A method for detecting octopamine and tyramine using a laccase-like Schiff base Cu-MOF nanozyme.

Abstract

This invention discloses a method for detecting octopamine and tyramine using a laccase-like Schiff base Cu-MOF nanozyme. The method uses a Schiff base formed from glutaraldehyde and 2-aminoterephthalic acid as starting reagents as ligands to synthesize a flower-like Cu-MOF nanozyme. This Cu-MOF exhibits near-infrared light-triggered laccase-like activity. Using octopamine and tyramine as substrates, Cu-MOF catalyzes the oxidation of octopamine and tyramine, conjugating 4-aminoantipyrine to form a red coupling product. This product exhibits a high absorption spectrum at 505 nm, demonstrating high detection efficiency with detection limits of 0.18 μg / mL and 0.60 μg / mL, respectively. X-ray photoelectron spectroscopy analysis shows that the Cu(I) / Cu(II) redox reaction participates in the catalytic process. This method is characterized by high sensitivity, strong specificity, simple operation, and rapid response.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and detection technology, specifically a method for detecting octopamine and tyramine using a NIR-enhanced laccase-like Schiff base Cu-MOF nanozyme. Background Technology

[0002] Microbial activity produces large amounts of biogenic amines (BAs), which can cause food spoilage during storage and processing. BAs have been found in a variety of foods, including raw meat, wine, and cheese. Octopamine (OA) and tyramine (Tyr) are typical examples of BAs, mediating various physiological processes in the nervous system. Therefore, developing inexpensive, rapid, and highly sensitive methods for detecting OA and Tyr to reduce the risk of food safety problems is particularly important. Currently, the effective national standard GB 5009.208-2016, "National Food Safety Standard - Determination of Biogenic Amines in Food," specifies the detection method for BAs in foods such as alcoholic beverages, condiments, aquatic products, and meat. This method utilizes pre-column derivatization combined with liquid chromatography for determination. The second method—spectrophotometry—specifically specifies the determination of histamine content in aquatic products, but reports on spectrophotometric determination of OA and Tyr are relatively few.

[0003] Laccase is a multi-copper oxidase with strong catalytic activity and good substrate adaptability. It can catalyze the oxidation of various phenols and aromatic compounds, and has wide applications in organic synthesis, environmental pollutants, and biotechnology processes. However, natural laccase exhibits some limitations, such as high cost, low decolorization efficiency, poor stability (pH, temperature, and storage time), and challenges in recyclability. Recently, some copper-containing nanomaterials have been explored for their laccase-like activity, but due to low enzyme activity, the phenols that can be oxidized are limited, mainly chlorophenols, phenols, and tetracyclines, with a few reports on the oxidation of bisphenol A. To improve the activity, selectivity, stability, and sensitivity of catalysts, novelty in nanomaterial preparation is essential. This invention utilizes the ability of Schiff base ligands to coordinate metal ions and exhibit specific functions to prepare copper Schiff base MOF nanosheets with high catalytic laccase-like activity and near-infrared response for the catalytic detection of OA and Tyr. Similar studies have not been reported. Summary of the Invention

[0004] This invention discloses a near-infrared (NIR) enhanced laccase-like activity Schiff base Cu-MOF nanozyme for the detection of octopamine and tyramine. The method uses a Schiff base formed from glutaraldehyde and 2-aminoterephthalic acid as starting reagents as ligands to synthesize a flower-like copper metal-organic framework (Cu-MOF). This Cu-MOF exhibits near-infrared light-triggered laccase-like activity. Compared with natural laccase, the Cu-MOF laccase-like activity shows excellent stability, including extreme pH values, ionic strength, temperature, storage time, and reusability. Using octopamine or tyramine as laccase substrates, the Cu-MOF nanozyme catalyzes the oxidation of octopamine and tyramine, binds to 4-aminoantipyrine, and couples to form a red coupling product. This product exhibits a high absorption spectrum at 505 nm, demonstrating high detection efficiency, with detection limits of 0.18 μg / mL and 0.60 μg / mL, respectively. X-ray photoelectron spectroscopy (XPS) analysis showed that the redox reaction of Cu(I) / Cu(II) was involved in the catalytic process. The method of this invention has the characteristics of high sensitivity, strong specificity, simple operation and speed.

[0005] The present invention provides a method for detecting octopamine and tyramine using a NIR-enhanced laccase-like Schiff base Cu-MOF nanozyme for the Schiff base assay:

[0006] (1) Mix 0.50-1.0g of 2-aminoterephthalic acid and 1-2mL of glutaraldehyde with a volume concentration of 20-30%, then add 20-30mL of 8-12mM NaOH methanol solution. Stir the mixture at room temperature for 60-90min and place it in an incubator at 40-50℃ for 24h to obtain Schiff base;

[0007] (2) Add 0.35-0.50g CuCl2·2H2O and 0.05-0.10g ascorbic acid to the Schiff base in step (1), stir for 15-20min, react in microwave at 170-200℃ for 1-2h, cool naturally to room temperature, centrifuge, wash the solid with ethanol and deionized water 2-3 times each, and vacuum dry to obtain Cu-MOF nanozyme;

[0008] Centrifugation is performed at 8000-10000 r / min for 10-15 min;

[0009] (3) After mixing Cu-MOF nanozyme solution, octopamine solution or tyramine solution of different concentrations and 2-aminoantipyrine solution, add pH 9.0 Tris–HCl buffer solution, irradiate with near-infrared light at 808nm for 10-20min, and measure absorbance at 510nm wavelength to determine the linear relationship between octopamine or tyramine concentration and absorbance value, and obtain the regression equation;

[0010] (4) The absorbance value of the sample solution to be tested is determined according to the method in step (3), and then substituted into the regression equation to obtain the concentration of octopamine or tyramine in the sample solution to be tested.

[0011] In the above method, the concentration of Cu-MOF nanozyme solution is 1 mg / mL, and the addition amount is 50-100 μL; the concentration of 4-aminoantipyrine solution is 20 mmol / L, and the addition amount is 100-200 μL; the concentration of Tris–HCl buffer solution is 0.1 mmol / L, and the addition amount is 1-3 mL; the near-infrared light power at 808 nm is 1.0-2.0 W / cm². 2 .

[0012] The advantages of this invention are:

[0013] 1. This invention synthesizes Schiff bases using 2-aminoterephthalic acid and glutaraldehyde, and then reacts them with Cu... 2+ The resulting Schiff base complex has a stable and specific structure. The copper organic framework material prepared using this complex as a precursor, with the addition of reducing ascorbic acid during the preparation process, enables the prepared Cu-MOF nanozyme to have a Cu(I) / Cu(II) coexisting valence state, exhibiting excellent laccase-like activity and near-infrared light-enhanced catalytic activity. It can use octopamine or tyramine as a catalytic substrate, combined with 4-aminoantipyrine, to form a red coupling product, which shows a high absorption spectrum at 505 nm. Thus, a simple, rapid, specific, and sensitive colorimetric method for the detection of octopamine and tyramine has been established.

[0014] 2. The established colorimetric method for the detection of octopamine and tyramine exhibits a wide linear range of 0.25–27.5 mg / L and 1–110 mg / L, respectively, with detection limits of 0.18 mg / L and 0.60 mg / L, which are 27 times and 3 times lower than the detection limits (5 mg / L and 2 mg / L) in GB 5009.208-2016, respectively. The established method was successfully applied to the detection of octopamine and tyramine in coffee and fermented foods. The spiked method was successfully used to detect octopamine and tyramine in samples, with recoveries ranging from 82.2% to 107.2%. The detection system provided by this invention has high detection accuracy. Attached Figure Description

[0015] Figure 1 This is a TEM image of the Cu-MOF synthesized in Example 1;

[0016] Figure 2 The image shows the XRD pattern of the Cu-MOF synthesized in Example 1.

[0017] Figure 3 This is a high-resolution XPS Cu 2p image of the synthesized Cu-MOF in Example 1;

[0018] Figure 4 shows the UV-Vis absorption spectra of Cu-MOF and Cu-MOF+NIR oxidation of OA, Tyr or 2,4-DP+4-AP in Example 1.

[0019] Figure 5 The Michaelis kinetic curves for the oxidation of 2,4-DP (a) and OA (b) by Cu-MOF and natural laccase in Example 1 are shown.

[0020] Figure 6 The Michaelis-Menten kinetic curves for the oxidation of Tyr (a) and OA-NIR (b) by Cu-MOF and natural laccase in Example 1 are shown.

[0021] Figure 7 The linear UV-Vis absorption spectrum (a) and linear regression equation (b) of Cu-MOF oxidation of OA in Example 1 are shown.

[0022] Figure 8 The linear UV-Vis absorption spectrum (a) and linear regression equation (b) of Cu-MOF oxidation of Tyr in Example 1 are shown.

[0023] Figure 9 The effects of coexisting biogenic amines (a) and coexisting metal ions (b) on OA and Tyr are presented. Detailed Implementation

[0024] The technical solution of the present invention will be described in further detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto;

[0025] Example 1: Determination of octopamine (OA) and tyramine (Tyr) in coffee samples

[0026] 1. Mix 1.0 g of 2-aminoterephthalic acid and 2 mL of 25% glutaraldehyde, then add 30 mL of 10 mM NaOH methanol solution. Stir the mixture at room temperature for 90 min and incubate at 47 °C for 24 h to obtain a Schiff base.

[0027] 2. In step (1), 0.50 g CuCl2·2H2O and 0.10 g ascorbic acid were added to the Schiff base. After stirring for 20 min, the mixture was reacted in a microwave at 200℃ for 1 h. After naturally cooling to room temperature, the mixture was centrifuged at 8000 rpm for 15 min. The solid was washed three times each with deionized water and ethanol, and then vacuum dried to obtain Cu-MOF nanozymes. The prepared Cu-MOF nanozymes were analyzed by transmission electron microscopy (TEM). Figure 1 As shown, the synthesized Cu-MOF nanozyme exhibits an irregular sheet-like structure, while also containing some particles. Powder X-ray diffraction (PXRD) analysis reveals Cu... 2+ The types of products generated by reactions with Schiff bases provide valuable insights, such as... Figure 2 As shown, the main diffraction peak 2 θ = 16°, 21°, 32° and 40°, consistent with previously reported PXRD patterns of Cu-MOF; X-ray photoelectron spectroscopy (XPS) analysis was used to determine the composition, valence states and binding energies of the elements present in Cu-MOF, Cu 2p pattern of Cu-MOF ( Figure 3 The results show that copper exists in the nanozyme structure in the forms of Cu(I) and Cu(II), and the peaks at binding energies of 932.0 eV and 933.8 eV can be attributed to Cu 2p of Cu(I) and Cu(II), respectively. 3 / 2 The peaks at 951.3 eV and 953.8 eV can be attributed to Cu 2p in Cu(I) and Cu(II), respectively. 1 / 2 The results showed that Cu(II) and Cu(I) coexisted in Cu-MOF.

[0028] 3. Evaluation of Cu-MOF nanozyme laccase activity: Using 2,4-chlorophenol (2,4-DP), octopamine (OA), and tyramine (Tyr) as substrates, natural laccase and Cu-MOF nanozyme as catalysts, and 4-aminoantipyrine (4-AP) as colorimetric reagent, the laccase mimic activity was determined. Figure 4 The results showed that in the presence of 4-AP, 2,4-DP, OA, and Tyr could be oxidized by Cu-MOF nanozymes and natural laccase to quinone analogs, producing a red product with a distinct absorption peak at 505 nm. The Cu-MOF nanozymes, after passing through a power density of 2.0 W / cm², exhibited this effect. 2 Irradiation with near-infrared light at 808 nm for 10 min significantly improved laccase activity. Michaelis-Menten catalytic kinetic parameters were also determined; results are shown below. Figure 5 , 6 As shown in Tables 1, 2, and 4, when 4-DP is used as the substrate, the Cu-MOF nanozyme exhibits stronger affinity and reaction rate than natural laccase. When OA is used as the substrate, the catalytic rate of the Cu-MOF nanozyme is 23.2 times higher than that of natural laccase. When Tyr is used as the substrate, the Cu-MOF nanozyme shows stronger affinity and reaction rate than natural laccase. Furthermore, the addition of 808 nm irradiation during OA oxidation enhances both the affinity for the OA substrate and the reaction rate. These results indicate that the Cu-MOF nanozyme synthesized in this invention possesses excellent laccase-like activity and exhibits infrared light-enhanced laccase-like activity.

[0029] Table 1 Michaelis catalytic kinetic parameters

[0030] ;

[0031] 4. Preparation of the octopamine (OA) working curve: Add 50 µL of 1 mg / mL Cu-MOF nanozyme, 100 µL of 20 mmol / L 4-aminopyridine (4-AP), and 1 mL of OA standard solution with a concentration of 0.25–27.5 mg / L to a 5 mL stoppered colorimetric tube. Add 0.1 mmol / L pH 9.0 Tris–HCl buffer to a final volume of 3 mL. (2.0 W / cm²) 2 Irradiation with near-infrared light at 808 nm for 10 min, absorbance value measured at 505 nm wavelength. Absorbance showed a linear relationship with OA concentration. The UV-Vis absorption spectrum is shown below. Figure 7 a. Plot a standard curve with OA concentration on the x-axis and absorbance values ​​on the y-axis to obtain the regression equation, see [link to curve]. Figure 7 b; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2;

[0032] 5. Preparation of Tyr (Tyr) working curve: Add 50µL of 1mg / mL Cu-MOF nanozyme, 100µL of 2mmol / L 4-aminopyridine (4-AP), and 1mL of Tyr standard solution with a concentration in the range of 1~110mg / L to a 5mL stoppered colorimetric tube. Add 0.1mmol / L pH 9.0 Tris–HCl buffer to a final volume of 3mL. (2.0 W / cm²) 2 Irradiation with near-infrared light at 808 nm for 10 min, followed by absorption measurement at 505 nm. The absorbance showed a linear relationship with Tyr concentration. The UV-Vis absorption spectrum is shown below. Figure 8 a. Plot a standard curve with Tyr concentration on the x-axis and absorbance values ​​on the y-axis to obtain the regression equation, see [link to curve]. Figure 8 b; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2;

[0033] Table 2. Linear equation, correlation coefficient, relative standard deviation, and linear range

[0034]

[0035] 6. Method Specificity Study: OA, Tyr, and other biogenic amines (histamine, tryptamine, cadaverine, putrescine, spermine, spermidine, phenylethylamine) were added to the above detection system for detection to study the specificity of the method of this invention. The concentration of OA or Tyr was 10 mg / kg, and the concentration of other biogenic amines was 50 mg / kg. The results are shown in […]. Figure 9 a;

[0036] Simultaneously, OA or Tyr is reacted with metal ions (Hg) 2+ Ni 2+ Cu 2+ Fe 2+ Fe3+ Zn 2+ Mg 2+ Na + Ca 2+ K + The mixture was analyzed to detect the effect of metal ions on OA or Tyr in the above detection system. The concentration of OA or Tyr was 10 mg / kg, and the concentration of metal ions was 50 mg / kg. The results are shown in [Figure number missing]. Figure 9 b;

[0037] The results above show that the detection system of the present invention has good selectivity and specificity for OA and Tyr.

[0038] 7. Determination of OA and Tyr in coffee samples

[0039] (1) Sample preparation: Accurately weigh 9g of the sample (accurate to 0.001g) and place it in a 25mL stoppered conical flask. Add 1.25mL of saturated borax solution and 6mL of ultrapure water (about 70℃). Mix well and heat in a boiling water bath for 15min. After removing it, cool it in a cold water bath and place it at room temperature. Transfer the above extract to a 20mL volumetric flask, add 0.5mL of 106g / L potassium ferrocyanide solution, shake well, add 0.5mL of 220g / L zinc acetate solution, add water to the mark, shake well, and let it stand for 30min. Remove the upper fat layer and filter the supernatant through a microporous membrane to obtain the sample solution to be tested.

[0040] (2) Sample determination: Add 100µL of 1mg / mL Cu-MOF nanozyme, 100µL of 20mmol / L 4-aminopyridine (4-AP), and 1mL of the sample solution from step (1) to a 5mL stoppered colorimetric tube, and add 0.1mmol / L pH 9.0 Tris–HCl buffer solution to a final volume of 3mL. (2.0W / cm²) 2 Irradiate with near-infrared light at 808 nm for 10 min, and measure absorbance at 505 nm wavelength. Substitute into the regression equations of steps 4 and 5, and OA and Tyr were not detected in the sample.

[0041] Example 2: Determination of OA and Tyr in pork and grass carp samples

[0042] 1. Mix 0.50g of 2-aminoterephthalic acid and 1mL of 25% glutaraldehyde, then add 20mL of 10mmol / L NaOH methanol solution. Stir the mixture at room temperature for 60min and incubate at 47℃ for 24h to obtain Schiff base.

[0043] 2. Add 0.35g CuCl2·2H2O and 0.05g ascorbic acid to the Schiff base in step (1), stir for 15min, react in microwave at 180℃ for 2h, cool naturally to room temperature, centrifuge at 8000rpm for 15min, wash the solid repeatedly with deionized water and ethanol 3 times, and dry under vacuum to obtain Cu-MOF nanozyme.

[0044] 3. Creation of OA and Tyr working curves: Same as in Example 1;

[0045] 4. Determination of OA and Tyr in pork and grass carp samples:

[0046] (1) Sample preparation: Accurately weigh 10g of minced solid sample (pork or grass carp) into a 100mL centrifuge tube, add 20mL of 5% trichloroacetic acid extraction solution, shake and extract for 30min, centrifuge at 5000rpm for 10min, take the supernatant, place the solid in a 50mL brown volumetric flask, extract twice consecutively, collect and combine the supernatants, and dilute to the mark with trichloroacetic acid; take 10mL of sample extract, add 0.5g of sodium chloride, vortex until the sodium chloride is completely dissolved, add 10mL of n-hexane to remove fat, vortex and mix well, centrifuge at 5000rpm for 5min, discard the upper organic phase, repeat twice, blow dry with nitrogen at 40℃, and redissolve with 1mL of methanol or ethanol to obtain the sample solution to be tested;

[0047] (2) The determination of OA and Tyr in pork or grass carp samples was the same as in Example 1. The contents of OA and Tyr in pork samples were 0.51 mg / kg and 2.47 mg / kg, respectively, and the contents of OA and Tyr in grass carp samples were 2.50 mg / kg and 1.34 mg / kg, respectively.

[0048] Example 3: Determination of OA and Tyr in Fermented Bean Curd

[0049] 1. Preparation of Cu-MOF nanozymes: Same as in Example 1;

[0050] 2. Creation of OA and Tyr working curves: Same as in Example 1;

[0051] 3. Determination of OA and Tyr in Fermented Bean Curd

[0052] (1) Accurately weigh 10g of sample, homogenize it, add 20mL of extraction solution (acetonitrile:water = 9:1, containing 2% perchloric acid), then extract with vortex oscillation for 10min, centrifuge at 5000rpm for 5min, repeat the extraction of sample residue once, centrifuge again, combine the two supernatants and make up to 25mL to obtain the sample solution to be tested;

[0053] (2) The determination of OA and Tyr in fermented bean curd was the same as in Example 1. The contents of OA and Tyr in fermented bean curd were 102.54 mg / kg and 25.32 mg / kg, respectively.

[0054] Example 4: Determination of OA and Tyr in Red Wine Samples

[0055] 1. The preparation of Cu-MOF nanozymes is the same as in Example 1;

[0056] 2. The creation of OA and Tyr working curves is the same as in Example 1;

[0057] 3. Determination of OA and Tyr in Red Wine Samples

[0058] (1) Processing of red wine sample: Take 6 mL of red wine and evaporate it at 40℃. Dissolve the remaining residue with 3 mL of ethanol to obtain the sample solution to be tested.

[0059] (2) The determination of OA and Tyr in the red wine sample was the same as in Example 1. The contents of OA and Tyr in the red wine sample were not detected and 1.67 mg / kg, respectively.

[0060] Example 5: Recovery and Precision Experiment

[0061] Three different concentrations of OA and Tyr standard solutions were added to the samples in Examples 1-4, respectively; each concentration was measured in triplicate, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 3. The spiked recoveries of OA and Tyr were found to be 82.2%–107.2%, and the RSDs were 1.98%–3.97%. This method has good accuracy and precision.

[0062] Table 3. Spike recoveries and RSDs of samples (n=3)

[0063]

[0064] Examples 1-4 were compared using the method of the present invention with the national standard GB 5009.208-2016 Food Safety Standard for the Determination of Biogenic Amines in Food. The results are shown in Table 4. The results show that the two methods yielded consistent results.

[0065] Table 4. Comparison results of the above methods: TBHQ values

[0066]

[0067] The method for determining OA and Tyr established in this invention has higher sensitivity, and the detection is rapid, simple, and quick, with low processing cost and easy operation. It does not require large instruments and equipment, and has strong advantages in actual detection.

Claims

1. A method for detecting octopamine and tyramine using a laccase-like Schiff base Cu-MOF nanozyme, characterized in that, Includes the following steps: (1) Mix 0.50-1.0g of 2-aminoterephthalic acid and 1-2mL of glutaraldehyde with a volume concentration of 20-30%, then add 20-30mL of 8-12mM NaOH methanol solution. Stir the mixture at room temperature for 60-90min and place it at 40-50℃ for 24h to obtain a Schiff base. (2) Add 0.30-0.50g CuCl2·2H2O and 0.05-0.10g ascorbic acid to the Schiff base in step (1), stir for 15-20min, react in microwave at 170-200℃ for 1-2h, cool naturally to room temperature, centrifuge, wash the solid with ethanol and deionized water 2-3 times each, and vacuum dry to obtain Cu-MOF nanozyme; (3) After mixing Cu-MOF nanozyme solution, octopamine solution or tyramine solution of different concentrations and 4-aminoantipyrine solution, add pH 9.0 Tris–HCl buffer solution, irradiate with near-infrared light at 808nm for 10-20min, and measure absorbance at 505nm wavelength to determine the linear relationship between octopamine or tyramine concentration and absorbance value, and obtain the regression equation; (4) The absorbance value of the sample solution to be tested is determined according to the method in step (3), and then substituted into the regression equation to obtain the concentration of octopamine or tyramine in the sample solution to be tested.

2. The method according to claim 1, characterized in that: The Cu-MOF nanozyme solution concentration was 1 mg / mL, with an addition volume of 50-100 μL; the 4-aminoantipyrine solution concentration was 20 mmol / L, with an addition volume of 100-200 μL; the Tris–HCl buffer solution concentration was 0.1 mmol / L, with an addition volume of 1-3 mL; and the near-infrared light power at 808 nm was 1.0-2.0 W / cm². 2 .

3. The method according to claim 1, characterized in that: Centrifugation is performed at 8000-10000 r / min for 10-15 min.

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