CN112782155A - Preparation method and application of electrochemiluminescence aflatoxin biosensor - Google Patents

Abstract

The invention provides a preparation method and application of an electrochemiluminescence aflatoxin biosensor, which comprises the following steps: polishing by using alumina slurry of 1.0 μm, 0.1 μm and 0.05 μm in sequence, and then carrying out ultrasonic treatment on the electrode in ethanol and ultrapure water for 60s respectively; dripping 2.5-5 mu L of g-CN-APTAMER/BSA onto the treated electrode, and drying at room temperature in nitrogen; and washing the dried electrode for 3 times by PBS, storing in a refrigerator at 4 ℃, and airing to obtain the electrochemiluminescence aflatoxin biosensor. The electrochemiluminescence aflatoxin biosensor obtained by the method can be used for quickly and sensitively detecting aflatoxin.

Description

Preparation method and application of electrochemiluminescence aflatoxin biosensor

Technical Field

The invention relates to the technical field of novel nanometer functional materials and electrochemical biosensing, in particular to a preparation method and application of an electrochemiluminescence aflatoxin biosensor.

Background

Aflatoxins are a group of carcinogenic mycotoxins produced by fungi of the aspergillus genus, aflatoxin B1 being the most carcinogenic of these, aflatoxins B1 can cause hepatocellular carcinoma and malnutrition, growth disorders and immune diseases, and in severe cases can even lead to death. Aflatoxins are widely present in soil, pollute many crops such as peanuts, corns and rice, and home-made fermentation products can also produce aflatoxins, and the detection rate of aflatoxins in grain and oil and products in high-temperature and high-humidity areas is higher.

At present, the commonly used aflatoxin detection methods mainly comprise thin-layer chromatography, liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry and immunochemical methods. Despite their safety and sensitivity, these chromatographic methods suffer from a significant disadvantage in terms of expensive, complex pre-processing of the instruments used and specialized training in the detection. Immunochemistry can simplify the processing process and save time, but antibodies are limited in stability and cost. Therefore, it is of great significance to develop an aflatoxin sensor with low cost, rapid detection, high sensitivity and strong specificity.

The development of photoluminescence, chemiluminescence and electrochemiluminescence is promoted by exploring a new method for detecting trace aflatoxin. Electrochemiluminescence enables control of the time and location of the luminescence reaction, which means that its reproducibility will be better. Moreover, it has the advantages of no background signal, high sensitivity and wide linear range. Under the optimal condition, the concentration change of the analyte can influence the size of the optical signal, and qualitative and quantitative analysis on the analyte can be realized according to the change of the optical signal by utilizing the biological immune combination. Therefore, the aflatoxin B1 trace detection method based on electrochemiluminescence has high value.

Disclosure of Invention

The invention provides a preparation method and application of an electrochemiluminescence aflatoxin biosensor, which are used for quickly and sensitively detecting aflatoxin.

In order to achieve the purpose, the invention adopts the following technical scheme.

The embodiment of the invention provides a preparation method of an electrochemiluminescence aflatoxin biosensor, which comprises the following steps:

11) polishing by using alumina slurry of 1.0 μm, 0.1 μm and 0.05 μm in sequence, and then carrying out ultrasonic treatment on the electrode in ethanol and ultrapure water for 60s respectively;

12) dripping 2.5-5 mu L of g-CN-APTAMER/BSA onto the treated electrode, and drying at room temperature in nitrogen;

13) and washing the dried electrode for 3 times by PBS, storing in a refrigerator at 4 ℃, and airing to obtain the electrochemiluminescence aflatoxin biosensor.

Preferably, the g-CN-APTAMER/BSA is prepared by the following steps:

21) dissolving carboxylated g-CN in a PBS solution to obtain a 0.01mg/mL solution, sequentially adding 1-2 parts of EDC with the concentration of 40mmol/L and 1 part of NHS with the concentration of 10mmol/L into 1-2 parts of the solution for activation, and stirring for 0.5-2 h to obtain a mixture;

22) adding 1 part of 0.4-1 μ Μ APTAMER to the mixture and incubating at 4 ℃ for 0.5h-1 h; 23) adding 1 part of BSA solution to the incubated mixture to block non-specific sites, and standing for 1-2 h after mixing;

24) centrifuging the mixed solution after standing, and washing and ultrasonically treating the mixed solution by using 0.01M PBS to obtain a uniform g-CN-APTAMER/BSA solution;

preferably, the preparation method of the carboxylated g-CN comprises the following steps:

31) putting 10-30 parts of urea into an alumina crucible, covering the alumina crucible with the same alumina crucible, heating the mixture to 530 ℃ and 570 ℃ in a muffle furnace at the speed of 3-10 ℃/min, keeping the mixture for 3-6 hours, and naturally cooling the mixture to room temperature;

32) grinding and screening the cooled product, soaking and carrying out ultrasound for 2 hours by adopting dilute nitric acid, and then centrifuging at 9000-12000rpm to obtain a precipitate;

33) 1 part of the precipitate was added to 100mL of 5mol/L HNO₃Refluxing and condensing at 125 deg.C for 24 hr, and naturally cooling at room temperature;

34) and centrifuging and washing the cooled mixture until the pH value reaches neutral, and drying the washed product in a vacuum oven at 35-50 ℃ for 8-12h to obtain carboxylated g-CN.

Preferably, PBS is 10mmol/L and pH is 7.4 phosphate buffer solution.

Preferably, the BSA solution is a bovine serum albumin aqueous solution with the mass concentration of 0.1% -1%.

Preferably, the APTAMER sequence is 5' -NH₂-C₆-CGTGTTGTCTCTCTGTGTCTCG-Fc-3′。

Preferably, the aflatoxin is aflatoxin B₁。

The embodiment of the invention also provides a method for detecting aflatoxin by applying the electrochemiluminescence aflatoxin biosensor, which comprises the following steps:

a. preparing a standard solution: preparing a group of aflatoxin standard solutions with different concentrations including blank standard samples;

b. modification of a working electrode: b, taking the electrochemiluminescence aflatoxin biosensor as a working electrode, respectively dripping aflatoxin standard solutions with different concentrations prepared in the step a onto the surface of the working electrode, and storing in a refrigerator at 4 ℃;

c. drawing a working curve: b, taking a silver/silver chloride electrode as a reference electrode, taking a platinum sheet electrode as an auxiliary electrode, and forming a three-electrode system with the modified working electrode in the step b, wherein the three-electrode system is connected to electrochemical fluorescence detection equipment; adding a phosphate buffer solution with the pH value of 20 mL7.4, 0.002mol of sodium persulfate and 0.002mol of potassium chloride into an electrolytic cell in sequence; when the solutions are fully mixed, a cyclic voltammetry test is adopted, and a working curve is drawn according to the relation between the obtained optical signal value and the concentration of the standard aflatoxin solution;

d. detection of aflatoxin: and (c) replacing the standard aflatoxin solution in the step (a) with the sample to be detected, detecting according to the methods in the steps (b) and (c), and obtaining the content of aflatoxin in the sample to be detected according to the obtained optical signal value and the working curve.

According to the technical scheme provided by the preparation method and the application of the electrochemiluminescence aflatoxin biosensor, the method has the following beneficial effects:

1) firstly, preparing a two-dimensional nano composite material graphite phase carbon nitride, then loading a nucleic acid aptamer with a specific sequence by utilizing good biocompatibility and large specific surface area of the modified material, then carrying out non-specific site closure on the material through bovine serum albumin, and modifying a glassy carbon electrode by using the electrode material to be used as a working electrode of an aflatoxin sensor, wherein the sensor can be applied to sensing determination of an actual rice sample;

2) when the detection is carried out, the ferrocene at the end of the aptamer is close to the graphite-phase carbon nitride of the luminescent substrate, so that the fluorescence quenching is carried out, and the intensity of an optical signal is correspondingly reduced; the electrode material of the method has simple synthesis method and low toxicity; applying a traditional three-electrode system as an aflatoxin sensing test system; the electrochemical sensor has very good AFB₁Electrochemical sensing activity, higher sensitivity, wider linear range and better selectivity of a detected object;

3) the electrochemiluminescence aflatoxin biosensor is simple to prepare, convenient to operate, low in cost and has certain market development prospect, and the sample is quickly, sensitively and selectively detected;

4) the invention prepares a novel two-dimensional nano composite material g-CN-APTAMER, combines a novel electrochemiluminescence substrate g-CN with a specificity recognition unit APTAMER APTAMER, gives full play to the characteristic of higher light yield of g-CN, utilizes the spatial deformation of the APTAMER to adjust the light signal output of g-CN under the condition of the existence of a detected object, solves the technical problem of light signal adjustment in electrochemiluminescence, and has certain scientific significance and application value;

5) the invention applies g-CN to the preparation of the electrochemiluminescence biosensor, improves the detection sensitivity of the electrochemiluminescence biosensor, realizes the application of the electrochemiluminescence biosensor in actual work, and has wide potential use value.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a schematic flow chart of a preparation method of an electrochemiluminescence aflatoxin biosensor provided by an embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other element or intervening elements may also be present. Further, "connected" or "coupled" as used herein may include wirelessly connected or coupled. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

For the convenience of understanding the embodiments of the present invention, the following description will be further explained by taking several specific embodiments as examples in conjunction with the drawings, and the embodiments are not to be construed as limiting the embodiments of the present invention.

Example one

Fig. 1 is a schematic flow chart of a preparation method of an electrochemiluminescence aflatoxin biosensor provided in this embodiment, and referring to fig. 1, the method specifically includes the following steps:

s1 preparation of carboxylated g-CN:

s11, uniformly putting 30g of urea into an alumina crucible, covering the alumina crucible with the same alumina crucible, heating the alumina crucible to 550 ℃ in a muffle furnace at the speed of 10 ℃/min, keeping the temperature for 4 hours at the same temperature, and naturally cooling the urea to room temperature;

s12, grinding the light yellow powder obtained after cooling in an agate mortar, screening the powder through a 325-mesh screen after grinding to enable the materials in the subsequent steps to fully react, soaking the materials in dilute nitric acid for 2 hours in an ultrasonic mode, and then centrifuging the materials at 9000rpm to obtain precipitates;

s13 adding 1g of the precipitate to 100mL of 5mol/LHNO₃Refluxing and condensing at 125 deg.C for 24 hr, and naturally cooling at room temperature;

s14 the cooled mixture was centrifuged at 12000rpm and washed several times thoroughly with water until the pH reached neutrality and the resulting product was dried in a vacuum oven at 35 ℃ for 12h to give carboxylated g-CN.

S2 preparation of g-CN-APTAMER/BSA

S21 dissolving 5mg of carboxylated g-CN in 500mL of Phosphate Buffer Solution (PBS) to obtain 0.01mg/mL solution, adding 2mL of 40mmol/L EDC and 1mL of 10mmol/L NHS into 2mL of the solution in turn for activation, and stirring for 0.5 hour to obtain a mixture;

s22 adding 1ml of 0.4 μm solution of APTAMER (APTAMER) to the resulting mixture and incubating at 4 ℃ for 1 h;

s23 adding 1mL of Bovine Serum Albumin (BSA) aqueous solution to the incubated mixture to block non-specific sites, and after mixing well, leaving the mixture to stand for 1 h;

s24, centrifuging the mixed solution after standing and washing the mixed solution with PBS for ultrasonic treatment to obtain a uniform g-CN-APTAMER/BSA solution;

wherein, the BSA solution is a bovine serum albumin aqueous solution with the mass ratio of 0.1%; the APTAMER sequence is 5' -NH₂-C₆-CGTGTTGTCTCTCTGTGTCTCG-Fc-3′。

S3 polishing a Glassy Carbon Electrode (GCE) by using alumina slurries of 1.0 μm, 0.1 μm and 0.05 μm in sequence, and then subjecting the electrode to ultrasonic treatment in ethanol and ultrapure water for 60S respectively; dripping 2.5-5 mu L of g-CN-APTAMER/BSA onto the treated electrode, and drying at room temperature in nitrogen; and washing the dried electrode for 3 times by PBS, storing in a refrigerator at 4 ℃, and airing to obtain the electrochemiluminescence aflatoxin biosensor.

Wherein, the PBS is a phosphate buffer solution of 10mmol/L, and the Ph value is 7.4.

Example two

S1 preparation of carboxylated g-CN:

s11, 20g of urea is uniformly put into an alumina crucible, covered by the same alumina crucible, heated to 570 ℃ in a muffle furnace at the speed of 5 ℃/min, kept at the same temperature for 3 hours and naturally cooled to room temperature;

s12, grinding the light yellow powder obtained after cooling in an agate mortar, screening the powder through a 325-mesh screen after grinding to enable the materials in the subsequent steps to fully react, soaking the materials in dilute nitric acid for 2 hours in an ultrasonic mode, and then centrifuging the materials at 10000rpm to obtain precipitates;

s13 adding 1g of precipitate to 100mL of 5mol/L HNO₃Refluxing and condensing at 125 deg.C for 24 hr, and naturally cooling at room temperature;

s14 the cooled mixture was centrifuged at 12000rpm and washed several times thoroughly with water until the pH reached neutrality and the resulting product was dried in a vacuum oven at 45 ℃ for 12h to give carboxylated g-CN.

S2 preparation of g-CN-APTAMER/BSA

S21 dissolving 1mg of carboxylated g-CN in 100mL of PBS to obtain a 0.01mg/mL solution, sequentially adding 1mL of 20mmol/L EDC and 1mL of 10mmol/L NHS into 1mL of the solution for activation, and stirring for 1 hour to obtain a mixture;

s22 Add 1mL of 1 μ M of APTAMER solution to the resulting mixture and incubate at 4 ℃ for 1 h;

s23 adding 1mL of BSA solution to the incubated mixture to block non-specific sites, and after mixing well, leaving the mixture for 1 h;

s24, centrifuging the mixed solution after standing and washing the mixed solution with PBS for ultrasonic treatment to obtain a uniform g-CN-APTAMER/BSA solution;

wherein, the BSA solution is a bovine serum albumin aqueous solution with the mass ratio of 0.5 percent; the APTAMER sequence is 5' -NH₂-C₆-CGTGTTGTCTCTCTGTGTCTCG-Fc-3′。

S3 polishing a Glassy Carbon Electrode (GCE) by using alumina slurries of 1.0 μm, 0.1 μm and 0.05 μm in sequence, and then subjecting the electrode to ultrasonic treatment in ethanol and ultrapure water for 60S respectively; dripping 2.5-5 mu L of g-CN-APTAMER/BSA onto the treated electrode, and drying at room temperature in nitrogen; and washing the dried electrode for 3 times by PBS, storing in a refrigerator at 4 ℃, and airing to obtain the electrochemiluminescence aflatoxin biosensor.

Wherein, the PBS is a phosphate buffer solution of 10mmol/L, and the Ph value is 7.4.

EXAMPLE III

S1 preparation of carboxylated g-CN:

s11, uniformly putting 10g of urea into an alumina crucible, covering the alumina crucible with the same alumina crucible, heating the alumina crucible to 530 ℃ in a muffle furnace at the speed of 3 ℃/min, keeping the temperature for 6 hours at the same temperature, and naturally cooling the alumina crucible to the room temperature;

s12, grinding the light yellow powder obtained after cooling in an agate mortar, screening the powder through a 325-mesh screen after grinding to enable the materials in the subsequent steps to fully react, soaking the materials in dilute nitric acid for 2 hours in an ultrasonic mode, and then centrifuging the materials at 12000rpm to obtain precipitates;

s13 adding 1g of precipitate to 100mL of 5mol/L HNO₃At 125 DEG CRefluxing and condensing for 24 hours, and then naturally cooling at room temperature;

s14 the cooled mixture was centrifuged at 12000rpm and washed thoroughly several times with water until the pH reached neutrality and the resulting product was dried in a vacuum oven at 50 ℃ for 8h to give carboxylated g-CN.

S2 preparation of g-CN-APTAMER/BSA

S21 dissolving 5mg of carboxylated g-CN in 500mL of LPBS to obtain a 0.01mg/mL solution, sequentially adding 1mL of 10mmol/L EDC and 1mL of 10mmol/L NHS into 1mL of the solution for activation, and stirring for 2 hours to obtain a mixture;

s22 adding 1ml of 0.5 μm APTAMER solution to the resulting mixture and incubating at 4 ℃ for 0.5 h;

s23 adding 1mL of BSA solution to the incubated mixture to block non-specific sites, and after mixing well, leaving the mixture to stand for 2 h;

s24, centrifuging the mixed solution after standing and washing the mixed solution with PBS for ultrasonic treatment to obtain a uniform g-CN-APTAMER/BSA solution;

wherein, the BSA solution is a bovine serum albumin aqueous solution with the mass ratio of 1 percent; the APTAMER sequence is 5' -NH₂-C₆-CGTGTTGTCTCTCTGTGTCTCG-Fc-3′。

S3 polishing a Glassy Carbon Electrode (GCE) by using alumina slurries of 1.0 μm, 0.1 μm and 0.05 μm in sequence, and then subjecting the electrode to ultrasonic treatment in ethanol and ultrapure water for 60S respectively; dripping 2.5-5 mu L of g-CN-APTAMER/BSA onto the treated electrode, and drying at room temperature in nitrogen; and washing the dried electrode for 3 times by PBS, storing in a refrigerator at 4 ℃, and airing to obtain the electrochemiluminescence aflatoxin biosensor.

Wherein, the PBS is a phosphate buffer solution of 10mmol/L, and the Ph value is 7.4.

Example four

The embodiment provides a method for detecting aflatoxin by applying an electrochemiluminescence aflatoxin biosensor, which comprises the following steps:

(1) preparing a standard solution: preparing a group of aflatoxin standard solutions with different concentrations including blank standard samples;

(2) modification of a working electrode: taking the photoelectrochemical aflatoxin biosensor prepared in the embodiment 1-3 as a working electrode, respectively dripping aflatoxin standard solutions with different concentrations prepared in the step (1) on the surface of the working electrode, and storing in a refrigerator at 4 ℃;

(3) drawing a working curve: forming a three-electrode system by using a silver/silver chloride electrode as a reference electrode, a platinum sheet electrode as an auxiliary electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; adding a phosphate buffer solution with the pH value of 20 mL7.4, 0.002mol of sodium persulfate and 0.002mol of potassium chloride into an electrolytic cell in sequence; when the solutions are fully mixed, a cyclic voltammetry test is adopted, and a working curve is drawn according to the relation between the obtained optical signal value and the concentration of the standard aflatoxin solution; wherein the linear detection range of the aflatoxin is 0.005-10 ng/ml, and the detection limit is as follows: 0.0037 ng/ml;

(4) and (3) actual sample detection: and (3) replacing the standard aflatoxin solution in the step (1) with the sample to be detected, detecting according to the methods in the steps (2) and (3), and obtaining the content of aflatoxin in the sample to be detected according to the obtained optical signal value and the working curve.

Wherein, the aflatoxin in the embodiment is aflatoxin B1.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (8)

1. A preparation method of an electrochemiluminescence aflatoxin biosensor is characterized by comprising the following steps:

11) polishing by using alumina slurry of 1.0 μm, 0.1 μm and 0.05 μm in sequence, and then carrying out ultrasonic treatment on the electrode in ethanol and ultrapure water for 60s respectively;

12) dripping 2.5-5 mu L of g-CN-APTAMER/BSA onto the treated electrode, and drying at room temperature in nitrogen;

13) and washing the dried electrode for 3 times by PBS, storing in a refrigerator at 4 ℃, and airing to obtain the electrochemiluminescence aflatoxin biosensor.

2. The method of claim 1, wherein the g-CN-APTAMER/BSA is prepared by the following steps:

21) dissolving carboxylated g-CN in a PBS solution to obtain a 0.01mg/mL solution, sequentially adding 1-2 parts of EDC with the concentration of 40mmol/L and 1 part of NHS with the concentration of 10mmol/L into 1-2 parts of the solution for activation, and stirring for 0.5-2 h to obtain a mixture;

22) adding 1 part of 0.4-1 μ Μ APTAMER to the mixture and incubating at 4 ℃ for 0.5h-1 h; 23) adding 1 part of BSA solution to the incubated mixture to block non-specific sites, and standing for 1-2 h after mixing;

24) the pooled solution after standing was centrifuged and sonicated with 0.01M PBS to give a homogeneous g-CN-APTAMER/BSA solution.

3. The method of claim 2, wherein the carboxylated g-CN is prepared by a process comprising:

31) putting 10-30 parts of urea into an alumina crucible, covering the alumina crucible with the same alumina crucible, heating the mixture to 530 ℃ and 570 ℃ in a muffle furnace at the speed of 3-10 ℃/min, keeping the mixture for 3-6 hours, and naturally cooling the mixture to room temperature;

32) grinding and screening the cooled product, soaking and carrying out ultrasound for 2 hours by adopting dilute nitric acid, and then centrifuging at 9000-12000rpm to obtain a precipitate;

33) 1 part of the precipitate was added to 100mL of 5mol/L HNO₃Refluxing and condensing at 125 deg.C for 24 hr, and naturally cooling at room temperature;

34) and centrifuging and washing the cooled mixture until the pH value reaches neutral, and drying the washed product in a vacuum oven at 35-50 ℃ for 8-12h to obtain carboxylated g-CN.

4. The method of claim 1, wherein the PBS is 10mmol/L phosphate buffered saline with a pH of 7.4.

5. The method of claim 2, wherein the BSA solution is 0.1% -1% by mass aqueous solution of bovine serum albumin.

6. The method of claim 2, wherein the APTAMER sequence is 5' -NH₂-C₆-CGTGTTGTCTCTCTGTGTCTCG-Fc-3′。

7. The method of claim 1, wherein the aflatoxin is aflatoxin B₁。

8. A method of detecting aflatoxins using the electrochemiluminescent aflatoxin biosensor of any of claims 1-7, comprising the steps of:

a. preparing a standard solution: preparing a group of aflatoxin standard solutions with different concentrations including blank standard samples;

b. modification of a working electrode: b, taking the electrochemiluminescence aflatoxin biosensor as a working electrode, respectively dripping aflatoxin standard solutions with different concentrations prepared in the step a onto the surface of the working electrode, and storing in a refrigerator at 4 ℃;

c. drawing a working curve: b, taking a silver/silver chloride electrode as a reference electrode, taking a platinum sheet electrode as an auxiliary electrode, and forming a three-electrode system with the modified working electrode in the step b, wherein the three-electrode system is connected to electrochemical fluorescence detection equipment; adding 20mL of phosphate buffer solution with the pH value of 7.4, 0.002mol of sodium persulfate and 0.002mol of potassium chloride into an electrolytic cell in sequence; when the solutions are fully mixed, a cyclic voltammetry test is adopted, and a working curve is drawn according to the relation between the obtained optical signal value and the concentration of the standard aflatoxin solution;

d. detection of aflatoxin: and (c) replacing the standard aflatoxin solution in the step (a) with the sample to be detected, detecting according to the methods in the steps (b) and (c), and obtaining the content of aflatoxin in the sample to be detected according to the obtained optical signal value and the working curve.

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