COF inverse opal photonic crystal and application thereof in label-free detection of AFB1

Abstract

The invention belongs to the field of small molecule toxin detection, and relates to a COF inverse opal photonic crystal and application thereof in label-free detection of AFB1. The COF inverse opal photonic crystal is prepared by a method comprising the following steps: (1) preparing a SiO2 photonic crystal by adopting a confinement self-assembly method; (2) enabling a COF solution to cover the SiO2 photonic crystal; and (3) after the SiO2 microspheres are etched with HF, the COF inverse opal photonic crystal is prepared. Compared with other detection methods, the COF inverse opal photonic crystal disclosed by the invention does not need to be additionally marked, the operation is simple, and the rapid detection of AFB1 can be realized only by a fiber optic spectrometer.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule toxin detection, and in particular, relates to a COF inverse opal photonic crystal and an application thereof, as well as a method for label-free detection of AFB1. Background Art

[0002] Aflatoxins (AFT) and their producing bacteria are widely distributed in nature, and some strains produce more than one type of aflatoxins. Aflatoxins can be detected in seeds and processed products of grains and oil crops, dried and fresh fruits, condiments, tobacco, milk and dairy products, meat, fish and shrimp, and animal feed. Peanuts and corn are the most easily contaminated, and the probability of aflatoxins appearing in food and feed in hot and humid areas is the highest.

[0003] AFT is highly toxic to both humans and animals, but the toxicity of different types of AFT varies greatly. AFB1 is the most carcinogenic poison known so far. Research by the International Agency for Cancer shows that there are sufficient human epidemiological and animal experiments to prove that AFB1 is highly carcinogenic, mutagenic and teratogenic. AFB1 is not only extremely acutely toxic, but also chronically toxic. If a large amount of food contaminated by AFB1 is ingested in a short period of time, it will cause severe liver damage and bile duct hyperplasia, endangering life; if a certain amount of AFB1 is continuously ingested over a period of time, it will manifest as growth disorders and chronic liver damage. Therefore, it is necessary to establish rapid detection technology for mycotoxins in food and its products to provide good protection for food safety and people's health.

[0004] At present, the commonly used methods for detecting aflatoxin include instrumental analysis: high performance liquid chromatography, liquid chromatography-mass spectrometry; immunoassay: enzyme-linked immunosorbent assay, colloidal gold immunochromatography; other detection methods: surface Raman spectroscopy, biosensor method, etc. The existing detection methods have high sensitivity and good accuracy, but the instruments are expensive and require professional operation. Therefore, it is urgent to build a simple-to-operate and fast-responding sensor detection technology to achieve rapid screening of AFB1. Summary of the invention

[0005] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a sensing detection technology with simple operation and rapid response to achieve rapid and label-free detection of fungal toxins in food.

[0006] In order to achieve the above object, a first aspect of the present invention provides a COF inverse opal photonic crystal (COF-IOPC), wherein the COF inverse opal photonic crystal is prepared by a method comprising the following steps:

[0007] (1) SiO2 photonic crystals were prepared by confined self-assembly method;

[0008] (2) allowing the COF solution to cover the SiO2 photonic crystal;

[0009] (3) After etching SiO2 microspheres with HF, the COF inverse opal photonic crystal is obtained.

[0010] The COF solution can be covered by a dripping method or a siphoning method, as long as the volume of the COF solution can cover the SiO2 photonic crystal.

[0011] Specifically, the preparation method of COF-IOPC includes the following steps:

[0012] (1) The PDMS fence is adhered to a glass slide, and then the SiO2 microsphere suspension is dropped into the PDMS fence. After drying, a tightly packed SiO2 photonic crystal is obtained, and the PDMS fence is removed;

[0013] (ii) dropping a COF solution onto one side of a glass slide on which a SiO2 photonic crystal is deposited, and then covering and fixing the slide with a piece of organic glass. After the slide is sealed and left to stand for a period of time at room temperature, the slide is immersed in an HF solution to etch the SiO2 microspheres. After the etching is completed, the slide is rinsed with deionized water to obtain the COF inverse opal photonic crystal.

[0014] According to a preferred embodiment of the present invention, the size of the SiO2 microspheres is 150 to 300 nm.

[0015] According to a preferred embodiment of the present invention, the COF solution is prepared from 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinylterephthalaldehyde; specifically, the preparation method of the COF solution comprises the following steps:

[0016] The method is prepared by mixing 1,3,5-tris(4-aminophenyl)benzene, 2,5-divinylterephthalaldehyde and acetonitrile, dissolving them by ultrasonication, and then adding glacial acetic acid to mix them evenly. The weight ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinylterephthalaldehyde is preferably 1.1 to 1.3:1.

[0017] According to a preferred embodiment of the present invention, the concentration of the SiO2 microsphere suspension is 0.05-2% (m:v).

[0018] According to a preferred embodiment of the present invention, the sealed and standing time at room temperature is 12 hours to 72 hours.

[0019] According to a preferred embodiment of the present invention, the concentration of the HF solution is 1-3% (v:v).

[0020] According to a preferred embodiment of the present invention, the etching time in the HF solution is 12 to 36 hours.

[0021] The second aspect of the present invention provides the use of the above COF inverse opal photonic crystal in label-free detection of AFB1.

[0022] The third aspect of the present invention provides a method for label-free detection of AFB1, comprising the following steps:

[0023] (1) Fixing the COF inverse opal photonic crystal at the bottom of a container, adding methanol aqueous solution to stabilize the material, using a fiber optic spectrometer to detect, and recording the reflection peak intensity;

[0024] (2) adding the sample to be tested to the above system, detecting it with a fiber optic spectrometer, recording the reflection peak intensity, and calculating the change in the reflection peak intensity before and after the addition of the sample to be tested;

[0025] (3) Substituting the change in the reflection peak intensity into the standard curve of the response of the COF inverse opal photonic crystal to AFB1, the content of AFB1 in the sample to be tested is calculated.

[0026] The sample to be tested may be a solid powder that may contain AFB1, including but not limited to corn flour, wheat flour, milk powder, etc.

[0027] When the sample to be tested is solid powder, the method further comprises pre-treating the sample to be tested: mixing the solid powder with a solvent by oscillation, centrifuging and taking the supernatant for testing. The solvent is an organic solvent capable of dissolving AFB1, such as methanol.

[0028] According to a preferred embodiment of the present invention, the method for preparing the response standard curve of the COF inverse opal photonic crystal to AFB1 comprises the following steps:

[0029] (1) using methanol and aqueous solution to prepare a series of AFB1 standard solutions with a concentration gradient;

[0030] (2) Fixing the COF inverse opal photonic crystal at the bottom of the container, adding methanol aqueous solution to stabilize the material, using a fiber optic spectrometer to detect, and recording the reflection peak intensity;

[0031] (3) adding AFB1 standard solution to the above system in order from low concentration to high concentration, detecting with a fiber optic spectrometer, recording the reflection peak intensity, and calculating the change in the reflection peak intensity before and after the solution is added;

[0032] (4) With the concentration of the AFB1 standard solution as the abscissa and the change in the reflection peak intensity as the ordinate, a standard curve of the response of the COF inverse opal photonic crystal to AFB1 is prepared.

[0033] Compared with other detection methods, the COF-IOPC of the present invention does not require additional labeling, is simple to operate, and can achieve rapid detection of AFB1 with only a fiber optic spectrometer. In the process of preparing the COF-IOPC material, the present invention optimizes the method used for synthesizing the material, the ratio of the ligand used in the COF solution, the volume used for dropping the COF solution, and the reaction time of the COF-PC. The COF-IOPC material prepared under the optimal conditions can more accurately and sensitively identify AFB1, and when immersed in a 10% methanol aqueous solution, it has good stability and can be stored for a long time.

[0034] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0036] Figure 1 Schematic diagram of PDMS fence.

[0037] Figure 2 SEM scanning electron microscope images, optical images and reflection spectra of SiO2 PC; (A) SEM image of SiO2 PC (top view); (B) SEM image of SiO2 PC (side view); (C) reflection spectrum of SiO2 PC (λ=503nm, inset: optical image of green SiO2 PC).

[0038] Figure 3 The SEM scanning electron microscope images, optical images and reflectance spectrum of COF-IOPC; (A) SEM image of COF-IOPC; (B) reflectance spectrum of COF-IOPC (inset: optical image of yellow-green COF-IOPC).

[0039] Figure 4 The response results of COF-IOPC to AFB1; (A) the response kinetics of COF-IOPC to AFB1; (B) the optical response of COF-IOPC to AFB1; (C) the standard curve of COF-IOPC detection of AFB1.

[0040] Figure 5 The response specificity results of COF-IOPC to (A) ZEN; (B) T-2; (C) OTA. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0042] Example 1

[0043] This example is used to illustrate a novel COF inverse opal photonic crystal (COF-IOPC) for label-free detection of AFB1 and its preparation, which specifically includes the following steps:

[0044] (1) Add 1 mL of 10% (m:v) 230 nm SiO2 microsphere suspension to 20 mL of deionized water, sonicate, and centrifuge (8000 rpm, 15 min);

[0045] (2) After washing three times, pour off the supernatant and add deionized water to prepare 10 mL of 1% (m:v) SiO2 microsphere suspension;

[0046] (3) The prepared PDMS fence was adhered to the glass slide substrate, and then 0.3 mL of 1% (m:v) microsphere suspension was dropped onto the PDMS fence using the confined self-assembly method ( Figure 1 ), put it in a 60℃ drying oven and dry it for 2h, remove the PDMS fence, and obtain SiO2 PC for use; the SEM scanning electron microscope image, optical image and reflectance spectrum of SiO2 PC are shown in Figure 2 As shown, it is shown that SiO2 photonic crystal is obtained;

[0047] (4) Preparation of COF solution: Accurately weigh 0.014 g TPB (1,3,5-tris(4-aminophenyl)benzene) and 0.011 g DVA (2,5-divinylterephthalaldehyde) into a brown glass bottle, then add 5 mL acetonitrile (ACN) and sonicate for 3 min. After the powder is completely dissolved, add 0.7 mL glacial acetic acid (HAc) and mix well. After thorough mixing, the solution turns milky yellow and contains floccules.

[0048] (5) Preparation of COF-IOPC: 0.1 mL of COF solution was added dropwise to the side of the glass slide with SiO2 PC, and then a piece of PMMA organic glass was placed to cover the SiO2 PC and fixed with a dovetail clip. After being sealed and left to stand at room temperature for 48 h, it was immersed in a HF solution with a mass volume fraction of 2% (v:v) for 24 h to etch the SiO2 microspheres; after etching, it was rinsed with deionized water for 3-5 times, and then immersed in a 10% methanol aqueous solution for use. The SEM scanning electron microscope image, optical image and reflectance spectrum of COF-IOPC are shown in Figure 2. Figure 3 As shown in (A) and (B) and the inset, COF-IOPC was prepared.

[0049] Example 2

[0050] This example is used to illustrate the establishment of a standard curve of the response of COF-IOPC to AFB1.

[0051] (1) Using 10% methanol aqueous solution (v:v), a 1 mg / mL methanol solution of AFB1 standard was diluted to 50 μg / mL, 40 μg / mL, 30 μg / mL, and 20 μg / mL; and then diluted to 10 μg / mL, 5 μg / mL, and 1 μg / mL using deionized water;

[0052] (2) The COF-IOPC material was fixed to the bottom of a glass dish with double-sided tape, 10 mL of 10% methanol aqueous solution (v:v) was added to stabilize the material, and optical fiber detection was performed until the spectral image was stable and unchanged, and the reflection peak intensity was recorded;

[0053] (3) Then, 0.1 mL of AFB1 solution was added sequentially from low concentration to high concentration. After each addition, 30 min was waited until the spectral image no longer changed. The reflection peak intensity was recorded and the change in reflection peak intensity was calculated.

[0054] (4) With the concentration of AFB1 in the detection system as the horizontal axis and the change in the reflection peak intensity as the vertical axis, a standard curve y = 0.24783x + 4.16388, R 2 =0.9872, linear range 1μg / mL-50μg / mL, minimum detection limit 4.05μg / mL.

[0055] Figure 4 The response results of COF-IOPC to AFB1; (A) Response kinetics of COF-IOPC to AFB1; (B) Optical response of COF-IOPC to AFB1; (C) Standard curve of COF-IOPC detection of AFB1. It can be seen that COF-IOPC has a good response to AFB1.

[0056] Example 3

[0057] This example is used to illustrate the response-specific detection of COF-IOPC to (A) ZEN; (B) T-2; (C) OTA.

[0058] (A) Specific detection of COF-IOPC response to ZEN

[0059] 1) Use 10% methanol aqueous solution (v:v) to dilute the 1 mg / mL ZEN standard methanol solution to 50 μg / mL, 40 μg / mL, 30 μg / mL, and 20 μg / mL; use deionized water to dilute to 10 μg / mL, 5 μg / mL, and 1 μg / mL;

[0060] 2) Fix the COF-IOPC material to the bottom of a glass dish with double-sided tape, add 10 mL of 10% methanol aqueous solution (v:v) to stabilize the material, and use a fiber optic spectrometer to detect the reflectance spectrum image of the COF-IOPC until the image is stable and unchanged, and record it;

[0061] 3) Then, 0.1 mL of ZEN solution was added in order from low concentration to high concentration. After each addition, wait for 30 minutes until the spectral image no longer changes, and then record the spectral image. Figure 5 As shown in A.

[0062] (B) Specific detection of COF-IOPC response to T-2

[0063] 1) Use 10% methanol aqueous solution (v:v) to dilute 1 mg / mL T-2 standard methanol solution to 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL; use deionized water to dilute to 10 μg / mL, 5 μg / mL, 1 μg / mL;

[0064] 2) Fix the COF-IOPC material to the bottom of a glass dish with double-sided tape, add 10 mL of 10% methanol aqueous solution (v:v) to stabilize the material, and use a fiber optic spectrometer to detect the reflectance spectrum image of the COF-IOPC until the image is stable and unchanged, and record it;

[0065] 3) Then, 0.1 mL of T-2 solution was added in order from low concentration to high concentration. After each addition, wait for 30 minutes until the spectral image no longer changes, and record the spectral image. Figure 5 As shown in B.

[0066] (C) Specific detection of COF-IOPC response to OTA

[0067] 1) Use 10% methanol aqueous solution (v:v) to dilute the 1 mg / mL OTA standard methanol solution to 50 μg / mL, 40 μg / mL, 30 μg / mL, and 20 μg / mL; and use deionized water to dilute to 10 μg / mL, 5 μg / mL, and 1 μg / mL;

[0068] 2) Fix the COF-IOPC material to the bottom of a glass dish with double-sided tape, add 10 mL of 10% methanol aqueous solution (v:v) to stabilize the material, and use a fiber optic spectrometer to detect the reflectance spectrum image of the COF-IOPC until the image is stable and unchanged, and record it;

[0069] 3) Then, 0.1 mL of OTA solution was added in order from low concentration to high concentration, and after each addition, 30 min was waited until the spectral image no longer changed, and the spectral image was recorded. Figure 5 As shown in C.

[0070] Depend on Figure 5 It can be seen that COF-IOPC has no response to other small molecule toxins ZEN, T-2, and OTA, which proves its specificity in detecting AFB1.

[0071] Example 4

[0072] This example is used to illustrate the use of COF-IOPC for actual sample detection.

[0073] (1) Sample pretreatment: Choose any solid powder (corn flour, wheat flour), weigh 5 g of the powder into a 50 mL centrifuge tube, add a known concentration of AFB1 methanol aqueous solution (70:30, v:v), vortex mix, place in a shaker at 140 rpm for 20 min, centrifuge at 6000 rpm for 10 min, and take the supernatant for later use;

[0074] (2) Actual sample detection: COF-IOPC was immersed in 10 mL of 10% methanol aqueous solution (v:v), and the fiber probe of the fiber optic spectrometer was immersed below the liquid surface. After the fiber optic spectrometer was stable, 0.1 mL of the above sample treatment solution was added to the detection system, and the sample was left for 30 min. The reflection peak intensity before and after the addition of the sample was recorded, and the change in the reflection peak intensity was calculated.

[0075] (3) Substitute the change in reflection peak intensity into the standard curve, calculate the concentration of AFB1 and compare it with the spiked concentration. The results are shown in Table 1.

[0076] (4) The GB5009.22-2016 method was used to perform spike recovery tests on the above samples. The results are listed in Table 1.

[0077] Table 1 Results of COF-IOPC testing of spiked samples (n=3)

[0078]

[0079] Nd: Not detected.

[0080] It can be seen from the results in Table 1 that the sample concentration measured by the method of the present invention is close to the actual concentration, and the relative standard deviation is significantly lower than that of the national standard method, indicating that the method of detecting AFB1 using the COF inverse opal photonic crystal of the present invention has good accuracy.

[0081] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A COF inverse opal photonic crystal, characterized in that: The COF inverse opal photonic crystal is prepared by a method comprising the following steps: (1) SiO2 photonic crystals were prepared by confined self-assembly method; (2) allowing the COF solution to cover the SiO2 photonic crystal; (3) After etching SiO2 microspheres with HF, the COF inverse opal photonic crystal is obtained.

2. The COF inverse opal photonic crystal according to claim 1, characterized in that: The COF inverse opal photonic crystal is prepared by a method comprising the following steps: (1) The PDMS fence is adhered to a glass slide, and then the SiO2 microsphere suspension is dropped into the PDMS fence. After drying, a tightly packed SiO2 photonic crystal is obtained, and the PDMS fence is removed; (ii) dropping a COF solution onto one side of a glass slide on which a SiO2 photonic crystal is deposited, and then covering and fixing the slide with a piece of organic glass. After the slide is sealed and left to stand for a period of time at room temperature, the slide is immersed in an HF solution to etch the SiO2 microspheres. After the etching is completed, the slide is rinsed with deionized water to obtain the COF inverse opal photonic crystal.

3. The COF inverse opal photonic crystal according to claim 1 or 2, characterized in that: The size of the SiO2 microspheres is 150-300 nm.

4. The COF inverse opal photonic crystal according to claim 1 or 2, characterized in that: The COF solution is prepared from 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinylterephthalaldehyde.

5. The COF inverse opal photonic crystal according to claim 4, characterized in that: The preparation method of the COF solution comprises the following steps: The method comprises mixing 1,3,5-tris(4-aminophenyl)benzene, 2,5-divinylterephthalaldehyde and acetonitrile, dissolving the mixture by ultrasonic, and then adding glacial acetic acid to mix the mixture evenly; the weight ratio of the 1,3,5-tris(4-aminophenyl)benzene to the 2,5-divinylterephthalaldehyde is 1.1-1.3:

1.

6. The COF inverse opal photonic crystal according to claim 1, characterized in that: The concentration of the SiO2 microsphere suspension is 0.05-2% (m:v); The sealed and standing time at room temperature is 12h to 72h; The concentration of the HF solution is 1 to 3% (v:v); the etching time in the HF solution is 12 to 36 hours.

7. Use of the COF inverse opal photonic crystal according to any one of claims 1 to 6 in label-free detection of AFB1.

8. A method for label-free detection of AFB1, characterized in that: The following steps are involved: (1) Fixing the COF inverse opal photonic crystal at the bottom of a container, adding methanol aqueous solution to stabilize the material, using a fiber optic spectrometer for detection, and recording the reflection peak intensity; (2) adding the sample to be tested to the above system, detecting it with a fiber optic spectrometer, recording the reflection peak intensity, and calculating the change in the reflection peak intensity before and after the addition of the sample to be tested; (3) Substituting the change in the reflection peak intensity into the standard curve of the response of the COF inverse opal photonic crystal to AFB1, the content of AFB1 in the sample to be tested is calculated.

9. The method according to claim 8, characterized in that The method for preparing the response standard curve of the COF inverse opal photonic crystal to AFB1 comprises the following steps: (1) using methanol and aqueous solution to prepare a series of AFB1 standard solutions with a concentration gradient; (2) Fixing the COF inverse opal photonic crystal at the bottom of the container, adding methanol aqueous solution to stabilize the material, using a fiber optic spectrometer to detect, and recording the reflection peak intensity; (3) adding AFB1 standard solution to the above system in order from low concentration to high concentration, detecting with a fiber optic spectrometer, recording the reflection peak intensity, and calculating the change in the reflection peak intensity before and after the solution is added; (4) With the concentration of the AFB1 standard solution as the abscissa and the change in the reflection peak intensity as the ordinate, a standard curve of the response of the COF inverse opal photonic crystal to AFB1 is prepared.

10. The method according to claim 8, characterized in that The sample to be tested is solid powder, and the method further comprises pre-treating the sample to be tested: mixing the solid powder with a solvent by oscillation, and centrifuging to obtain a supernatant for testing.

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