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

Abstract

The application 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 using a confined self-assembly method; (2) covering the SiO2 photonic crystal with a COF solution; and (3) preparing the COF inverse opal photonic crystal after etching the SiO2 microspheres with HF. Compared with other detection methods, the COF inverse opal photonic crystal of the application does not need additional labeling, is simple to operate, and can realize rapid detection of AFB1 only by using a fiber spectrometer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of small molecule toxin detection, and particularly relates to a COF inverse opal photonic crystal and application thereof, and a method for label-free detection of AFB1. BACKGROUND

[0002] AFT and its producing bacteria are widely distributed in nature, and some strains produce more than one type of AFT. AFT can be detected in the seeds and processed products of grain and oil crops, dried and fresh fruits, condiments, tobacco, milk and dairy products, meat, fish and animal feed. Among them, peanuts and corn are most easily contaminated, and AFT is most likely to occur in food and feed in hot and humid areas.

[0003] AFT is highly toxic to humans and animals, but the toxicity of different types of AFT varies greatly, and AFB1 is the most carcinogenic poison known. International Cancer Agency studies have shown that there is sufficient human epidemiological and animal experimental evidence that AFB1 has high carcinogenicity, mutagenicity and teratogenicity. AFB1 not only has strong acute toxicity, but also has chronic toxicity. If a large amount of food contaminated with AFB1 is ingested in a short period of time, it can cause severe liver damage and bile duct hyperplasia, which is life-threatening. If a certain amount of AFB1 is ingested continuously over a period of time, it will show growth retardation and chronic liver damage. Therefore, it is necessary to establish a rapid detection technology for mycotoxins in food and its products to provide good protection for food safety and human health.

[0004] The commonly used detection methods for AFT at present include instrument analysis methods: high performance liquid chromatography, liquid chromatography-mass spectrometry; immunoassay methods: 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 need to be operated by professionals. Therefore, it is urgent to develop a simple and rapid sensor detection technology to realize the rapid screening of AFB1. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a simple and rapid sensor detection technology to realize the rapid label-free detection of mycotoxins in food.

[0006] To achieve the above-mentioned purpose, the first aspect of the present application provides a COF inverse opal photonic crystal (COF-IOPC), which is prepared by a method comprising the following steps:

[0007] (1) SiO2 photonic crystal is prepared by using a confined self-assembly method;

[0008] (2) covering the SiO2 photonic crystal with a COF solution;

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

[0010] The covering of the COF solution with the SiO2 photonic crystal can be achieved by dropwise addition or siphoning, as long as the volume of the COF solution can cover the SiO2 photonic crystal.

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

[0012] (1) adhering a PDMS fence to a glass slide, then dropwise adding a SiO2 microsphere suspension into the PDMS fence, drying to obtain a close-packed SiO2 photonic crystal, and removing the PDMS fence;

[0013] (ii) dropwise adding a COF solution to the side of the glass slide on which the SiO2 photonic crystal is deposited, then covering with a piece of organic glass and fixing, sealing at room temperature for a period of time, then immersing in a HF solution to etch the SiO2 microspheres, and after etching, washing with deionized water to obtain the COF inverse opal photonic crystal.

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

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

[0016] Mixing 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl terephthaldehyde with acetonitrile, ultrasonically dissolving, then adding glacial acetic acid and mixing to obtain. The weight ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinyl terephthaldehyde is preferably 1.1-1.3:1.

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

[0018] According to a preferred embodiment of the present application, the time for sealing at room temperature is 12-72 h.

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

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

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

[0022] The third aspect of the present application 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 stabilizing material, and detecting by using a fiber spectrometer to record the reflection peak intensity;

[0024] (2) adding the sample to be detected to the above system, detecting by using a fiber spectrometer to record the reflection peak intensity, and calculating the change of the reflection peak intensity before and after adding the sample to be detected;

[0025] (3) substituting the change of the reflection peak intensity into the standard curve of the COF inverse opal photonic crystal responding to AFB1 to calculate the content of AFB1 in the sample to be detected.

[0026] The sample to be detected can be a solid powder possibly containing AFB1, including but not limited to corn flour, wheat flour, milk powder, etc.

[0027] When the sample to be detected is a solid powder, the method further comprises pretreatment of the sample to be detected: oscillating mixing the solid powder with a solvent, and centrifuging to take supernatant for detection. The solvent is an organic solvent capable of dissolving AFB1, for example, methanol.

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

[0029] (1) preparing AFB1 standard samples into a series of AFB1 standard sample solutions with concentration gradient by using methanol aqueous solution;

[0030] (2) fixing the COF inverse opal photonic crystal at the bottom of a container, adding methanol aqueous solution stabilizing material, and detecting by using a fiber spectrometer to record the reflection peak intensity;

[0031] (3) adding the AFB1 standard sample solution to the above system in order from low concentration to high concentration, detecting by using a fiber spectrometer to record the reflection peak intensity, and calculating the change of the reflection peak intensity before and after adding the solution;

[0032] (4) taking the concentration of the AFB1 standard sample solution as the abscissa and the change of the reflection peak intensity as the ordinate to make the standard curve of the COF inverse opal photonic crystal responding to AFB1.

[0033] Compared with other detection methods, the COF-IOPC of the application does not need additional labeling, is simple to operate, and can realize rapid detection of AFB1 only by using a fiber spectrometer. The application optimizes the method for preparing the COF-IOPC material, the proportion of the ligand for the COF solution, the volume of the COF solution for dropwise addition, and the reaction time of the COF-PC in the process of preparing the COF-IOPC material, and the COF-IOPC material prepared under the optimal conditions can more accurately and sensitively identify AFB1, is better in stability and can be stored for a long time after being soaked in 10% methanol aqueous solution.

[0034] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0035] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0036] Figure 1 Schematic diagram of PDMS fence.

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

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

[0039] Figure 4 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 for detecting AFB1.

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

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

[0042] Example 1

[0043] The present embodiment is used to illustrate a new COF inverse opal photonic crystal (COF-IOPC) for label-free detection of AFB1 and its preparation, which specifically comprises the following steps:

[0044] (1) 1 mL of 10% (m:v) 230 nm SiO2 microsphere suspension was added to 20 mL of deionized water, ultrasonic, centrifugation (8000 rpm, 15 min);

[0045] (2) After washing with water for three times, the supernatant was poured out, and 10 mL of 1% (m:v) SiO2 microsphere suspension was prepared by adding deionized water;

[0046] (3) The prepared PDMS fence was adhered on the glass slide substrate, then 0.3 mL of 1% (m:v) microsphere suspension was added dropwise in the PDMS fence (as shown in Figure 1 ) and placed in a 60°C drying oven for 2 h, the PDMS fence was removed, and SiO2 PC was obtained for standby; the SEM scanning electron microscope image, optical image and reflection spectrum of SiO2 PC are shown in Figure 2 , which illustrates that SiO2 photonic crystal is obtained;

[0047] (4) Preparation of COF solution: 0.014 g of TPB (1,3,5-tris (4-aminophenyl) benzene) and 0.011 g of DVA (2,5-divinyl benzene formaldehyde) were accurately weighed in a brown glass bottle, then 5 mL of acetonitrile (ACN) was added, ultrasonic was dissolved for 3 min, after the powder was completely dissolved, 0.7 mL of glacial acetic acid (HAc) was added and mixed, and the solution turned to milk yellow color and contained flocculation after sufficient mixing;

[0048] (5) Preparation of COF-IOPC: 0.1 mL of COF solution was added dropwise on the side of the glass slide with SiO2 PC, then a piece of PMMA organic glass was covered on the SiO2 PC and fixed with a dovetail clamp, after 48 h of sealed standing at room temperature, the SiO2 microspheres were etched in 2% (v:v) HF solution for 24 h; after etching, the sample was washed with deionized water for 3-5 times, then it was immersed in 10% methanol aqueous solution for standby. The SEM scanning electron microscope image, optical image and reflection spectrum of COF-IOPC are shown in Figure 3 (A) and (B) and the inserted image, which illustrates that COF-IOPC is prepared.

[0049] Example 2

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

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

[0052] (2) COF-IOPC material was fixed on 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 sequentially added from low concentration to high concentration, and after each addition, it was waited for 30 min until the spectral image no longer changed, the reflection peak intensity was recorded, and the change of the reflection peak intensity was calculated;

[0054] (4) The concentration of AFB1 in the detection system was taken as the abscissa, and the change of the reflection peak intensity was taken as the ordinate, to draw the standard curve y=0.24783x+4.16388, R 2 =0.9872, the linear range was 1 μg / mL-50 μg / mL, and the lowest detection limit was 4.05 μg / mL.

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

[0056] Example 3

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

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

[0059] 1) 1 mg / mL ZEN standard methanol solution was diluted to 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL with 10% methanol aqueous solution (v:v), and then diluted to 10 μg / mL, 5 μg / mL, 1 μg / mL with deionized water;

[0060] 2) Fix the COF-IOPC material to the bottom of a glass petri 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 of COF-IOPC until the image stabilizes and remains unchanged, and record the result.

[0061] 3) Subsequently, add 0.1 mL of ZEN solution sequentially from low to high concentration, waiting 30 min after each addition until the spectral image no longer changes, and then record the spectral image. The results are as follows: 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 serially dilute the 1 mg / mL T-2 standard methanol solution to 50 μg / mL, 40 μg / mL, 30 μg / mL, and 20 μg / mL; use deionized water to serially dilute to 10 μg / mL, 5 μg / mL, and 1 μg / mL.

[0064] 2) Fix the COF-IOPC material to the bottom of a glass petri 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 of COF-IOPC until the image stabilizes and remains unchanged, and record the result.

[0065] 3) Subsequently, add 0.1 mL of T-2 solution sequentially from low to high concentration, waiting 30 min after each addition until the spectral image no longer changes, and record the spectral image. The results are as follows: Figure 5 As shown in B.

[0066] (C) COF-IOPC response specificity detection for OTA

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

[0068] 2) Fix the COF-IOPC material to the bottom of a glass petri 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 of COF-IOPC until the image stabilizes and remains unchanged, and record the result.

[0069] 3) Subsequently, 0.1 mL of OTA solution was added sequentially from low to high concentration, waiting 30 minutes after each addition until the spectral image no longer changed, and then the spectral image was recorded. The results are as follows: Figure 5 As shown in C.

[0070] By Figure 5 It can be seen that COF-IOPC has no response to other small molecule toxins ZEN, T-2 and OTA, proving the specificity of AFB1 detection.

[0071] Example 4

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

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

[0074] (2) Actual sample detection: immerse COF-IOPC in 10mL 10% methanol aqueous solution (v:v), immerse the optical fiber probe of the optical fiber spectrometer below the liquid surface, after the optical fiber spectrometer is stable, add 0.1mL of the above sample treatment liquid dropwise to the detection system, wait for 30min, record the reflection peak intensity before and after the sample is added, calculate the change of the reflection peak intensity;

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

[0076] (4) Use GB5009.22-2016 method to test the sample for added standard recovery, the results are listed in Table 1.

[0077] Table 1 COF-IOPC detection of added standard sample results (n=3)

[0078]

[0079] Nd: not detected.

[0080] As can be seen from the results in Table 1, the sample concentration determined by the method of the present application 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 COF inverse opal photonic crystal of the present application has good accuracy.

[0081] The above has described various embodiments of the present application, 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 method for label-free detection of AFB1, characterized in that, The method comprises the following steps: (1) fixing the COF inverse opal photonic crystal at the bottom of a container, adding methanol aqueous solution stabilizing material, detecting by using a fiber spectrometer, and recording the reflection peak intensity; (2) adding the sample to be detected into the above system, detecting by using the fiber spectrometer, recording the reflection peak intensity, and calculating the change of the reflection peak intensity before and after adding the sample to be detected; (3) substituting the change of the reflection peak intensity into the standard curve of the COF inverse opal photonic crystal responding to AFB1, and calculating the content of AFB1 in the sample to be detected; The COF inverse opal photonic crystal is prepared by a method comprising the following steps: (1) adhering a PDMS fence on a glass slide, then dropping a SiO2 microsphere suspension into the PDMS fence, and obtaining a SiO2 photonic crystal in a close-packed form after drying, and removing the PDMS fence; (ii) dropping the COF solution onto the side of the glass slide on which the SiO2 photonic crystal is deposited, then covering an organic glass and fixing, after sealing at room temperature for a period of time, etching the SiO2 microspheres in a HF solution, washing with deionized water after etching, and obtaining the COF inverse opal photonic crystal.

2. The method of claim 1, wherein, The method for preparing the standard curve of the COF inverse opal photonic crystal responding to AFB1 comprises the following steps: (1) preparing AFB1 standard samples into a series of AFB1 standard sample solutions with concentration gradients by using methanol aqueous solution; (2) fixing the COF inverse opal photonic crystal at the bottom of a container, adding methanol aqueous solution stabilizing material, detecting by using a fiber spectrometer, and recording the reflection peak intensity; (3) adding the AFB1 standard sample solution into the above system in order from low concentration to high concentration, detecting by using the fiber spectrometer, recording the reflection peak intensity, and calculating the change of the reflection peak intensity before and after adding the solution; (4) taking the concentration of the AFB1 standard sample solution as the abscissa and the change of the reflection peak intensity as the ordinate, and preparing the standard curve of the COF inverse opal photonic crystal responding to AFB1.

3. The method of claim 1, wherein, The sample to be detected is a solid powder, and the method further comprises pretreating the sample to be detected: mixing the solid powder with a solvent by oscillation, centrifuging to take supernatant for detection.

4. The method of claim 1, wherein, The size of the SiO2 microspheres is 150-300 nm.

5. The method of claim 1, wherein, The COF solution is prepared from 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl terephthalaldehyde.

6. The method of claim 1, wherein, The method for preparing the COF solution comprises the following steps: mixing 1,3,5-tris(4-aminophenyl)benzene, 2,5-divinyl terephthalaldehyde and acetonitrile, dissolving under ultrasonic, then adding glacial acetic acid and mixing uniformly to obtain; the weight ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinyl terephthalaldehyde is 1.1-1.3:

1.

7. The method of claim 1, wherein, The concentration of the SiO2 microsphere suspension is 0.05-2% (m:v); The time for sealing at room temperature is 12-72 h; The concentration of the HF solution is 1-3% (v:v); the etching time in the HF solution is 12-36 h.

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