Determination of Aflatoxin B1 in Food: ELISA & TLC Methods for Accurate Detection

Aflatoxins are a group of secondary metabolites produced by various fungi such as Aspergillus flavus and Aspergillus parasiticus. They are highly toxic and can cause liver enlargement, lesions, and even cancer in animals, posing a significant threat to the health of humans and livestock. Aflatoxin B1 (AFB1) is a derivative of difuranocoumarin and has been listed by the World Health Organization (WHO) as the most toxic compound. In 2002, the International Agency for Research on Cancer (IARC) classified it as a Group I strong carcinogen.

👉 For professional support in ensuring food safety, laboratories often rely on specialized microbiology testing services to accurately detect contaminants such as aflatoxins.

1. Enzyme-Linked Immunosorbent Assay (ELISA) Screening Method

Experimental Principle

Aflatoxin B1 in the sample is extracted with a methanol-water solution. After homogenization, vortexing, and centrifugation (or filtration), the supernatant is obtained. Aflatoxin B1 labeled with horseradish peroxidase or immobilized in the reaction wells competitively binds with the specific antibody against aflatoxin B1 in the sample supernatant or standard solution. After washing, a corresponding chromogenic substrate is added for color development. The reaction is terminated by an inorganic acid, and the absorbance is measured at 450 nm or 630 nm. The concentration of aflatoxin B1 in the sample is inversely proportional to the absorbance within a certain concentration range.

Scope of Application

Detection of aflatoxin B1.

Main Reagents

(1) All reagents required for solution preparation should be of analytical grade.

(2) Prepare the necessary solutions according to the instructions provided in the reagent kit.

Main Experimental Instruments and Equipment

Microplate reader [equipped with 450 nm and 630 nm (optional) filters], grinder, shaker, electronic balance (sensitivity 0.01 g), centrifuge (speed ≥ 6000 r/min), rapid quantitative filter paper (pore size 11 μm), sieve (pore size 1–2 mm), and other instruments required by the kit.

Experimental Procedure

(1) Sample Pretreatment

1) Liquid samples (oils and seasonings): Weigh 100 g of the test sample and mix thoroughly. Transfer 5.0 g of the sample into a 50 mL centrifuge tube, add the extraction solution required by the kit, and perform the test according to the instructions provided with the test kit.

2) Solid samples (grains, nuts, and special dietary foods): Weigh at least 100 g of the sample and grind it using a grinder. Pass the ground sample through a test sieve with a pore size of 1–2 mm. Transfer 5.0 g of the sample into a 50 mL centrifuge tube, add the extraction solution required by the kit, and perform the test according to the instructions provided with the test kit.

(2) Sample Testing

Perform quantitative testing of the sample (solution) according to the operating steps described in the enzyme-linked immunosorbent assay (ELISA) kit.

Experimental Results Calculation

(1) Standard Calibration Curve Plotting for ELISA Quantitative Detection

According to the calculation method provided in the kit instructions or computer software, plot the standard calibration curve based on the relationship between the concentration of the standard solution and absorbance.

(2) Calculation of Sample Concentration

According to the calculation method provided in the kit instructions and computer software, substitute the absorbance of the test solution into the obtained standard curve equation to calculate the concentration (ρ) of the test solution. The content of aflatoxin B1 in food is calculated using Formula (1):

In the formula:

X — Content of aflatoxin B1 in the sample, expressed in micrograms per kilogram (μg/kg).

ρ — Concentration of aflatoxin B1 in the test solution, expressed in micrograms per liter (μg/L).

V — Volume of extract (for solid samples, the volume of extraction solvent added; for liquid samples, the total volume of the sample and extraction solvent), expressed in liters (L).

f — Dilution factor during the pretreatment process.

m — Sample mass, expressed in kilograms (kg).

The calculation results should be reported to two decimal places. Positive samples must undergo further confirmation.

Experimental Notes

(1) Precision

Each sample should be weighed in duplicate for parallel determination, and the arithmetic mean shall be taken as the analytical result. The relative deviation between the results should not exceed 20%.

(2) Other

• For grains, nuts, oils, condiments, etc., when a 5 g sample is taken, the method detection limit is 1 μg/kg, and the quantification limit is 3 μg/kg.
• For special dietary food samples, when a 5 g sample is taken, the method detection limit is 0.1 μg/kg, and the quantification limit is 0.3 μg/kg.

2. Thin Layer Chromatography (TLC)

Experimental Principle

After extraction, concentration, and thin-layer separation of the sample, aflatoxin B1 produces a blue-violet fluorescence under ultraviolet light (wavelength 365 nm). The content is determined based on the minimum detectable fluorescence intensity observed on the thin layer.

Main Reagents

Unless otherwise specified, all reagents used in this method are of analytical grade.

(1) Reagents

Methanol (CH₃OH), n-Hexane (C₆H₁₄), Petroleum ether (boiling range 30–60 ℃ or 60–90 ℃), Chloroform (CHCl₃), Benzene (C₆H₆), Acetonitrile (CH₃CN), Anhydrous ether (C₂H₆O), Acetone (C₃H₆O).

Note: Before use in experiments, the above reagents should first undergo a blank test. If no interference is observed in the determination, they may be used directly; otherwise, they should be redistilled individually.

Silica gel G (for thin-layer chromatography), Trifluoroacetic acid (CF₃COOH), Anhydrous sodium sulfate (Na₂SO₄), Sodium chloride (NaCl).

(2) Preparation of Reagents

• Benzene–acetonitrile solution (98+2): Add 2 mL of acetonitrile into 98 mL of benzene and mix well.
• Methanol–water solution (55+45): Add 550 mL of methanol into 450 mL of water and mix well.
• Methanol–chloroform solution (4+96): Add 4 mL of methanol into 96 mL of chloroform and mix well.
• Acetone–chloroform solution (8+92): Add 8 mL of acetone into 92 mL of chloroform and mix well.
• Sodium hypochlorite solution (for disinfection): Weigh 100 g of bleaching powder, add into 500 mL of water, and stir thoroughly. Separately dissolve 80 g of industrial sodium carbonate (Na₂CO₃·10H₂O) in 500 mL of warm water. Mix the two solutions together, stir, allow to clarify, and filter. The filtrate contains sodium hypochlorite at a concentration of approximately 25 g/L. If refined bleaching powder is used, the amount of sodium carbonate can be doubled, resulting in a solution concentration of about 50 g/L. Contaminated glassware can be detoxified by soaking in 10 g/L sodium hypochlorite solution for half a day or in 50 g/L sodium hypochlorite solution for a short time.

(3) Standards

Aflatoxin B₁ standard (C₁₇H₁₂O₆, CAS No. 1162-65-8), purity ≥ 98%.

1) Aflatoxin B₁ standard stock solution (10 μg/mL): Accurately weigh 1–1.2 mg of aflatoxin B₁ standard, dissolve in 2 mL of acetonitrile, then dilute with benzene to 100 mL. Store in a refrigerator at 4 ℃, protected from light. The resulting concentration is about 10 μg/mL.

2) Purity determination: Spot 5 μL of the 10 μg/mL aflatoxin B₁ standard solution onto a silica gel G TLC plate (coating thickness 0.25 mm). Develop using methanol–chloroform and acetone–chloroform as mobile phases. Under UV light, the fluorescence should meet the following criteria: only a single fluorescent spot is observed after development, with no additional impurity spots; no residual fluorescent material remains at the origin.

3) Aflatoxin B₁ standard working solutions:

• Accurately pipette 1 mL of the stock solution into a 10 mL volumetric flask, dilute to volume with benzene–acetonitrile solution, and mix well. This solution corresponds to 1 μg aflatoxin B₁ per mL.
• Pipette 1.0 mL of this solution into a 5 mL volumetric flask, dilute to volume with benzene–acetonitrile solution. This solution corresponds to 0.2 μg aflatoxin B₁ per mL.
• Pipette 1.0 mL of the 0.2 μg/mL solution into a 5 mL volumetric flask, dilute to volume with benzene–acetonitrile solution. This solution corresponds to 0.04 μg aflatoxin B₁ per mL.

Main Instruments and Equipment

Round-hole sieve (aperture 2.0 mm), small grinder, electric oscillator, all-glass concentrator, glass plates (5 cm × 20 cm), thin-layer plate coater (commercial TLC plates suitable for aflatoxin detection may also be used), developing tank (25 cm × 6 cm × 4 cm), ultraviolet lamp (100–125 W, with 365 nm filter), microsyringe or hemoglobin pipette.

Experimental Procedure

(1) Preparation of Thin-Layer Plates

Weigh approximately 3 g of silica gel G, add 2–3 times the volume of water relative to the silica gel, and grind vigorously for 1–2 minutes until a paste is formed. Immediately pour the paste into the coater and spread it into three thin-layer plates of 5 cm × 20 cm with a thickness of about 0.25 mm. Air-dry for about 15 minutes, then activate at 100 °C for 2 hours. Remove the plates and store them in a desiccator. Generally, they can be preserved for 2–3 days. If stored for a longer time, reactivate before use.

(2) Spotting

Scrape off the adsorbent attached to the edges of the thin-layer plate. On a baseline 3 cm from the lower edge of the plate, apply the sample solution with a microsyringe or hemoglobin pipette. Up to four spots can be applied on one plate, with a spacing of about 1 cm between the edge and between each spot, and a spot diameter of about 3 mm. The spot sizes on the same plate should be uniform. During spotting, a blower with cold air may be used to aid drying. The spotting scheme is as follows:

• Spot 1: 0 μL aflatoxin B1 standard working solution (0.04 μg/mL).
• Spot 2: 20 μL sample solution.
• Spot 3: 20 μL sample solution + 10 μL aflatoxin B1 standard working solution (0.04 μg/mL).
• Spot 4: 20 μL sample solution + 10 μL aflatoxin B1 standard working solution (0.2 μg/mL).

(3) Development and Observation

Add 10 mL of anhydrous ether to the developing chamber, pre-develop for 12 cm, then remove and evaporate to dryness. Next, add 10 mL of acetone–chloroform (8+92) to another developing chamber and develop for 10–12 cm, then remove the plate. Observe the results under UV light as follows:

Since aflatoxin B1 standard working solution is added to the sample spots, the standard aflatoxin B1 spot overlaps with the fluorescence spot of aflatoxin B1 in the sample solution.

• If the sample is negative, the third spot on the plate will contain 0.0004 μg aflatoxin B1, which can be used to verify whether the minimum detectable level of aflatoxin B1 in the sample appears normally.
• If the sample is positive, the result serves as qualitative evidence.
• The fourth spot contains 0.002 μg aflatoxin B1, which mainly serves as a reference for positioning.
• If the second spot does not show a blue-violet fluorescent spot at the corresponding position of the aflatoxin B1 standard, it indicates that the aflatoxin B1 content in the sample is below 5 μg/kg. If a blue-violet fluorescent spot appears at the corresponding position, a confirmatory test is required.

(4) Confirmation Test

To confirm that the fluorescence observed on the TLC plate is produced by aflatoxin B1, a few drops of trifluoroacetic acid are added to generate the aflatoxin B1 derivative. After development, the Rf value of this derivative is about 0.1. Two points are successively spotted on the left side of the TLC plate:

• First point: 10 μL of 0.04 μg/mL aflatoxin B1 standard working solution.
• Second point: 20 μL of the sample solution.

A small drop of trifluoroacetic acid is added to cover each of the above two points. After reacting for 5 minutes, blow hot air for 2 minutes using a hair dryer, ensuring the plate temperature does not exceed 40°C. Then, the following two points are spotted on the TLC plate:

• Third point: 10 μL of 0.04 μg/mL aflatoxin B1 standard working solution.
• Fourth point: 20 μL of the sample solution.

After development, observe under a UV lamp whether the sample produces the same derivative as the aflatoxin B1 standard spots. The third and fourth points, to which trifluoroacetic acid was not added, can serve as blank controls for the sample and standard derivatives.

(5) Quantitative Dilution

If the fluorescence intensity of the aflatoxin B1 spot in the sample solution matches the fluorescence intensity of the minimum detectable amount of the aflatoxin B1 standard (0.0004 μg), then the aflatoxin B1 content in the sample is 5 μg/kg.

If the fluorescence intensity of the sample spot is higher than the minimum detectable amount, adjust by either reducing the volume of sample added or diluting the sample and spotting different volumes until the fluorescence intensity of the sample spot matches that of the minimum detectable amount.

Spots are added as follows:

• First point: 10 μL of 0.04 μg/mL aflatoxin B1 standard working solution.
• Second point: 10 μL of sample solution, adjusted as needed.
• Third point: 15 μL of sample solution, adjusted as needed.
• Fourth point: 20 μL of sample solution, adjusted as needed.

Calculation of Experimental Results

The content of aflatoxin B1 in the sample is calculated according to equation (2):

In the formula:

X — Content of aflatoxin B1 in the sample, in micrograms per kilogram (μg/kg).

0.0004 — Limit of detection for aflatoxin B1, in micrograms (μg).

V₁ — Volume of the added benzene–acetonitrile mixture, in milliliters (mL).

f — Total dilution factor of the sample solution.

V₂ — Volume of sample solution added when the minimum fluorescence appears, in milliliters (mL).

m — Mass of the sample equivalent dissolved in the benzene–acetonitrile mixture, in grams (g).

1000 — Conversion factor.

Results are expressed rounded to the nearest whole number.

Experimental Precautions

Ensure that the entire procedure is carried out under darkroom conditions.