Aflatoxin detection method based on surface plasma resonance effect, kit and application

Abstract

The invention belongs to the technical field of aflatoxin detection, and relates to an aflatoxin detection method based on a surface plasma resonance effect, a kit and application. Comprising the following steps: S1, preparing a lysine functionalized gold / silver nanoparticle solution; s2, adding an aflatoxin standard solution with known concentration into the Hg < 2 + > standard solution, oscillating and incubating, and then adding the lysine functionalized gold / silver nanoparticle solution to construct an aggregation colorimetric reaction system; and a series of aggregation colorimetric reaction systems. S3, acquiring color data by a machine image recognition technology, wherein the color data comprises an R value, a G value and a B value; s4, constructing a standard curve; s5, color data of a to-be-detected sample are obtained, and the content of aflatoxin in the to-be-detected sample is obtained through the standard curve. Visual quantitative analysis detection of aflatoxin is realized, the sensitivity is ensured, the detection process is simple, and the test efficiency is high.

Description

Technical Field

[0001] This invention belongs to the field of aflatoxin detection technology, and relates to an aflatoxin detection method, kit, and application based on surface plasmon resonance effect. Background Technology

[0002] Aflatoxins (AFs) are toxic secondary metabolites produced by Aspergillus parasiticus and Aspergillus flavus. More than 20 different aflatoxins have been identified, with aflatoxins B1, B2, G1, and G2 being the most common. AFs can widely contaminate agricultural products such as peanuts, corn, and their derivatives, posing a harmful threat to human health through the food chain. Therefore, reliable detection and early warning of AF contamination in food are crucial for ensuring food safety.

[0003] A method for detecting aflatoxin in grains is disclosed in Chinese invention patent document CN117110511B, which uses thin-layer chromatography (TLC) to detect aflatoxin in grains. The documents "Discussion on the Detection of Aflatoxin in Grains by Enzyme-Linked Immunosorbent Assay and High-Performance Liquid Chromatography" and "Application of High-Performance Liquid Chromatography in the Detection of Aflatoxin" also disclose several methods for detecting aflatoxins (AFs). In summary, conventional analytical methods for AF detection include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and enzyme-linked immunosorbent assay (ELISA). However, these detection methods have the following problems:

[0004] (1) Aflatoxin content in samples is extremely low (usually μg / kg or even ng / kg level), and the matrix (such as fat, protein and pigment in peanuts and corn) has strong interference, which can easily lead to measurement errors. At the same time, thin-layer chromatography requires multiple pretreatment steps, which makes the process cumbersome. Moreover, manual operation is time-consuming (it takes 3-6 hours from processing to detection of a single sample), and the entire detection process is time-consuming and the detection efficiency is extremely low, which indirectly increases the time cost.

[0005] (2) High performance liquid chromatography (HPLC) requires very high sample purity, which requires extraction and purification of the test sample, resulting in complex pretreatment. Moreover, HPLC testing requires the use of a special aflatoxin purification column (such as an immunoaffinity column IAC), which is costly. In addition, the natural fluorescence of aflatoxin B1 and G1 is weak, and derivatization is required to enhance the signal when using HPLC, resulting in long detection time and low testing efficiency.

[0006] (3) In high performance liquid chromatography-mass spectrometry, mass spectrometry has poor tolerance to sample matrix (matrix effect will inhibit ionization), and impurities are easy to contaminate ion source (such as ESI) or mass analyzer. Ultra-purification treatment is required before detection, and sample pH and salinity must be strictly controlled (otherwise it will affect ionization). This makes the process more complicated, time-consuming, and costly.

[0007] (4) Enzyme-linked immunosorbent assay (ELISA) is based on the specific binding of antigen (aflatoxin) and antibody, and the substrate is quantified by enzyme catalysis; this requires 8-10 steps to detect a single sample, which is time-consuming and has low testing efficiency.

[0008] In summary, due to the low content and complex matrix of aflatoxin, existing aflatoxin detection methods suffer from cumbersome procedures, long processing times, and low efficiency in order to meet the requirements of high sensitivity and interference resistance. Therefore, there is an urgent need to develop a rapid, efficient, and highly sensitive method for aflatoxin detection. Summary of the Invention

[0009] In view of the existing technical problems of cumbersome procedures, long testing time, and low testing efficiency in the detection of aflatoxin, this invention provides a method, kit, and application for the detection of aflatoxin based on surface plasmon resonance effect.

[0010] This invention uses lysine-functionalized gold / silver nanoparticles with surface plasmon resonance properties as sensitive color probes to construct an aggregation colorimetric reaction system. Then, machine image recognition technology is used to achieve visualized quantitative analysis and detection of aflatoxin. The detection method of this invention ensures sensitivity without requiring sample pretreatment, and the detection process is simple and efficient, achieving rapid and efficient detection of aflatoxin.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A method for detecting aflatoxin based on surface plasmon resonance effect includes the following steps:

[0013] S1. Preparation of Lys-Au / Ag NPs solution

[0014] A lysine-functionalized gold / silver nanoparticle solution with surface plasmon resonance effect was formed by self-assembling gold / silver nanoparticle solution with lysine. This solution is denoted as Lys-Au / Ag NPs solution.

[0015] S2. Constructing an aggregation colorimetric reaction system

[0016] To Hg 2+ A known concentration of aflatoxin standard solution was added to the standard solution, and after shaking and incubation, the Lys-Au / Ag NPs solution from step S1 was added and shaken to obtain the aggregation colorimetric reaction system; the Lys-Au / Ag NPs solution competitively binds Hg to aflatoxin. 2+This causes changes in the position and intensity of the surface plasmon resonance absorption peak of the Lys-Au / Ag NPs solution in the aggregation colorimetric reaction system, thereby changing the color.

[0017] By varying the concentration of the aflatoxin standard solution, a series of aggregation colorimetric reaction systems based on surface plasmon resonance effects were obtained using the method described above.

[0018] S3, Machine image recognition technology to acquire color data

[0019] Machine image recognition technology was used to acquire color data corresponding to a series of aggregated colorimetric reaction systems. The color data included R values, G values, and B values.

[0020] S4. Constructing the standard curve

[0021] A three-dimensional curve, i.e., a standard curve, was constructed using the concentration, R value, G value, and B value of the aflatoxin standard solution.

[0022] S5, Aflatoxin Content Detection

[0023] Take the sample to be tested, refer to steps S2 and S3 to obtain the color data corresponding to the sample to be tested, and then substitute it into the standard curve in step S4 to obtain the content of aflatoxin in the sample to be tested.

[0024] Further specifying, the gold / silver nanoparticle solution in step S1 is synthesized using HAuCl4 and AgNO3 as raw materials via a sodium citrate solution reduction method; in every 4.0 mg AgNO3, the amount of HAuCl4 and sodium citrate solution added is 1 mL, the concentration of HAuCl4 is 20 mmol / L, and the mass fraction of sodium citrate solution is 1%.

[0025] Further specified, the gold / silver nanoparticle solution was mixed with lysine at a volume ratio of 9:1, and the concentration of lysine was 10 mmol / L.

[0026] Further specifying that in step S2, the mixture is incubated at 37°C with shaking for 30 min, and after adding Lys-Au / Ag NP solution, it is shaken for at least 30 s.

[0027] Further specifying, in step S2, the Hg 2+ The volume ratio of the standard solution, the aflatoxin standard solution, and the Lys-Au / Ag NPs solution from step S1 is 0.1:1:1; the Hg 2+ The concentration of the standard solution is 1 μmol / L.

[0028] Further specifying, the specific process in step S3 is as follows: first, a series of standardized images corresponding to the aggregation colorimetric reaction system are collected respectively; then, the color data of each standardized image is obtained using machine image recognition technology.

[0029] A kit for detecting aflatoxin, including Hg 2+ Standard solutions, aflatoxin standard solutions, and lysine-functionalized gold / silver nanoparticle solutions; the Hg 2+ The volume ratio of the standard solution, aflatoxin standard solution, and lysine-functionalized gold / silver nanoparticle solution was 0.1:1:1; Hg 2+ The concentration of the standard solution is 1 μmol / L.

[0030] Further specifying, the lysine-functionalized gold / silver nanoparticle solution is obtained by thoroughly mixing gold / silver nanoparticle solution and lysine at a volume ratio of 9:1 at room temperature for 10 minutes.

[0031] Further specified, the gold / silver nanoparticle solution is synthesized using HAuCl4 and AgNO3 as raw materials via a sodium citrate solution reduction method; in every 4.0 mg AgNO3, the amount of HAuCl4 and sodium citrate solution added is 1 mL, the concentration of HAuCl4 is 20 mmol / L, and the mass fraction of sodium citrate solution is 1%.

[0032] The application of the aflatoxin detection kit in aflatoxin detection.

[0033] The detection method and aflatoxin detection kit provided by this invention are based on the principle of using lysine-functionalized gold / silver nanoparticles with distance-responsive surface plasmon resonance (LSPR) properties as sensitive color probes, and introducing mercury ions (Hg) into the system. 2+ As an aggregation inducer, AFs competitively bind to Hg in the presence of aflatoxins (AFs). 2+ This causes changes in the position and intensity of the LSPR absorption peak, and the color of the solution gradually changes from gray to orange-yellow. Then, images of the solution are collected using a portable camera, and the RGB values ​​(including R, G, and B values) of the collected images are analyzed using machine image recognition technology to construct a three-dimensional quantitative relationship curve (also known as a three-dimensional curve) between the R, G, and B values ​​and the concentration of AFs, and to form a sampling database to achieve visualized quantitative detection of AFs.

[0034] Compared with the prior art, the technical solution adopted by the present invention is as follows:

[0035] 1. Based on surface plasmon resonance (LSPR) and machine image recognition technology, this invention can simultaneously detect multiple aflatoxins through intuitive color signals. It does not require complex sample pretreatment, but only uses a portable imaging device to acquire images, achieving accurate quantification of the target analyte. It is convenient to operate, fast to detect, and highly sensitive.

[0036] 2. Through analysis and research, this invention has found that lysine-functionalized gold / silver nanoparticles (Lys-Au / Ag NPs) have sensitive LSPR response characteristics, which effectively improves the sensitivity and efficiency of aflatoxin analysis and ensures high accuracy in detection.

[0037] 3. The aggregation colorimetric reaction system of the present invention has good specificity in the detection of aflatoxin and does not require the addition of recognition probes such as antibodies or aptamers, which significantly reduces the analysis cost and makes the detection method more convenient and improves the detection efficiency.

[0038] 4. The machine image recognition technology of this invention is highly compatible with the aggregation colorimetric reaction system. In the detection of aflatoxin, the visual signal can be effectively captured and the visual quantitative signal can be quickly output, thereby improving the analysis efficiency while significantly reducing the labor cost. Attached Figure Description

[0039] Figure 1 High-resolution transmission electron microscopy images of Au / Ag NPs and solution color (inset);

[0040] Figure 2 The particle size distribution histogram of Au / Ag NPs;

[0041] Figure 3 This is the Fourier transform infrared spectrum of Au / Ag NPs;

[0042] Figure 4 The (B) Au and (C) Ag elemental mapping diagrams and (D) energy-dispersive X-ray (EDS) spectra of Au / Ag NPs are shown.

[0043] Figure 5 These are the (A) XPS spectra, (B) high-resolution XPS energy spectrum of Au 4f, and (C) high-resolution XPS energy spectrum of Ag 3d for Au / Ag NPs.

[0044] Figure 6 For colorimetric reaction systems of different concentrations of Aspergillus flavus;

[0045] Figure 7 A three-dimensional curve created using RGB values;

[0046] Figure 8It is a detection system for the specific analysis of AFs and other non-targeting toxins. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compounds of the present invention, with some conventional modifications to the reaction conditions according to the present invention, are considered to be within the scope of the present invention.

[0048] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0049] Techniques, methods, and apparatus known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.

[0050] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or changes made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0051] The principle of this invention for detecting aflatoxin is as follows: Gold / silver nanoparticles (Au / Ag NPs) were synthesized via a sodium citrate solution reduction method. Lysine was then self-assembled onto the surface of the gold / silver nanoparticles to form lysine-functionalized gold / silver nanoparticles (Lys-Au / Ag NPs) exhibiting surface plasmon resonance (LSPR) effect. These lysine-functionalized nanoparticles were then used as a color probe to design an aggregation colorimetric reaction based on the LSPR effect. The aggregation colorimetric reaction based on the LSPR effect is based on mercury ions (Hg... 2+ Lys-Au / Ag NPs aggregate in the presence of aflatoxin AFs, and in the presence of aflatoxin AFs, they aggregate with Hg. 2+ Chelation occurs, breaking the aggregation of Lys-Au / Ag NPs; due to Hg 2+ It can induce the aggregation of Lys-Au / Ag NPs within the system. Due to the change in interparticle distance, the intensity of its LSPR characteristic absorption peak decreases and undergoes a red shift, with new absorption peaks appearing in the high-wavelength range. Correspondingly, the solution color gradually changes from orange to green and then to gray. When aflatoxin is present, Hg... 2+ It can chelate with aflatoxin to form a stable chelate, Hg 2+The resulting Lys-Au / Ag NPs aggregation effect is weakened, and the solution color in the system changes with the aflatoxin concentration. Standardized images are recorded using a camera; the color data of the standardized images are analyzed using machine image recognition technology, thereby achieving quantitative analysis of aflatoxin AFs.

[0052] The technical solution provided by this invention will be described in detail below.

[0053] It should be noted that, unless otherwise specified, the chemicals and reagents used in the following embodiments are all commercially available products commonly used in the field.

[0054] It should be noted that, unless otherwise specified, the operations used in the following embodiments are all conventional operations; for example, the operating temperature is always at room temperature unless otherwise specified. The test methods are all existing standard test methods in the art unless otherwise specified.

[0055] Example 1

[0056] This embodiment synthesizes a lysine-functionalized gold / silver nanoparticle solution exhibiting surface plasmon resonance (LSPR) properties. The synthesis method includes the following steps:

[0057] Step 1: Synthesis of gold / silver nanoparticle solution

[0058] The gold / silver nanoparticle solution was synthesized using HAuCl4 and AgNO3 as raw materials via a sodium citrate solution reduction method.

[0059] The aqueous solution of 1 mL HAuCl4 (20 mmol / L) and 4.0 mg AgNO3 was heated to boiling to form a mixed solution. Then, 1 mL of sodium citrate solution with a mass fraction of 1% was added to the mixed solution, and the mixture was heated until the color of the mixed solution changed from colorless to orange. The mixed solution was then cooled to room temperature to obtain a gold / silver nanoparticle solution.

[0060] Step 2: Preparation of Lys-Au / Ag NPs solution

[0061] A lysine-functionalized gold / silver nanoparticle solution with surface plasmon resonance effect was formed by self-assembling gold / silver nanoparticle solution with lysine, denoted as Lys-Au / Ag NPs solution.

[0062] The specific steps are as follows: Take 9 mL of the gold / silver nanoparticle solution prepared in step one and mix it with 1 mL of 10 mmol / L lysine (Lys) aqueous solution by vortex mixing to obtain a lysine-functionalized gold / silver nanoparticle solution with LSPR effect, denoted as Lys-Au / Ag NPs solution, and store it in the dark and refrigerated.

[0063] In this embodiment, the morphology of the above-mentioned gold / silver nanoparticle solution was characterized by high-resolution transmission electron microscopy, and the results are as follows: Figure 1 As shown. The gold / silver nanoparticle solution is orange-yellow to the naked eye. Figure 1 (Illustration); and the gold / silver nanoparticles in the solution are uniformly spherical particles with a particle size between 42.44 nm and 43.29 nm, basically following a normal distribution (). Figure 2 The average size is 42.86 nm.

[0064] The characteristic functional groups of gold / silver nanoparticles were determined by Fourier transform infrared spectroscopy, such as... Figure 3 As shown, 3455cm -1 and 1589cm -1 The peaks at the specified locations are attributed to the stretching vibration of the OH bond and the asymmetric vibration of the C-OO bond, respectively, proving that gold / silver nanoparticles were successfully synthesized using the sodium citrate reduction method.

[0065] To further confirm the experimental results, the elemental composition and elemental states of the synthesized material were characterized using an EDS spectrometer and an X-ray photoelectron spectrometer.

[0066] EDS spectrum shows that the synthesized material is mainly composed of two elements, Au and Ag. Figure 4 (B, C, D). X-ray photoelectron spectroscopy of gold / silver nanoparticle powder ( Figure 5 The characteristic peaks at 531.42 eV, 367.93 eV, 284.84 eV, and 83.84 eV (A) are attributed to O1s, Ag 3d, C 1s, and Au 4f, respectively. Peak fitting was performed on the Au 4f and Ag 3d spectra. Figure 5 The peaks at binding energies of 87.5 eV and 83.8 eV (B and C) correspond to characteristic peaks of elemental Au, further confirming the presence of Au. The peaks at binding energies of 374.0 eV and 367.9 eV correspond to characteristic peaks of elemental Ag, indicating the presence of Ag.

[0067] Example 2

[0068] The purpose of this embodiment is to develop an aflatoxin detection kit using the lysine-functionalized gold / silver nanoparticle solution prepared in Example 1, so as to achieve rapid and efficient detection of aflatoxin.

[0069] In this embodiment, the aflatoxin detection kit includes Hg 2+ Standard solutions, aflatoxin standard solutions, and lysine-functionalized gold / silver nanoparticle solutions.

[0070] Hg 2+The volume ratio of the standard solution, aflatoxin standard solution, and lysine-functionalized gold / silver nanoparticle solution was 0.1:1:1; Hg 2+ The concentration of the standard solution is 1 μmol / L.

[0071] The aflatoxin detection kit provided in this embodiment is based on surface plasmon resonance (LSPR) and machine image recognition technology to achieve visualized quantitative analysis of total aflatoxin. It is not only highly sensitive, but also requires no sample processing and has simple operation steps. At the same time, the machine image recognition technology can directly use a camera to collect image samples, making the detection fast and low-cost.

[0072] Example 3

[0073] This embodiment mainly combines the lysine-functionalized gold / silver nanoparticle solution (Lys-Au / Ag NP solution) with LSPR properties prepared in Example 1 with machine image recognition technology to achieve visual detection of aflatoxin (AFs) content. The specific detection method is as follows:

[0074] Step 1: Draw the standard curve

[0075] Take 100 μL of Hg 2+ Add 1 mL of aflatoxin standard solutions (AFs solutions) of different concentrations (0 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL) to a standard solution (1 μmol / L), shake and incubate at 37 °C for 30 min, take the reaction solution into a colorimetric glass bottle, add 1 mL of Lys-Au / Ag NP solution and shake and react for 30 s to obtain an aggregation colorimetric reaction system based on surface plasmon resonance effect.

[0076] Because Lys-Au / Ag NPs competitively bind to Hg in the presence of AFs. 2+ The position and intensity of the LSPR absorption peak of Lys-Au / Ag NPs in the aggregation colorimetric reaction system change with the concentration of AFs, resulting in sensitive changes in colorimetry from a visual perspective.

[0077] Next, a portable camera was used to capture standardized images of all aggregated colorimetric reaction systems; then, machine image recognition technology was used to obtain the color data of each standardized image.

[0078] Finally, a digital database for aflatoxin detection was established based on standardized image samples. The color data of the standardized images, i.e., RGB values ​​(including R, G, and B values), were analyzed to plot a three-dimensional curve of the concentration of the aflatoxin standard solution versus its RGB values. The specific method is as follows:

[0079] (1) After taking a standardized image, since the standardized image is composed of pixels. In the color picture of the collected standardized image, the RGB value is used for precise image classification and distinction. The RGB value is a numerical system for representing colors.

[0080] (2) Method for obtaining the RGB value

[0081] To obtain the RGB value of the pixel at the middle position of the collected standardized picture, interpolation calculation is performed based on the surrounding pixel values. Set the positions and values of the four surrounding pixels as R11 (upper left corner), R21 (upper right corner), R12 (lower left corner), and R22 (lower right corner), and the R value of the pixel at the middle position is R mid The calculation formula is:

[0082] R mid = 1 / 4(R11 + R21 + R12 + R22)

[0083] The finally obtained RGB value ( Figure 6 ). [[ID=]))

[0084] (3) Establish a three-dimensional curve

[0085] a. Each color is composed of three components: red (R), green (G), and blue (B). The value range of each component is 0 - 255. Therefore, a three-dimensional space is constructed, where R, G, and B represent the three coordinate axes respectively.

[0086] For example, pure red can be represented as (255, , 0), pure green is (0, 255, 0), and pure blue is (0, 0, 255). And white is (255, 255, 255), black is (0, 0, 0). The RGB value and the color distinction curve (illustrated by a two-dimensional example). Assume only two color components (such as R and G) are considered, and B = 0 is fixed. Different combinations of R values and G values are plotted as a curve to distinguish colors. For example, on the R - G plane, the colors on the line where R = G will start from black (0, 0, 0), pass through gray (such as 128, 128, 0), and reach white (255, 255, 0). If R > G, the color will tend towards red; if R < G, the color will tend towards green. Just like on this two-dimensional plane, different regions represent different color tendencies.

[0087] b. Color Distinction Curve in 3D RGB Space (Real-world Situation) The situation becomes more complex when considering the complete 3D RGB space. Using the concept of vectors, a vector pointing from black (0, 0, 0) to (255, 255, 255) represents a gradient from black to white. Different colors have different "trajectories" in this 3D space. For example, the change from red (255, 0, 0) to yellow (which can be seen as a mixture of red and green, such as 255, 255, 0) in RGB space is a "curve" (more like a path in 3D space) where the R-axis remains at its maximum value while the G-axis increases from 0 to 255.

[0088] c. In the practical application of this invention, by establishing a three-dimensional curve for the RGB values, different properties of colors are distinguished using curves in the color space, thereby differentiating different samples. The three-dimensional curve is established based on experimental data results, as shown below. Figure 7 As shown.

[0089] Step 2: Detect aflatoxin AFs in the sample

[0090] (1) The peanut sample was crushed by a high-speed grinder and passed through a 24-mesh sieve. Then, 5.00g of the sample was placed in a 50mL centrifuge tube and 25mL of methanol-water (volume ratio 7:3) mixed solution was added. The mixture was vortexed and extracted in a shaker for 20min. The supernatant was obtained by centrifugation at 6000r / min for 10min. The performance of this method in the detection of AFs in peanut samples was evaluated by the spiked recovery method, as shown in Table 1.

[0091] (2) Take corn samples and process them using the above method. The performance of this method in the detection of AFs in corn samples is evaluated by the spiked recovery method, as shown in Table 1.

[0092] (3) For the above peanut and corn samples, the national standard method (high performance liquid chromatography) was also used for detection. The test results of the method in this embodiment and the high performance liquid chromatography were compared, as shown in Table 1.

[0093] Table 1. Detection results of AFs in samples by this method and high performance liquid chromatography.

[0094]

[0095] As shown in Table 1, the recovery rate of the method in this embodiment is 96.3%–105.6% (relative standard deviation (RSD) of 2.6%–8.8%); the recovery rate of the high-performance liquid chromatography (HPLC) method is 98.3%–104.7% (relative standard deviation (RSD) of 1.0%–6.0%). It is evident that the detection results of the present invention are largely consistent with those of the HPLC method, indicating that the detection method has high accuracy.

[0096] To further demonstrate the selective detection of AFs based on surface plasmon resonance (LSPR) and machine image recognition technology, the concentration of mycotoxins in the control groups was 10 times that in the AFs group to make the experimental results more convincing. 100 μL of Hg... 2+ 1 mL of different types of mycotoxins (ochratoxin A (OTA), zearalenone (ZEN), vomitoxin (DON), aflatoxin B1 / B2 (AFB1 / B2), aflatoxin G1 / G2 (AFG1 / G2), total aflatoxin (AFs), and a mixture of AFs and other non-targeted toxins, as well as a total aflatoxin (AFs) solution (non-targeted toxin concentration of 1000 ng / mL and aflatoxin concentration of 100 ng / mL), were added to the standard solution. The solution was incubated at 37°C for 30 min, and a Lys-Au / Ag NPs probe was added. The solution color was recorded, and images were acquired using a portable camera. The color data was then analyzed.

[0097] Depend on Figure 8 As can be seen, the color changes of the AFs group, AFB1 / B2 group, AFG1 / G2 group, and Mixed group are significantly different from those of the OTA, ZEN, and DON groups, indicating that other non-targeting toxins do not interfere with the detection of aflatoxin. Therefore, the surface plasmon resonance (LSPR) and machine image recognition system of this invention for the visual detection of total aflatoxin levels demonstrate good selectivity.

[0098] The above are several preferred embodiments of the preparation method of the present invention, but they should not be regarded as limitations on the technical solutions protected by the present invention. Any alternative solutions obtained by those skilled in the art based on the technical ideas of the present invention without creative labor should fall within the protection scope of the present invention.

Claims

1. A method for detecting aflatoxin based on surface plasmon resonance effect, characterized in that, Includes the following steps: S1. Preparation of Lys-Au / Ag NPs solution A lysine-functionalized gold / silver nanoparticle solution with surface plasmon resonance effect was formed by self-assembling gold / silver nanoparticle solution with lysine. This solution is denoted as Lys-Au / Ag NPs solution. S2. Constructing an aggregation colorimetric reaction system To Hg 2+ A known concentration of aflatoxin standard solution was added to the standard solution, and after shaking and incubation, the Lys-Au / Ag NPs solution from step S1 was added and shaken to obtain the aggregation colorimetric reaction system; the Lys-Au / Ag NPs solution competitively binds Hg to aflatoxin. 2+ This causes changes in the position and intensity of the surface plasmon resonance absorption peak of the Lys-Au / Ag NPs solution in the aggregation colorimetric reaction system, thereby changing the color. By varying the concentration of the aflatoxin standard solution, a series of aggregation colorimetric reaction systems based on surface plasmon resonance effects were obtained using the method described above. S3, Machine image recognition technology to acquire color data Machine image recognition technology was used to acquire color data corresponding to a series of aggregated colorimetric reaction systems. The color data included R values, G values, and B values. S4. Constructing the standard curve A three-dimensional curve, i.e., a standard curve, was constructed using the concentration, R value, G value, and B value of the aflatoxin standard solution. S5, Aflatoxin Content Detection Take the sample to be tested, refer to steps S2 and S3 to obtain the color data corresponding to the sample to be tested, and then substitute it into the standard curve in step S4 to obtain the content of aflatoxin in the sample to be tested.

2. The aflatoxin detection method based on surface plasmon resonance effect according to claim 1, characterized in that, In step S1, the gold / silver nanoparticle solution is synthesized using HAuCl4 and AgNO3 as raw materials via a sodium citrate solution reduction method. For every 4.0 mg AgNO3, the amount of HAuCl4 and sodium citrate solution added is 1 mL, the concentration of HAuCl4 is 20 mmol / L, and the mass fraction of sodium citrate solution is 1%.

3. The aflatoxin detection method based on surface plasmon resonance effect according to claim 2, characterized in that, The gold / silver nanoparticle solution was mixed with lysine at a volume ratio of 9:1, and the concentration of lysine was 10 mmol / L.

4. The aflatoxin detection method based on surface plasmon resonance effect according to claim 1, characterized in that, In step S2, the mixture is incubated at 37°C with shaking for 30 minutes, and then the Lys-Au / Ag NP solution is added and shaken for at least 30 seconds.

5. The aflatoxin detection method based on surface plasmon resonance effect according to claim 3, characterized in that, In step S2, the Hg 2+ The volume ratio of the standard solution, the aflatoxin standard solution, and the Lys-Au / Ag NPs solution from step S1 is 0.1:1:1; the Hg 2+ The concentration of the standard solution is 1 μmol / L.

6. The aflatoxin detection method based on surface plasmon resonance effect according to claim 1, characterized in that, The specific process in step S3 is as follows: first, a series of standardized images corresponding to the aggregation colorimetric reaction system are collected; then, machine image recognition technology is used to obtain the color data of each standardized image.

7. An aflatoxin detection kit, characterized in that, Including Hg 2+ Standard solutions, aflatoxin standard solutions, and lysine-functionalized gold / silver nanoparticle solutions; the Hg 2+ The volume ratio of the standard solution, aflatoxin standard solution, and lysine-functionalized gold / silver nanoparticle solution was 0.1:1:1; Hg 2+ The concentration of the standard solution is 1 μmol / L.

8. The aflatoxin detection kit according to claim 7, characterized in that, The lysine-functionalized gold / silver nanoparticle solution was obtained by thoroughly mixing gold / silver nanoparticle solution and lysine at a volume ratio of 9:1 at room temperature for 10 minutes.

9. The aflatoxin detection kit according to claim 8, characterized in that, The gold / silver nanoparticle solution was synthesized using HAuCl4 and AgNO3 as raw materials via a sodium citrate solution reduction method. In every 4.0 mg AgNO3, the amount of HAuCl4 and sodium citrate solution added was 1 mL, the concentration of HAuCl4 was 20 mmol / L, and the mass fraction of sodium citrate solution was 1%.

10. The application of the aflatoxin detection kit according to claim 7 in the detection of aflatoxin.

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