the mass ratio of Fe(OH)3 colloid to montmorillonite is 1:0.0012.

[0007] Preferably, the test paper is made of nitrocellulose membrane.

[0008] Ideally, the purity of montmorillonite should be 90%.

[0009] The preparation method of the above-mentioned colorimetric test strip for aflatoxin detection includes the following steps:

[0010] Step 1: Add a certain amount of montmorillonite powder to the Fe(OH)3 colloidal solution and mix it evenly by ultrasonic vibration;

[0011] Step 2: Add the solution from Step 1 onto the test paper and dry it thoroughly at 40℃~60℃.

[0012] Preferably, in step 1, the density of the Fe(OH)3 colloid is 3.4~3.9 g / cm³. 3 .

[0013] Preferably, in step 1, ultrasonic vibration is used to mix the materials evenly for a period of 20 minutes or more; more preferably, the ultrasonic vibration time is 30 minutes.

[0014] The present invention also provides a colorimetric reagent kit for the detection of aflatoxin.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] (1) This invention is the first to discover that when a mixed solution of ferric hydroxide colloid and montmorillonite is dropped onto nitrocellulose and dried, it will adsorb and aggregate aflatoxin, thus producing a colorimetric effect. This can be observed directly with a camera or through an optical microscope. Compared with conventional gold standard test strips, the preparation process is simpler, eliminating the need for setting detection T lines and quality control C lines on the nitrocellulose membrane. This makes detection more convenient, greatly reducing the difficulty of aflatoxin detection and realizing simplified detection of aflatoxin.

[0017] (2) The method of the present invention can be further extended to test strips for other high molecular organic compounds, and then to the detection of other toxins, realizing the visual detection of aflatoxin and other toxins, with broad application prospects. Attached Figure Description

[0018] Figure 1(a) and (b) in the figure show the color development effect of the test paper made from the mixed solution of Fe(OH)3 colloid and montmorillonite on aflatoxin. The red circles indicate the color block aggregation effect after color development.

[0019] Figure 2 (a) and (b) are images of the test paper prepared by mixing Fe(OH)3 colloid and montmorillonite solution under a colorimetric optical microscope for aflatoxin. The red circles represent the aggregated color patches.

[0020] Figure 3 (a) and (b) in the figure show the color development effect of the test paper made from the mixed solution of Fe(OH)3 colloid and montmorillonite on water.

[0021] Figure 4 (a) and (b) in the figure show the colorimetric effect of the test paper prepared by mixing Fe(OH)3 colloid with montmorillonite after increasing the montmorillonite content on aflatoxin.

[0022] Figure 5 (a) and (b) in the figure show the colorimetric effect of the test paper prepared by mixing Fe(OH)3 colloid with montmorillonite after increasing the content of ferric hydroxide colloid on aflatoxin.

[0023] Figure 6 (a) and (b) in the figure show the colorimetric effect of the test paper prepared using only Fe(OH)3 colloid on aflatoxin. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0025] The ferric hydroxide colloid used in the following examples can be commercially available or prepared by conventional methods. The specific preparation method is as follows:

[0026] Step 1: Prepare reactants and reagents: Prepare ferric chloride solution (FeCl3) and sodium hydroxide solution (NaOH) as reactants, and prepare an appropriate amount of deionized water for dilution.

[0027] Step 2: Mixing the reactants: Slowly mix appropriate amounts of ferric chloride solution and sodium hydroxide solution in a container. Note that the molar ratio of the reactants should be appropriate; generally, the molar ratio of ferric chloride to sodium hydroxide should be 1:2.

[0028] Step 3: Stirring and Heating: Use a magnetic stirrer to stir the mixture thoroughly and heat it to an appropriate temperature. The heating temperature can be adjusted according to the specific experimental conditions, generally between 60-80 degrees Celsius.

[0029] Step 4: Observe the changes in the reaction solution during stirring and heating. Initially, the reaction solution may appear yellow or brown. As the reaction proceeds, the color will gradually change to brownish-black, which is due to the formation of ferric hydroxide colloid.

[0030] Step 5: Cooling and Dilution: After the reaction is complete, cool the reaction solution to room temperature and dilute it with deionized water to the required concentration. The purpose of dilution is to adjust the concentration of the colloid to suit the specific application requirements. Example 1

[0031] Step 1: Take 1.5 ml of commercial ferric hydroxide colloid (Hebei Hongzhuangyuan Education Technology Co., Ltd., density 3.7 g / L). Add 0.0067g of montmorillonite powder (Guzhang County Shanlin Stone Mineral Products Co., Ltd., purity 90%) to an argon-protected glove box and mix thoroughly by ultrasonic vibration for 0.5 hours.

[0032] Step 2: Add (cover) the solution mixed in Step 1 onto the nitrocellulose membrane and dry it at 55°C until fully dry to obtain the aflatoxin detection colorimetric test strip.

[0033] Characterization experiments and results:

[0034] (1) Drop the purchased 5 mg / ml aflatoxin B1 antigen onto the above aflatoxin detection colorimetric strip. After standing for 10 minutes, you can see that the sample with the added aflatoxin solution shows particle aggregation and color development, such as... Figure 1 As shown in (a) and (b), the red circles indicate the presence of color patch aggregation after color development, i.e., obvious aggregation occurs after adding aflatoxin solution, which is not present before adding aflatoxin. Images under a color-developing optical microscope are also provided, such as... Figure 2 As shown in (a) and (b), Figure 2 The red circle in (b) indicates that obvious aggregation occurs when observed under an optical microscope.

[0035] Comparative Example 1

[0036] This comparative example is largely the same as Example 1, except that the aflatoxin solution was replaced with water. It can be observed that the sample before adding water, such as... Figure 3 In (a), compared to after adding water, as shown in the example... Figure 3 (b) in the equation did not show any significant change.

[0037] Comparative Example 2

[0038] This comparative example is largely the same as Example 1, except that the amounts of ferric hydroxide colloid and montmorillonite used are 1.5 ml each (density 3.7 g / L). A colorimetric test strip for aflatoxin detection was prepared by adding 0.015g of montmorillonite, i.e., increasing the montmorillonite content. It was observed that the change in aflatoxin content before and after adding it to the test strip was not significant, failing to achieve the desired detection effect. Figure 4 As shown in (a) and (b) in the figure.

[0039] Comparative Example 3

[0040] This comparative example is largely the same as Example 1, except that the amounts of ferric hydroxide colloid and montmorillonite used are 2.5 ml and 0.0067 g, respectively, i.e., the aflatoxin detection colorimetric test strip is prepared by increasing the ferric hydroxide colloid content. It can be observed that the change before and after adding aflatoxin to this test strip is not significant, failing to achieve the ideal detection effect. Figure 5 As shown in (a) and (b) in the figure.

[0041] Comparative Example 4

[0042] This comparative example is largely the same as Example 1, except that montmorillonite was not added to the ferric hydroxide colloid; that is, only the ferric hydroxide colloid was used to prepare the aflatoxin detection colorimetric test strip. It can be observed that the change before and after adding aflatoxin to this test strip is not significant, failing to achieve the desired detection effect. Figure 6 As shown in (a) and (b) in the figure.

Claims

1. A colorimetric test strip for aflatoxin detection, characterized in that, The test paper is coated with a mixture of Fe(OH)3 colloid and montmorillonite; wherein the mass ratio of Fe(OH)3 colloid to montmorillonite is 1:0.0012. Prepared by the following steps: Step 1: Add a certain amount of montmorillonite powder to the Fe(OH)3 colloidal solution and mix it evenly by ultrasonic vibration; Step 2: Add the solution from Step 1 onto the test paper and dry it thoroughly at 40℃~60℃.

2. The colorimetric test paper as described in claim 1, characterized in that, The test strip is made of nitrocellulose membrane.

3. The colorimetric test paper as described in claim 1, characterized in that, The purity of montmorillonite is greater than 90%.

4. A method for preparing a colorimetric test strip for aflatoxin detection as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Add a certain amount of montmorillonite powder to the Fe(OH)3 colloidal solution and mix it evenly by ultrasonic vibration; Step 2: Add the solution from Step 1 onto the test paper and dry it thoroughly at 40℃~60℃.

5. The method as described in claim 4, characterized in that, The density of Fe(OH)3 colloid is 3.4~3.9 g / cm³. 3 .

6. The method as described in claim 4, characterized in that, Ultrasonic vibration is used to ensure uniform mixing, and the ultrasonic vibration time is more than 20 minutes.

7. The method as described in claim 4, characterized in that, Ultrasonic vibration was used to ensure uniform mixing, and the ultrasonic vibration time was 30 minutes.

8. A kit for detecting aflatoxin comprising a chromogenic test strip as described in any one of claims 1-3.

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