A fluorescent bionic identification test paper for detecting tetracycline and its application

Abstract

The present invention discloses a fluorescent bionic identification test paper for detecting tetracycline and its application, belonging to the technical field of food detection. The preparation method of the fluorescent bionic identification test paper for detecting tetracycline comprises the following steps: (1) synthesis of luminescent metal organic framework FP@BA‑Eu functionalized with boric acid; (2) synthesis of luminescent metal organic framework molecular imprinting polymer FP@BA‑Eu@MIP / NIP functionalized with boric acid. The fluorescent bionic identification test paper for detecting tetracycline prepared by the present invention has high sensitivity and can quickly detect the content of tetracycline in food.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a fluorescent bionic identification test paper for detecting tetracycline and an application thereof. Background Art

[0002] Tetracyclines have broad-spectrum and highly effective bactericidal and antibiotic effects, can be mixed with most drugs or feed additives, and have been widely used in livestock and poultry production. However, excessive use inevitably causes serious drug residues, becoming one of the food safety risks that seriously endangers human health.

[0003] Traditional methods for the detection of tetracycline antibiotics mainly include liquid chromatography-spectrometry, capillary electrophoresis, high performance liquid chromatography, gas chromatography-mass spectrometry and supercritical fluid chromatography. These methods can analyze and identify specific group structures in antibiotic molecules, thereby achieving qualitative and quantitative analysis and detection of antibiotic content in food. However, there are limitations such as cumbersome pretreatment methods, long time consumption, high cost, and the need for professional skills, which is not conducive to rapid detection. Therefore, how to simply, quickly and accurately detect tetracycline antibiotics is an urgent problem to be solved. In recent years, in order to solve the defects of traditional methods, corresponding methods such as electrochemistry, fluorescence sensing, enzyme-linked immunosorbent assay and colorimetry have been studied and applied to the detection of tetracycline antibiotics; however, these methods still have the disadvantages of complex operation, high cost and high toxicity. Summary of the invention

[0004] One of the purposes of the present invention is to provide a method for preparing a fluorescent bionic identification test paper for detecting tetracycline, the method for preparing the fluorescent bionic identification test paper for detecting tetracycline comprising the following steps:

[0005] (1) Synthesis of boronic acid functionalized luminescent metal organic framework FP@BA-Eu

[0006] ① Soak the qualitative filter paper in a 0.1-0.3 M hydrochloric acid solution, then rinse it with ultrapure water and dry it in a vacuum drying oven to obtain FP; then place FP in a sodium poly(p-styrene sulfonate) solution for ultrasonic treatment for 20-30 minutes, then rinse it with ultrapure water and dry it in a vacuum drying oven to obtain FP@PSS;

[0007] ②Synthesis of FP@BA-Eu: Eu(NO 3 ) 3 6H 2 O and BBDC were added to DMF / H 2 In the mixed solution of DMF and H 2 O volume ratio is 6-8:2-4, Eu(NO 3 ) 3 6H 2The final concentrations of O and BBDC were both 8-12 mmol / L, and ultrasonic treatment was performed for 10-20 min. The mixed solution was then transferred into a polytetrafluoroethylene-lined stainless steel reactor, and FP@PSS was added. The mixture was heated at 115-125°C for 10-14 hours, and cooled to room temperature to obtain FP@BA-Eu. The obtained material FP@BA-Eu was washed with DMF and ethanol, and dried in a vacuum drying oven.

[0008] (2) Synthesis of Boronic Acid Functionalized Luminescent Metal-Organic Framework Molecular Imprinting Polymer FP@BA-Eu@MIP / NIP

[0009] FP@BA-Eu is added to a TC solution prepared with an ammonium bicarbonate buffer solution, wherein the concentration of the TC solution is 0.5-1.5 mg / mL, and the mixture is shaken at room temperature for 1-3 hours. After the reaction is completed, the mixture is rinsed with an ammonium bicarbonate buffer solution and ethanol, and after naturally drying, the mixture is immersed in a mixed solution of ethanol and ammonia water, and a certain amount of prepolymer solution is added. The mixture is shaken at room temperature for a certain period of time, and the mixture is rinsed with ultrapure water and ethanol respectively, and dried in a vacuum drying oven. Subsequently, the mixture is repeatedly eluted with an acetic acid solution until the template molecule TC is undetectable, and the mixture is rinsed with ultrapure water and dried in a vacuum drying oven, thereby obtaining a fluorescent bionic identification test paper FP@BA-Eu@MIP for detecting tetracycline. The prepolymer solution refers to a mixed solution of a crosslinking agent TEOS, a functional monomer IBTES, and ethanol.

[0010] Preferably, the concentration of the hydrochloric acid solution in step (1) is 0.2M.

[0011] More preferably, in step (1), Eu(NO 3 ) 3 6H 2 The final concentrations of O and BBDC were both 10 mmol / L.

[0012] More preferably, in step (1), DMF and H 2 The volume ratio of O is 7:3.

[0013] More preferably, in step (1), the heating temperature is 120° C. and the heating time is 12 h.

[0014] More preferably, the concentration of the TC solution in step (2) is 1 mg / mL.

[0015] More preferably, the concentration of the ammonium bicarbonate buffer solution in step (2) is 0.1 M, and the pH value is 8.5.

[0016] The second object of the present invention is to provide a fluorescent bionic identification test paper for detecting tetracycline prepared by the above preparation method.

[0017] The third object of the present invention is to provide the use of the above-mentioned fluorescent bionic identification test paper in detecting tetracycline.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The fluorescent bionic identification test paper for detecting tetracycline prepared by the invention has high sensitivity and can quickly detect the content of tetracycline in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the synthesis flow chart of FP@BA-Eu@MIP in Example 1.

[0021] Figure 2 This is a diagram of the assembly and detection process of the test strip in Example 1.

[0022] Figure 3 The fluorescence spectra and standard curves of FP@BA-Eu@MIP under different concentrations of TC standard solutions in Example 1. DETAILED DESCRIPTION Example

[0023] 1. Experimental part

[0024] 1.1 Synthesis of Boric Acid Functionalized Luminescent Metal-Organic Framework (FP@BA-Eu)

[0025] The Whatman No. 1 qualitative filter paper was designed using CorelLASER software and cut into multiple circular paper pieces with a diameter of 6 mm using a laser engraving machine. It was soaked in 0.2 M hydrochloric acid solution for 30 min, rinsed with ultrapure water, and dried in a vacuum drying oven to obtain FP. Then, FP was placed in an aqueous solution of sodium poly(p-styrene sulfonate) (average molecular weight Mw-70000) and ultrasonicated for 20-30 min. After that, it was rinsed with ultrapure water and dried in a vacuum drying oven to obtain FP@PSS.

[0026] Synthesis of FP@BA-Eu: Eu(NO 3 ) 3 6H 2 O and BBDC (3,5-dicarboxyphenylboronic acid) were added to DMF (N,N-dimethylformamide) / H 2 O (DMF and H 2 O with a volume ratio of 7:3) in a mixed solution, Eu(NO 3 ) 3 6H 2The final concentrations of O and BBDC were both 10 mmol / L. Ultrasonication was performed for 15 min, and then the mixed solution was transferred into a polytetrafluoroethylene-lined stainless steel reactor. An appropriate number of FP@PSS sheets were added, and the mixture was heated at 120 °C for 12 h. After cooling to room temperature, FP@BA-Eu was obtained. The obtained material FP@BA-Eu was thoroughly washed with DMF and ethanol, and then dried in a vacuum drying oven.

[0027] 1.2 Synthesis of Boronic Acid Functionalized Luminescent Metal-Organic Framework Molecular Imprinting Polymer (FP@BA-Eu@MIP / NIP)

[0028] FP@BA-Eu was added to a TC solution (tetracycline solution, concentration 1 mg / mL) prepared with an ammonium bicarbonate buffer solution (0.1 M, pH = 8.5), and shaken at room temperature for 2 hours. After the reaction, it was rinsed with an ammonium bicarbonate buffer solution (0.1 M, pH = 8.5) and ethanol. After waiting for natural drying, it was immersed in a mixed solution of ethanol and ammonia water (the volume ratio of ethanol and ammonia water was 400:7), and 10 ml of prepolymer solution was added (the prepolymer solution refers to a mixed solution of crosslinker TEOS, functional monomer IBTES, and ethanol; the mixed concentration of crosslinker TEOS and functional monomer IBTES in 10 mL ethanol is 10 mM, and the molar ratio of IBTES to TEOS is 1:100), shaken at room temperature for a certain period of time, rinsed with ultrapure water and ethanol respectively, and placed in a vacuum drying oven for drying. Subsequently, elution was repeated with acetic acid solution until the template molecule TC could not be detected by HPLC method, rinsed with ultrapure water and placed in a vacuum drying oven for drying to obtain FP@BA-Eu@MIP. The specific assembly principle is as follows Figure 1 shown.

[0029] Meanwhile, the non-molecularly imprinted polymer FP@BA-Eu@NIP was obtained under the same polymerization conditions except that the template molecule TC was not added.

[0030] 1.3 Assembling the test strip and fluorescence sensor

[0031] First, assemble the fluorescent test strips, paste the FP@BA-Eu@MIP paper-based chip on one end of the PVC adhesive backing board, then cover the PVC backing board with polyester fiber film, and finally cut the assembled long PVC adhesive backing board into test strips with a length and width of 40×6mm to obtain tetracycline rapid detection test strips.

[0032] For the fluorescence detection of TC, 300 μL of standard solutions of different concentrations were placed in a 96-well plate, and one end of the test strip with the FP@BA-Eu@MIP paper-based chip was placed in the solution. The test strip was left to adsorb for 30 min at room temperature. After the reaction, the strip was taken out and rinsed with buffer three times. The fluorescence signal value was determined at Ex=360 nm using a fluorescence microplate reader. The specific process is as follows: Figure 2 shown.

[0033] 1.4 Detection of TC in actual samples

[0034] Milk, honey, and chicken were purchased from Yonghui Supermarket (Hangzhou, China). The pretreatment process was carried out according to the Chinese national standards (GB / T22990−2008, GB / T 18932.23−2003, Announcement No. 1025 of the Ministry of Agriculture and Rural Affairs−12−2008). Briefly, different concentrations of TC solution were added to the above samples, and Na 2 EDTA-Mcllvaine buffer solution was mixed on a vortex oscillator for 2 min, centrifuged at 8000 r / min for 10 min, the supernatant was taken and the pH was adjusted to 8.5, and then the supernatant was filtered through a 0.22 µm needle filter. The rest of the detection steps were the same as 1.3.

[0035] 2 Results and Discussion

[0036] 2.1 Fluorescence detection results

[0037] The fluorescence spectra after adding different concentrations of TC are shown in Figure 2. Figure 3 As shown in the figure, in the concentration range of 0.01-30 mg / L, an R 2 The linear curve is 0.969, indicating that F 630 / F 440 There is a good linear relationship between the concentration of tetracycline. According to the formula, the detection limit (LOD) and quantification limit (LOQ) of the fluorescence sensor are calculated to be 8.47 and 28.2 μg / L, respectively. (LOD=3SD / S, LOQ=10 SD / S, SD is the standard deviation of the blank measurement value, S is the slope of the standard curve).

[0038] 2.2 Actual samples

[0039] Different concentrations of TC solution (200, 400 and 600 μg / kg) were added to the pretreated milk, honey and chicken samples to investigate the recovery rate. As shown in Table 1, the spiked recoveries of the three different samples ranged from 73.02 to 119.33%.

[0040] Table 1 Determination of TC in real samples. (n=3)

[0041]

[0042] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a fluorescent bionic identification test paper for detecting tetracycline, characterized in that: The preparation method of the fluorescent bionic identification test paper for detecting tetracycline comprises the following steps: (1) Synthesis of Boric Acid Functionalized Luminescent Metal-Organic Framework FP@BA-Eu ① Soak the qualitative filter paper in a 0.1-0.3M hydrochloric acid solution, then rinse it with ultrapure water and dry it in a vacuum drying oven to obtain FP; then place FP in a sodium poly(p-styrene sulfonate) solution for ultrasonic treatment for 20-30 minutes, then rinse it with ultrapure water and dry it in a vacuum drying oven to obtain FP@PSS; ②Synthesis of FP@BA-Eu: Add Eu(NO3)3·6H2O and BBDC to a DMF / H2O mixed solution, the volume ratio of DMF to H2O is 6-8:2-4, the final concentrations of Eu(NO3)3·6H2O and BBDC are both 8-12mmol / L, ultrasonicate for 10-20min, then transfer the mixed solution into a polytetrafluoroethylene-lined stainless steel reactor, add FP@PSS, heat at 115-125°C for 10-14 hours, cool to room temperature to obtain FP@BA-Eu, wash the obtained material FP@BA-Eu with DMF and ethanol, and dry it in a vacuum drying oven; (2) Synthesis of Boronic Acid Functionalized Luminescent Metal-Organic Framework Molecular Imprinting Polymer FP@BA-Eu@MIP / NIP FP@BA-Eu is added to a TC solution prepared with an ammonium bicarbonate buffer solution, wherein the concentration of the TC solution is 0.5-1.5 mg / mL, and the mixture is shaken at room temperature for 1-3 hours. After the reaction is completed, the mixture is rinsed with an ammonium bicarbonate buffer solution and ethanol, and after naturally drying, the mixture is immersed in a mixed solution of ethanol and ammonia water, and a certain amount of prepolymer solution is added. The mixture is shaken at room temperature for a certain period of time, and the mixture is rinsed with ultrapure water and ethanol respectively, and dried in a vacuum drying oven. Subsequently, the mixture is repeatedly eluted with an acetic acid solution until the template molecule TC is undetectable, and the mixture is rinsed with ultrapure water and dried in a vacuum drying oven, thereby obtaining a fluorescent bionic identification test paper FP@BA-Eu@MIP for detecting tetracycline. The prepolymer solution refers to a mixed solution of a crosslinking agent TEOS, a functional monomer IBTES, and ethanol.

2. The preparation method according to claim 1, characterized in that: The concentration of the hydrochloric acid solution in step (1) is 0.2M.

3. The preparation method according to claim 2, characterized in that: The final concentrations of Eu(NO3)3·6H2O and BBDC in step (1) are both 10 mmol / L.

4. The preparation method according to claim 3, characterized in that: The volume ratio of DMF to H2O in the step (1) is 7:

3.

5. The preparation method according to claim 4, characterized in that: In the step (1), the heating temperature is 120° C. and the heating time is 12 h.

6. The preparation method according to claim 5, characterized in that: The concentration of the TC solution in step (2) is 1 mg / mL.

7. The preparation method according to claim 6, characterized in that: The concentration of the ammonium bicarbonate buffer solution in step (2) is 0.1 M, and the pH value is 8.

5.

8. A fluorescent bionic identification test paper for detecting tetracycline prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the fluorescent bionic identification test paper according to claim 8 in detecting tetracycline.

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