A method for near-infrared driven plasma enhanced nanoenzyme colorimetric detection of histamine

Abstract

The present invention discloses a method for near-infrared driven plasma enhanced nanoenzyme colorimetric detection of histamine, wherein the method synthesizes graphite-like carbon nitride (g-C3N4) by pyrolysis, and then synthesizes composite nanomaterial Cu / MOF / C3N4 by Zn(NO3)2·6H2O, 2-hydroxyterephthalic acid and g-C3N4. Under near-infrared driven plasmon excitation, Cu / MOF / C3N4 exhibits significantly enhanced laccase-like activity, and the photothermal effect induced by localized surface plasmon resonance improves the laccase-like activity. When Cu / MOF / C3N4 oxidizes 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP), histamine can be adsorbed on the surface of Cu / MOF / C3N4, inhibiting the generation of semiquinone free radicals, thereby inhibiting the activity of Cu / MOF / C3N4-type laccase. The histamine concentration is in the linear range of 0.005~22.5 mg / L, causing the red color to become lighter. Therefore, a new highly sensitive colorimetric detection method for histamine was developed with a detection limit of 1 μg / L. The method of the present invention can only selectively detect histamine, and there is no interference from other substances.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis and detection, and specifically to a method for near-infrared driven plasma enhanced nanoenzyme colorimetric detection of histamine. Background Art

[0002] Histamine is the most toxic nitrogenous compound among all biogenic amines. It is usually produced from histidine in the presence of the enzyme decarboxylase, which makes it susceptible to histamine overload in foods with high protein content. High levels of histamine ingested by the body usually cause symptoms such as nausea, vomiting, diarrhea, rash, and skin itching. Histamine poisoning has attracted more and more attention, and many countries and organizations have established allowable limits for histamine in aquatic products. my country's national food safety standard for the determination of histamine in food GB5009.200-2016 stipulates that the content of histamine in aquatic products, soy sauce and its products shall not exceed 50-200 mg / kg. Therefore, it is urgent to establish a sensitive, reliable, economical and simple method for detecting histamine in food. At present, the commonly used methods for histamine detection include high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS) and high performance capillary electrophoresis. These methods usually have high sensitivity and reliable results, but require expensive large instruments and specialized technicians, and are not suitable for on-site detection of large quantities of samples. Recently, researchers have developed colorimetric, fluorescence, enzyme-linked immunosorbent, and electrochemical methods based on antibody recognition for the detection of histamine in food. However, antibodies have disadvantages such as high preparation cost, poor stability, and difficulty in purification. Therefore, it is of great significance to find a new generation of biorecognition molecules with low price and good stability for the specific detection of histamine.

[0003] As a nanozyme, graphite-like carbon nitride (g-C3N4) has attracted widespread attention in the field of photocatalysis due to its simple preparation method, wide and cheap raw material sources, intrinsic visible light absorption and unique conjugated π electron structure. However, the two-dimensional layered structure of g-C3N4 gives it a strong exciton effect, resulting in low separation efficiency of photogenerated carriers and poor catalytic performance. In addition, g-C3N4 cannot utilize most visible light and near-infrared light that accounts for about 50% of the solar spectrum. Among various nanozymes, plasmonic nanozymes have recently attracted the attention of researchers due to their tunable catalytic activity and unique localized surface plasmon resonance (LSPR) characteristics, that is, using light as an external stimulus to enhance the enzymatic activity of nanozymes. Compared with other modulation methods, these plasmonic nanozymes with light-enhanced activity have the advantages of non-invasiveness, adjustability, and spatial controllability. Summary of the invention

[0004] The present invention provides a method for colorimetric detection of histamine by near-infrared driven plasma enhanced nanozyme, wherein melamine is used as a precursor, graphite-like carbon nitride (g-C3N4) is synthesized by thermal decomposition, and then Zn(NO3)2·6H2O, 2-hydroxyterephthalic acid and g-C3N4 are used to synthesize the composite nanomaterial Cu / MOF / C3N4. Under near-infrared driven plasmon excitation, Cu / MOF / C3N4 exhibits significantly enhanced laccase-like activity, and the photothermal effect of Cu / MOF / C3N4 plasma nanozyme induced by localized surface plasmon resonance greatly improves the reaction kinetics, thereby significantly improving the laccase-like activity. When Cu / MOF / C3N4 oxidizes 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP), histamine can be adsorbed on the surface of Cu / MOF / C3N4, inhibiting the production of semiquinone free radicals, thereby inhibiting the activity of Cu / MOF / C3N4-type laccase. The histamine (Hi) concentration is in the linear range of 0.005 ~ 22.5 mg / L, causing the red color to become lighter. Therefore, a new highly sensitive colorimetric detection method for histamine was developed with a detection limit of 1 μg / L. The method of the present invention can only selectively detect histamine without interference from other substances. The method has the characteristics of high sensitivity, strong specificity, simple operation and rapidity.

[0005] The method of the present invention for near-infrared driven plasma enhanced nanoenzyme colorimetric detection of histamine is as follows:

[0006] (1) Take 3.0-3.5 g of melamine, calcine at 500-550 °C for 4-5 h, grind into powder, add 30-40 mL of anhydrous ethanol, ultrasonically treat for 2-2.5 h, centrifuge, collect the precipitate, wash with anhydrous ethanol 3-4 times, and vacuum dry to obtain g-C3N4;

[0007] (2) Take 0.2-0.3 g of Zn(NO3)2·6H2O and 0.05-0.1 g of 2-hydroxyterephthalic acid and dissolve them in 50-80 mL of DMF, then add 0.5-0.8 g of g-C3N4 and stir at room temperature for 12-15 h; then add 1-2 mL of 10 mg / mL CuCl2 and continue stirring for 1-2 h. Centrifuge and collect the precipitate. Wash it alternately with ethanol and ultrapure water three times and dry it in vacuum to obtain Cu / MOF / C3N4.

[0008] (3) Different concentrations of histamine standard solution, 2,4-dichlorophenol (2,4-DP) solution and 4-aminoantipyrine (4-AP) solution were added to the Cu / MOF / C3N4 nanomaterial solution to generate red oxidation products. The solution was diluted to 3 mL with pH 6.8 MES buffer solution and shaken well. After irradiation with 808 nm laser for 5-10 min, the absorbance was measured at a wavelength of 510 nm to establish a linear relationship between histamine concentration and absorbance A. A standard curve was drawn to obtain a regression equation.

[0009] (4) Extract Hi from the sample to be tested to obtain a sample assay solution, add the Cu / MOF / C3N4 nanozyme solution to the sample assay solution, then add the 2,4-DP solution and the 4-AP solution, dilute to 3 mL with a pH 6.8 MES buffer solution, shake well, irradiate with an 808 nm laser for 5-10 min, measure the absorbance at a wavelength of 510 nm, substitute the absorbance into the regression equation in step (3), and obtain the Hi content in the sample.

[0010] The concentration of the Cu / MOF / C3N4 nanozyme solution is 2 mg / mL, and the addition amount is 50-100 μL; the concentration of 2,4-DP is 1 mg / mL, and the volume is 50-100 μL; the concentration of 4-AP is 1 mg / mL, and the volume is 50-100 μL; the laser power used is 0.75-1.0 W / cm 2 .

[0011] In the step (1), the centrifugation is carried out at 8000-10000 r / min for 5-10 min.

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

[0013] 1. The present invention prepares Cu / MOF / C3N4 nanozymes, which exhibit significantly enhanced laccase-like activity under near-infrared driven plasmon excitation. The photothermal effect of Cu / MOF / C3N4 plasma nanozymes induced by localized surface plasmon resonance greatly improves the reaction kinetics, thereby significantly improving the laccase-like activity. When Cu / MOF / C3N4 oxidizes 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP), histamine can be adsorbed on the surface of Cu / MOF / C3N4, inhibiting the generation of semiquinone free radicals, thereby inhibiting the activity of Cu / MOF / C3N4-like laccases. The histamine concentration is in the linear range of 0.005 ~ 22.5 mg / L, resulting in a lighter red color. Therefore, a new method for highly sensitive histamine colorimetric detection has been developed.

[0014] 2. The colorimetric detection method for histamine established in the present invention has a wide linear range, high detection sensitivity, and a detection limit of 1 μg / L. The coexisting substances do not interfere with the determination, and the method has good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 TEM image of the Cu / MOF / C3N4 nanozyme prepared in Example 1 of the present invention;

[0016] Figure 2 This is the UV-vis-NIR absorption spectrum of the Cu / MOF / C3N4 nanozyme in Example 1;

[0017] Figure 3 The effect of near infrared light (808 nm) and Hi on the activity of Cu / MOF / C3N4 laccase in Example 1;

[0018] Figure 4 The effect of N2 on the activity of Cu / MOF / C3N4 laccase in Example 1;

[0019] Figure 5 This is a graph showing the determination results of semiquinone free radicals in Example 1;

[0020] Figure 6 The linear regression equation for detecting Hi in the Cu / MOF / C3N4 +2,4-DP +4-AP system irradiated at 808 nm for 10 min in Example 1;

[0021] Figure 7 The results of the effects of other biogenic amines and amino acids on the Hi detection system in Example 1;

[0022] Figure 8 This is the result of the effect of other coexisting ions on the Hi detection system in Example 1. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1: Determination of histamine in fermented bean curd samples

[0025] 1. Preparation of g-C3N4: Take 3.0 g of melamine, calcine it at 500℃ for 5 h, grind it into powder, add 30 mL of anhydrous ethanol, ultrasonically treat it for 2 h, centrifuge it, collect the precipitate, wash it with anhydrous ethanol 3-4 times, and vacuum dry it to obtain g-C3N4.

[0026] 2. Preparation of Cu / MOF / C3N4: 0.2 g Zn(NO3)2·6H2O and 0.05 g 2-hydroxyterephthalic acid were dissolved in 50 mL DMF, and then 0.5 g g-C3N4 was added and stirred at room temperature for 12 h. Then 1 mL 10 mg / mL CuCl2 was added and stirred for 1 h. The mixture was centrifuged at 8000 r / min for 5 min, and the precipitate was collected and washed alternately with ethanol and ultrapure water for three times. The precipitate was dried in vacuum to obtain Cu / MOF / C3N4. The TEM characterization results are shown in the figure. Figure 1 The figure shows the flake state of g-C3N4 and its aggregation state after being composited with Cu / MOF.

[0027] 3. Evaluation of the laccase-like activity of Cu / MOF / C3N4 enhanced by plasma excitation: The UV-vis-NIR absorption spectrum of Cu / MOF / C3N4 shows that Cu / MOF / C3N4 exhibits a wide absorption region as a plasma, even extending to the near infrared ( Figure 2 ). The laccase-like activity of the enzyme was evaluated using 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) as substrates for the catalytic reaction. Figure 3 It shows that in the presence of Cu,Ce-PTs, 2,4-DP and 4-AP are oxidized to generate corresponding red products with a maximum absorption peak at 510 nm. 2 , 808 nm near-infrared light irradiation for 10 min, the absorbance at 510 nm was significantly increased, and after adding Hi, the absorbance decreased ( Figure 3 ); At the same time, in order to evaluate the activity of Cu / MOF / C3N4-type laccase, N2 was used to replace air, and the absorbance decreased significantly, indicating that the activity of Cu / MOF / C3N4-type laccase oxidase ( Figure 4 ).

[0028] 4. Determination of semiquinone free radicals: A mixture containing 100 μL of 2 mg / mL Cu / MOF / C3N4 and 100 μL of 5 mg / L histamine was incubated at 30°C for 20 min. Then, 200 μL of 7.0 HEPES buffer was added to the mixture and mixed immediately, followed by sampling and EPR detection. Figure 5 The results showed that significant EPR signals were captured in the Cu / MOF / C3N4 laccase-mimicking catalytic system with substrate 2,4-DP, indicating the generation of semiquinone radicals. When Hi solution was added to the solution, the EPR signal in the sample decreased significantly, which was attributed to the adsorption of Hi on the surface of Cu / MOF / C3N4, covering the active sites and inhibiting the generation of semiquinone radicals.

[0029] 5. Preparation of histamine (Hi) working curve: add 100 μL of 2 mg / mL Cu / MOF / C3N4 (Hi standard solution with a concentration of 0.005 ~ 22.5 mg / L) to a 5 mL stoppered colorimetric tube, add 100 μL of 20 mmol / L 2,4-dichlorophenol (2,4-DP), 100 μL of 20 mmol / L 4-aminopyrine (4-AP), add 0.1 mmol / L pH 6.8 MES buffer solution to 3 mL, shake well, and incubate at a power of 1.0 W / cm 2 , 808nm near-infrared light irradiation for 10min, and measure the absorbance A at a wavelength of 510nm. With Hi concentration as the abscissa and absorbance A as the ordinate, standard curves were drawn to obtain the regression equation, see Figure 6 ; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0030]

[0031] 6. Method specificity investigation: Histamine (Hi) was replaced by other biogenic amines and amino acids, including cadaverine (Cad), tyramine (Ty), putrescine (Put), tryptamine (Tr), 2-hydroxyphenylethylamine (Ta), phenylalanine (Phe), proline (Pro), leucine (Leu), tyrosine (Tyr), serine (Ser), histidine (His), ascorbic acid (AA), and coexisting ions (Na + , K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ , Fe 2+ , Cl - 、SO4 2- 、NO3 - ), detect the effects of Hi and other substances on Cu / MOF / C3N4 nanozyme in the detection system of step 4; the concentration of Hi and other preservatives was 5 μg / mL, and the concentration of coexisting ions was 50 μg / mL. The results are shown in Figure 7 , 8 ,As can be seen from the figure, the Cu / MOF / C3N4 nanozyme detection system has good ,selectivity. Hi obviously inhibits the oxidation reaction, while other ,substances have almost no effect. The method for determining Hi has good ,selectivity.

[0032] 7. Determination of histamine in fermented bean curd samples

[0033] (1) Sample processing for Hi determination

[0034] Weigh 2.0 g of sample (accurate to 0.001 g), place in a 50 mL centrifuge tube, add 20 mL of 0.1 mol / L HCl solution, homogenize with a stirring separator (16000 r / min) for 1-2 min, centrifuge at 6500 r / min for 30 min (20 g / L), take the supernatant and filter through a 0.45 μm organic filter membrane to obtain the sample test solution;

[0035] (2) Determination of Hi in fermented bean curd samples: 100 μL of 2 mg / mL Cu / MOF / C3N4 was added to a 5 mL stoppered colorimetric tube. 200 μL of the sample solution from step (1) was added. 100 μL of 20 mmol / L 2,4-dichlorophenol (2,4-DP) and 100 μL of 20 mmol / L 4-aminopyrine (4-AP) were added. 0.1 mmol / L pH 6.8 MES buffer solution was added to make up to 3 mL. The mixture was shaken and incubated at a power of 1.0 W / cm 2 , 808 nm near-infrared light irradiation for 10 min, measure the absorbance A at a wavelength of 510 nm, and substitute it into the regression equation in step 5, the Hi content is 205.5 mg / kg.

[0036] Example 2: Determination of histamine in ham samples

[0037] 1. Preparation of g-C3N4: Take 3.5 g of melamine, calcine it at 550℃ for 4 h, grind it into powder, add 40 mL of anhydrous ethanol, ultrasonically treat it for 2.5 h, centrifuge it, collect the precipitate, wash it with anhydrous ethanol 3-4 times, and vacuum dry it to obtain g-C3N4.

[0038] 2. Preparation of Cu / MOF / C3N4: Take 0.3 g of Zn(NO3)2·6H2O and 0.1 g of 2-hydroxyterephthalic acid and dissolve them in 80 mL of DMF, then add 0.8 g of g-C3N4 and stir at room temperature for 15 h; then add 2 mL of 10 mg / mL CuCl2 and continue stirring for 2 h. Centrifuge at 8000 r / min for 10 min, collect the precipitate, wash it alternately with ethanol and ultrapure water three times, and dry it in vacuum to obtain Cu / MOF / C3N4.

[0039] 3. Preparation of histamine (Hi) working curve: Same as Example 1.

[0040] 4. Determination of Hi in ham samples

[0041] (1) Sample processing for Hi determination

[0042] Weigh 5 g of the minced and mixed sample, add 15 mL of trichloroacetic acid solution (100 g / L), ultrasonically extract for 30 min, and filter. Pipette 2 mL of the filtrate into a centrifuge tube, adjust the pH value to alkaline with sodium hydroxide solution, add 3 mL of n-pentanol each time, vortex for 1 min, extract 3 times, combine the n-pentanol extracts and dilute to 10.0 mL. Pipette 2.0 mL of n-pentanol extract into a centrifuge tube, add 3 mL of hydrochloric acid (0.1 mol / L) each time, shake and extract 3 times, combine the hydrochloric acid extracts and dilute to 10.0 mL to obtain the sample test solution;

[0043] (2) Determination of Hi in ham samples: Same as Example 1, the Hi content was 68.8 mg / kg.

[0044] Example 3: Determination of histamine in wine samples

[0045] 1. Preparation of g-C3N4: Take 3.2 g of melamine, calcine it at 550℃ for 4 h, grind it into powder, add 35 mL of anhydrous ethanol, ultrasonically treat it for 2.5 h, centrifuge it, collect the precipitate, wash it with anhydrous ethanol 3-4 times, and vacuum dry it to obtain g-C3N4.

[0046] 2. Preparation of Cu / MOF / C3N4: Take 0.3 g of Zn(NO3)2·6H2O and 0.08 g of 2-hydroxyterephthalic acid and dissolve them in 60 mL of DMF, then add 0.6 g of g-C3N4 and stir at room temperature for 14 h; then add 2 mL of 10 mg / mL CuCl2 and continue stirring for 2 h. Centrifuge at 10000 r / min for 5 min, collect the precipitate, wash it alternately with ethanol and ultrapure water three times, and dry it in vacuum to obtain Cu / MOF / C3N4.

[0047] 3. Preparation of histamine (Hi) working curve: Same as Example 1.

[0048] 4. Determination of Hi in wine samples

[0049] (1) Determination of Hi in wine samples: Same as Example 1, Hi content is 0.5 mg / L.

[0050] (2) Recovery and precision experiments

[0051] Three different concentrations of Hi standard solutions were added to three samples respectively; each concentration was measured three times in parallel, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2; the spiked recovery rate of DHA was measured to be between 93.6% and 105.0%, and the RSD was between 2.8% and 4.6%. This method has good accuracy and precision.

[0052]

[0053] The histamine determination method established by the present invention has the advantages of fewer processing steps, shorter time, lower processing cost, simple operation, and no need for large-scale instruments and equipment, and has great advantages in actual detection.

Claims

1. A method for near-infrared driven plasma enhanced nanozyme colorimetric detection of histamine, characterized in that: The following steps are involved: (1) Take 3.0-3.5 g of melamine, calcine at 500-550 °C for 4-5 h, grind into powder, add 30-40 mL of anhydrous ethanol, ultrasonically treat for 2-2.5 h, centrifuge, collect the precipitate, wash with anhydrous ethanol 3-4 times, and vacuum dry to obtain g-C3N4; (2) Take 0.2-0.3 g of Zn(NO3)2·6H2O and 0.05-0.1 g of 2-hydroxyterephthalic acid and dissolve them in 50-80 mL of DMF, then add 0.5-0.8 g of g-C3N4 and stir at room temperature for 12-15 h; then add 1-2 mL of 10 mg / mL CuCl2 and continue stirring for 1-2 h. Centrifuge and collect the precipitate. Wash it alternately with ethanol and ultrapure water for three times and dry it in vacuum to obtain Cu / MOF / C3N4. (3) Add different concentrations of histamine standard solution, 2,4-dichlorophenol solution and 4-aminoantipyrine solution to the Cu / MOF / C3N4 nanomaterial solution to generate red oxidation products. Use pH 6.8 MES buffer solution to make the volume 3 mL, shake well, irradiate with 808 nm laser for 5-10 min, measure the absorbance at 510 nm, establish the linear relationship between histamine concentration and absorbance A, draw the standard curve, and obtain the regression equation. (4) Extract histamine from the sample to be tested to obtain a sample assay solution, add the Cu / MOF / C3N4 nanozyme solution to the sample assay solution, then add the 2,4-dichlorophenol solution and the 4-aminoantipyrine solution, dilute to 3 mL with a pH 6.8 MES buffer solution, shake well, irradiate with an 808 nm laser for 5-10 min, and then measure the absorbance at a wavelength of 510 nm. Substitute the absorbance into the regression equation in step (3) to obtain the histamine content in the sample.

2. The method for colorimetric detection of histamine by near-infrared driven plasma enhanced nanozyme according to claim 1, characterized in that: The concentration of Cu / MOF / C3N4 nanozyme solution was 2 mg / mL, and the addition amount was 50-100 μL; the concentration of 2,4-dichlorophenol was 20 mmol / L, and the volume was 100-150 μL; the concentration of 4-aminoantipyrine was 20 mmol / L, and the volume was 100-150 μL; the laser power used was 0.75-1.0 W / cm 2 .

3. The method for colorimetric detection of histamine by near-infrared driven plasma enhanced nanoenzyme according to claim 1, characterized in that: In step (1), the centrifugation is carried out at 8000-10000 r / min for 5-10 min.

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