A method for rapid detection of antioxidant BHA by near-infrared enhanced MOF / g-C3N5 / Cu laccase

Abstract

The present invention discloses a method for rapidly detecting antioxidant BHA by near-infrared enhanced MOF / g‑C3N5 / Cu laccase. The invention synthesizes a MOF and g‑C3N5 / Cu complex (MOF / g‑C3N5 / Cu). MOF / g‑C3N5 / Cu has laccase-like and peroxidase-like activities and photocatalytic properties. MOF increases its active sites and charge transfer ability, while g‑C3N5 / Cu has a wider visible light absorption region (> 600nm) and fast photogenerated carrier separation efficiency. In the presence of 4-aminoantipyrine (4-AP), H2O2 and near-infrared light, MOF / g-C3N5 / Cu can selectively catalyze the oxidation of butylated hydroxyanisole (BHA) to produce a typical color change from colorless to wine red. Therefore, a new colorimetric probe method for rapid detection of BHA was established. The method is used to detect BHA in food and cosmetics with a detection limit of 0.1 mg / kg, which can meet the relevant requirements of national food safety and other products. This method has the characteristics of simple operation, strong specificity, high sensitivity and rapidity.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis and detection, and specifically to a method for rapidly detecting antioxidant BHA by near-infrared enhanced MOF / g-C3N5 / Cu laccase. Background Art

[0002] Nitrogen-rich graphitic carbon nitride (g-C3N5) is a new type of semiconductor material with high nitrogen content, narrow band gap width (~1.9 eV), and stable chemical properties. Therefore, g-C3N5 has excellent performance in the field of photocatalysis. In addition, the P orbital on the azo bond (-N = N-) in the g-C3N5 structure is delocalized with the π-π* electron. This result shows that g-C3N5 has a narrower band gap width than g-C3N4. Therefore, g-C3N5 has attracted the attention of researchers in terms of photocatalytic performance. However, g-C3N5 also has shortcomings similar to g-C3N4, such as high photogenerated electron-hole recombination rate and low visible light utilization rate, which limits its application in photocatalytic performance. To this end, researchers have explored the incorporation of various elements into the framework of C3N5, which can change its band gap and electronic structure. Metals, non-metals and their compounds have been used in the study of enhancing the photoresponsiveness of C3N5 and promoting charge transfer.

[0003] Laccases are an important class of multi-copper oxidases that catalyze the single-electron oxidation of many important biological and environmental organic compounds. 2+ Complexes such as Cu 2+ -Imidazole, Cu 2+ -Porphyrin, Cu 2+ -metal organic frameworks (MOFs) have been developed to mimic laccase. Although great progress has been made in the research of laccase-like nanozymes, most of them are based on Cu 2+ The covalent coordination of laccase-like nanozymes is constructed and mainly concentrated on the active center Cu; however, the role of cofactors in laccase-like nanozymes has been neglected.

[0004] The present invention synthesizes a MOF and g-C3N5 / Cu complex (MOF / g-C3N5 / Cu), MOF / g-C3N5 / Cu has laccase-like and peroxidase-like activities and photocatalytic properties, MOF increases its active sites and charge transfer ability, and g-C3N5 / Cu has a wide visible light absorption region (> 600nm) and fast photogenerated carrier separation efficiency. In the presence of 4-aminoantipyrine (4-AP), H2O2 and near-infrared light, MOF / g-C3N5 / Cu can selectively catalyze the oxidation of butylated hydroxyanisole (BHA) to produce a typical color change from colorless to wine red, so a new colorimetric probe rapid detection method for BHA is established, the method is used to detect BHA in food and cosmetics, the detection limit is 0.1mg / kg, and can meet the relevant requirements of national food safety and other products; the method has the characteristics of simple operation, strong specificity, high sensitivity, rapidity, etc. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for rapid detection of antioxidant BHA by near-infrared enhanced MOF / g-C3N5 / Cu laccase. The MOF / g-C3N5 / Cu nanomaterial synthesized by the present invention has laccase-like and peroxidase-like properties, and can oxidize BHA to produce red in the presence of 4-AP, H2O2 and near-infrared light, thereby establishing a colorimetric rapid detection method for BHA.

[0006] The present invention relates to a method for rapidly detecting antioxidant BHA by near infrared enhanced MOF / g-C3N5 / Cu laccase, comprising the following steps:

[0007] (1) 3.0-4.0 g of 3-amino-1,2,4-triazole was heated to 500°C in a tube furnace at 5°C / min and calcined for 2-3 h. After cooling to room temperature, the powder was ground and 1.0-2.0 g of the powder and 0.36-0.60 g of NaOH were added to a beaker. 80-150 mL of deionized water were added and stirred for 30-40 min to fully mix the mixture so that the solid particles were evenly dispersed in the water. The mixed solution was then transferred to a polytetrafluoroethylene tank in a stainless steel autoclave and calcined at 120°C for 10-12 h. The obtained product was centrifuged at 8000-10000 rpm for 10-15 min, washed with deionized water for 3-5 times, dried at 100°C for 0.5-1 h, ground into powder, and placed in a tube furnace and heated to 500°C at 5°C / min for calcination for 2-3 h, centrifuge the product at 8000-10000 rpm for 10-15 min, and wash with deionized water 3-5 times to obtain g-C3N5;

[0008] (2) Dissolve 0.2-0.3 g of Zn(NO3)2·6H2O and 0.05-0.1 g of 2-hydroxyterephthalic acid in 50-80 mL of DMF, add 0.5-0.8 g of g-C3N5 and stir for 10-12 h; then add 1-2 mL of 10 mg / mL CuCl2 and continue stirring for 1-2 h. Finally, wash with anhydrous ethanol and ultrapure water alternately for three times, and dry in a vacuum oven at 60 °C to obtain MOF / g-C3N5 / Cu.

[0009] (3) Add different concentrations of BHA standard solution, 4-aminopyrine (4-AP) and H2O2 to the MOF / g-C3N5 / Cu nanomaterial solution, dilute to 3 mL with pH 6.8 MES buffer solution, shake well, irradiate under 808 nm near-infrared light for 10-20 min, measure the absorbance at a wavelength of 510 nm, and determine the linear relationship between BHA concentration and absorbance;

[0010] (4) Extract BHA from the sample to be tested to obtain a sample test solution, measure the absorbance according to step (3), substitute it into the regression equation, and calculate the BHA concentration in the sample solution to be tested.

[0011] The concentration of the BHA standard solution is 10-1500 μg / L, the concentration of the MOF / g-C3N5 / Cu nanomaterial solution is 2 mg / mL, the addition amount is 50-100 μL, the concentration of 4-AP is 1 mg / mL, the volume is 50-100 μL, the concentration of H2O2 is 50 mmol / L, and the volume is 50-100 μL.

[0012] Advantages and technical effects of the present invention:

[0013] 1. In the MOF / g-C3N5 / Cu nanomaterial synthesized by the present invention, MOF increases its active sites and charge transfer ability, while g-C3N5 / Cu has a wide visible light absorption region (> 600nm) and fast photogenerated carrier separation efficiency. MOF / g-C3N5 / Cu has laccase-like activity and photocatalytic performance. In the presence of 4-aminoantipyrine (4-AP), H2O2 and near-infrared light, MOF / g-C3N5 / Cu can selectively catalyze the oxidation of butylated hydroxyanisole (BHA) to produce a typical color change from colorless to wine red. Based on this, a rapid detection method for BHA is established.

[0014] 2. The BHA colorimetric detection method established in the present invention has good specificity and high detection sensitivity, with a detection limit of 0.1 mg / kg, which can meet the relevant national food safety requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 TEM image of MOF / g-C3N5 / Cu prepared in Example 1 of the present invention;

[0016] Figure 2 This is the UV spectrum of MOF / g-C3N5 / Cu oxidizing TMB and H2O2 in Example 1 of the present invention;

[0017] Figure 3 The effect of NIR on the activity of MOF / g-C3N5 / Cu laccase in Example 1;

[0018] Figure 4 is the Michaelis-Menten equation of (a) 4-AP+H2O2+MOF / g-C3N5 / Cu+BHA+NIR, (b) 4-AP+H2O2+MOF / g-C3N5 / Cu+BHA+natural light in Example 1;

[0019] Figure 5 The ultraviolet absorption spectrum and linear equation of BHA detected by the system of 4-AP+H2O2+MOF / g-C3N5 / Cu under irradiation of infrared light at 808nm for 10min in Example 1;

[0020] Figure 6 This is the effect of the coexisting substances on BHA in Example 1. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0022] Example 1: Determination of BHA in edible oil

[0023] 1. Preparation of g-C3N5: 3.0-4.0 g of 3-amino-1,2,4-triazole was heated to 500°C at 5°C / min in a tube furnace and calcined for 2-3 h. After cooling to room temperature, the powder was ground and 1.0-2.0 g of the powder and 0.36-0.60 g of NaOH were added to a beaker. 80-150 mL of deionized water were added and stirred for 30-40 min to fully mix the mixture so that the solid particles were evenly dispersed in the water. The mixed solution was then transferred to a polytetrafluoroethylene tank in a stainless steel autoclave and calcined at 120°C for 10-12 h. The obtained product was centrifuged at 8000-10000 rpm for 10-15 min, washed with deionized water for 3-5 times, dried at 100°C for 0.5-1 h, ground into powder, and placed in a tube furnace and heated to 500°C at 5°C / min for calcination for 2-3 h, centrifuge the product at 8000-10000 rpm for 10-15 min, and wash with deionized water 3-5 times to obtain g-C3N5.

[0024] 2. Preparation of MOF / g-C3N5 / Cu: Dissolve 0.2-0.3 g of Zn(NO3)2·6H2O and 0.05-0.1 g of 2-hydroxyterephthalic acid in 50-80 mL of DMF, add 0.5-0.8 g of g-C3N5 and stir for 10-12 h; then add 1-2 mL of 10 mg / mL CuCl2 and continue stirring for 1-2 h. Finally, wash alternately with anhydrous ethanol and ultrapure water for three times, and dry in a vacuum drying oven at 60°C to obtain MOF / g-C3N5 / Cu. Figure 1 This is the TEM image of MOF / g-C3N5 / Cu. The material has a rough surface and certain irregular wrinkles and stacking structures.

[0025] 3. Evaluation of peroxidase activity of MOF / g-C3N5 / Cu nanozyme: 100 μL of 10 mmol / L TMB, 100 μL of 2 mg / mL MOF / g-C3N5 / Cu and 100 μL of 50 mmol / L H2O2 were added to a 5 mL stoppered colorimetric tube, diluted to 3 mL with 0.1 mmol / L pH 4.0 HAc-NaAc buffer solution, shaken and mixed, and allowed to stand for 10 min. The absorbance was measured at a wavelength of 654 nm. The results are shown in Table 2. Figure 2 , MOF / g-C3N5 / Cu exhibits strong peroxidase activity.

[0026] 3. Evaluation of MOF / g-C3N5 / Cu laccase nanozyme activity: BHA was used as the catalytic reaction substrate. Its laccase activity was evaluated in the presence of H2O2 and 4-aminopyrine (4-AP). 100 μL of 50 μg / mL BHA, 100 μL of 1 mg / mL 4-AP, 100 μL of 2 mg / mL MOF / g-C3N5 / Cu and 100 μL of 50 mmol / L H2O2 were added to a 5 mL stoppered colorimetric tube. The pH 6.8 MES buffer solution was adjusted to 3 mL and shaken. The tube was irradiated under 808 nm near-infrared light or natural light for 10 min, and the absorbance was measured at a wavelength of 505 nm. The results are shown in Figure 2. Figure 3 Compared with no irradiation under 808 nm near-infrared light, irradiation under near-infrared light significantly enhanced the laccase activity of MOF / g-C3N5 / Cu, and MOF / g-C3N5 / Cu exhibited strong laccase activity.

[0027] The Michaelis-Menten catalytic kinetic parameters were also determined ( Figure 4 (ab) and Table 1), Michaelis constant of MOF / g-C3N5 / Cu for substrate 2,4-DP K mare 0.468 mmol / L, respectively, and the reaction rate constant is 3.15×10 -9 mol / Ls, 2,4-DP after infrared light irradiation K m is 0.339 mmol / L, and the reaction rate constant is 1.68×10 -8 mol / L·s, indicating that irradiation increased the affinity and reaction rate of MOF / g-C3N5 / Cu nanozyme with the substrate.

[0028]

[0029] 4. Preparation of BHA working curve: Add 100 μL of 2 mg / mL MOF / g-C3N5 / Cu and 0.5-100 g / L BHA standard solution to a 5 mL stoppered colorimetric tube, add 100 μL of 50 mmol / L H2O2, 100 μL of 1 mg / mL 4-aminopyrine (4-AP), add 0.1 mmol / L pH 6.8 MES buffer solution to 3 mL, shake well, irradiate under 808 nm near-infrared light for 10 min, measure the absorbance A at a wavelength of 510 nm, plot the standard curve with BHA concentration as the horizontal axis and absorbance A as the vertical axis, and obtain the regression equation, see Figure 5 ; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.

[0030]

[0031] 5. Method specificity investigation: Mix BHA with other coexisting ions and detect the effect of coexisting ions on BHA in the above detection system. 15 times ascorbic acid, 20 times vitamin E and 60 times citric acid concentrations do not interfere with the determination of BHA. A large amount of inorganic ions, such as Mg 2+ , Ca 2+ , Zn 2+ , Cu 2+ and SO4 2- The influence on the determination is negligible. The same concentrations of propyl gallate (PG), 2,4,5-trihydroxybutyrophenone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT), and dodecyl gallate (DG) interfere with the detection system. The results show that TBHQ has a certain interference, while other antioxidants have less interference.

[0032] 6. Determination of BHA in walnut oil samples

[0033] (1) Sample pretreatment: Weigh 1 g (accurate to 0.01 g) of sample into a 50 mL centrifuge tube, add 5 mL of acetonitrile-saturated n-hexane solution to dissolve the sample, vortex for 1 min, let stand for 10 min, vortex extract with 5 mL of n-hexane-saturated acetonitrile solution for 2 min, centrifuge at 3000 r / min for 5 min, collect the acetonitrile layer in a test tube, and repeat the extraction with 5 mL of n-hexane-saturated acetonitrile solution for 2 times. Combine the three extracts to obtain the sample test solution. Perform a blank test at the same time.

[0034] (2) Determination of BHA in walnut oil: In a 5 mL stoppered colorimetric tube, add 200 μL of the sample solution in step (1), 100 μL of 2 mg / mL MOF / g-C3N5 / Cu, 100 μL of 50 mmol / L H2O2, 100 μL of 1 mg / mL 4-AP, and 0.1 mmol / L pH 6.8 MES buffer solution to 3 mL. Shake well and irradiate under 808 nm near-infrared light for 10 min. Measure the absorbance A at a wavelength of 510 nm. Substituting it into the regression equation, the BHA content is 30.2 mg / kg.

[0035] Example 2: Determination of BHA in nut samples

[0036] 1. Preparation of g-C3N5: 3.0-4.0 g of 3-amino-1,2,4-triazole was heated to 500°C at 5°C / min in a tube furnace and calcined for 2-3 h. After cooling to room temperature, the powder was ground, 1.0-2.0 g of the powder and 0.36-0.60 g of NaOH were added to a beaker, and 80-150 mL of deionized water were added. The mixture was stirred for 30-40 min to fully mix the mixture so that the solid particles were evenly dispersed in the water. The mixed solution was then transferred to a polytetrafluoroethylene tank in a stainless steel autoclave and calcined at 120°C for 10-12 h. The obtained product was centrifuged at 8000-10000 rpm for 10-15 min, washed with deionized water for 3-5 times, dried at 100°C for 0.5-1h, ground into powder, and placed in a tube furnace and heated to 500°C at 5°C / min for calcination for 2-3 h, centrifuge the product at 8000-10000 rpm for 10-15 min, and wash with deionized water 3-5 times to obtain g-C3N5.

[0037] 2. Preparation of MOF / g-C3N5 / Cu: Dissolve 0.2-0.3 g of Zn(NO3)2·6H2O and 0.05-0.1 g of 2-hydroxyterephthalic acid in 50-80 mL of DMF, add 0.5-0.8 g of g-C3N5 and stir for 10-12 h; then add 1-2 mL of 10 mg / mL CuCl2 and continue stirring for 1-2 h. Finally, wash alternately with anhydrous ethanol and ultrapure water for three times, and dry in a vacuum drying oven at 60°C to obtain MOF / g-C3N5 / Cu. Figure 1 This is the TEM image of MOF / g-C3N5 / Cu. The material has a rough surface and certain irregular wrinkles and stacking structures.

[0038] 3. Preparation of BHA working curve: same as Example 1.

[0039] 4. Determination of BHA in nut samples

[0040] (1) Sample pretreatment method: Accurately weigh 1.0 g of sample (accurate to 0.01 g), place in a 50 mL stoppered centrifuge tube, add 10 mL acetonitrile, vortex for 2 min, sonicate for 10 min, centrifuge at 3000 r / min for 5 min, collect the supernatant in a centrifuge tube, and repeat extraction once with 10 mL acetonitrile solution. Combine the two supernatants, load all the supernatants onto the SPE column, collect the effluent, and elute with 10 mL solution (V (methanol) ∶ V (acetonitrile) = 1∶2) to collect the effluent. Combine the collected solutions in a rotary evaporator, spin dry at 40°C, and re-dissolve with acetonitrile to a constant volume of 2 mL to prepare the sample test solution.

[0041] (2) Determination of BHA in nut samples: Same as in Example 1, the BHA content in the nut samples was 12.6 mg / kg.

[0042] 5. Recovery and precision experiment: Three different concentrations of BHA standard solutions were added to the sample; each concentration was measured three times in parallel, the spiked recovery was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 3; the spiked recovery of BHA was measured to be between 93.1% and 103.6%, and the RSD was between 2.13% and 3.93%. This method has good accuracy and precision.

[0043]

[0044] The BHA determination method established in the present invention has the advantages of fewer processing steps, shorter time, lower processing cost, easier operation, and no need for large-scale instruments and equipment, thus having great advantages in actual detection.

Claims

1. A method for rapid detection of antioxidant BHA by near-infrared enhanced MOF / g-C3N5 / Cu laccase, characterized in that: The following steps are involved: (1) 3.0-4.0 g of 3-amino-1,2,4-triazole was heated to 500°C in a tube furnace at 5°C / min and calcined for 2-3 h. After cooling to room temperature, the powder was ground and 1.0-2.0 g of the powder and 0.36-0.60 g of NaOH were added to a beaker. 80-150 mL of deionized water were added and stirred for 30-40 min to fully mix the mixture so that the solid particles were evenly dispersed in the water. The mixed solution was then transferred to a polytetrafluoroethylene tank in a stainless steel autoclave and calcined at 120°C for 10-12 h. The obtained product was centrifuged at 8000-10000 rpm for 10-15 min, washed with deionized water for 3-5 times, dried at 100°C for 0.5-1 h, ground into powder, placed in a tube furnace and heated to 500°C at 5°C / min for calcination for 2-3 h. rpm centrifugation for 10-15 min, and washed with deionized water 3-5 times to obtain g-C3N5; (2) Dissolve 0.2-0.3 g of Zn(NO3)2·6H2O and 0.05-0.1 g of 2-hydroxyterephthalic acid in 50-80 mL of DMF, add 0.5-0.8 g of g-C3N5 and stir for 10-12 h; then add 1-2 mL of 10 mg / mL CuCl2 and continue stirring for 1-2 h. Finally, wash with anhydrous ethanol and ultrapure water alternately for three times, and dry in a vacuum oven at 60°C to obtain MOF / g-C3N5 / Cu. (3) Add different concentrations of BHA standard solution, 4-aminopyrine and H2O2 to the MOF / g-C3N5 / Cu nanomaterial solution, dilute to 3 mL with pH 6.8 MES buffer solution, shake well, irradiate under 808 nm near-infrared light for 10-20 min, measure the absorbance at a wavelength of 510 nm, and determine the linear relationship between BHA concentration and absorbance; (4) Extract BHA from the sample to be tested to obtain a sample test solution, measure the absorbance according to step (3), substitute it into the regression equation, and calculate the BHA concentration in the sample solution to be tested.

2. The method according to claim 1, characterized in that: The concentration of BHA standard solution is 10~1500 μg / L, the concentration of MOF / g-C3N5 / Cu nanomaterial solution is 2 mg / mL, the addition amount is 50-100 μL, the concentration of 4-AP is 1 mg / mL, the volume is 50-100 μL, and the concentration of H2O2 is 50 mmol / L, the volume is 50-100 μL.

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