A histamine and spermine colorimetric detection method based on pro-ag mimicking laccase activity

Abstract

This invention discloses a colorimetric detection method for histamine and spermine based on Pro-Ag nanozyme-mimicking laccase activity. The method involves first plotting standard absorbance curves for histamine and spermine, then constructing linear regression equations to determine the relationship between the concentrations of histamine and spermine and their absorbance values. The concentrations of histamine and spermine in the sample are then determined using these linear regression equations. This invention is the first to apply the laccase-mimicking activity of Pro-Ag nanozymes to the detection of biogenic amines. In HEPES buffer solution, histamine and spermine can inhibit the laccase activity of Pro-Ag nanozymes, causing the reaction solution to appear light pink, thus enabling rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of colorimetric detection technology of histamine and spermine, and in particular relates to a colorimetric detection method for histamine and spermine based on Pro-Ag simulating laccase activity. Background Technology

[0002] Biogenic amines are nitrogen-containing organic compounds produced during protein breakdown. Histamine is a typical example, formed by the decarboxylation of L-histidine under the catalysis of decarboxylases. Various biogenic amines are commonly found in beer; they are natural byproducts of alcoholic fermentation. The formation of these low-molecular-weight nitrogenous compounds mainly stems from the decarboxylation of amino acids in the raw materials under the action of decarboxylases secreted by microorganisms (especially yeast and lactic acid bacteria). Whether these microorganisms are introduced intentionally for specific fermentation processes or due to accidental contamination, their metabolic activities affect the sensory characteristics of beer and contribute to the formation of biogenic amines. Among the many biogenic amines, histamine is the most typical and of particular concern. It is formed by the decarboxylation of L-histidine in the raw materials under the catalysis of histidine decarboxylases secreted by microorganisms, and its content increases significantly, especially when the freshness of the raw materials is insufficient or the hygiene conditions of the production process are poor. Therefore, detecting biogenic amines in beer is of great significance. Firstly, it is a key food safety and quality control indicator; in particular, the histamine content effectively reflects the freshness of raw materials such as malt and the level of hygiene management during the production process. Secondly, testing is also to protect consumers' health, because excessive intake of histamine may cause discomfort such as headaches and palpitations. By controlling the content within a safe range through testing, the safety of drinking the product can be ensured, especially for people who are sensitive to histamine. Summary of the Invention

[0003] The purpose of this invention is to provide a colorimetric detection method for histamine and spermine based on Pro-Ag-mimicking laccase activity. This method offers advantages such as visual identification, ease of operation, rapid detection, and low cost.

[0004] The technical solution of this invention is as follows: A colorimetric detection method for histamine and spermine based on Pro-Ag simulating laccase activity. First, standard absorbance curves for histamine and spermine are plotted separately. Then, linear regression equations are constructed based on these standard absorbance curves to determine the relationship between the concentrations of histamine and spermine and their absorbance values. The concentrations of histamine and spermine in the test sample are determined using these linear regression equations. The specific steps are as follows: (1) Preparation of Pro-Ag nanozyme solution: 9-11 mL of 5 mM silver nitrate aqueous solution was slowly added to 9-11 mL of 10 mM proline alkaline solution. The reaction was carried out at 22-28°C for 11-13 hours, centrifuged for 8-12 min, the precipitate was collected, washed 4 times with ultrapure water, and finally dried at 55-65°C to obtain monodisperse nanozyme. Ultrapure water was added to prepare a solution of 0.9-1.1 mg·mL⁻¹. -1 Pro-Ag nanozyme solution, for later use; (2) Preparation of blank control solution: Take one centrifuge tube and add 1 mg·mL -1 Add 28-32 μL of Pro-Ag nanozyme, then add 8-12 μL of ultrapure water, mix well, and incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution with a concentration of 28-32 mM, then add 390-410 μL of HEPES buffer with a concentration of 8-12 mM, and finally add 48-52 μL of 4-aminoantipyrine solution with a concentration of 4.5-5.5 mM to obtain the blank control solution. (3) Preparation of the histamine standard solution to be tested: Take 8 portions of histamine reference standard and add ultrapure water to prepare solutions of 23-27 mg·L⁻¹. -1 Histamine standard solution, 48-52 mg / L -1 Histamine standard solution, 73-77 mg / L -1 Histamine standard solution, 98-102 mg / L -1 Histamine standard solution, 198-202 mg / L -1 Histamine standard solution, 298-302 mg·L -1 Histamine standard solution, 398-402 mg·L -1 Histamine standard solution and 498-502 mg·L -1 Take 8-12 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 25-35 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 0.9-1.1 mg·mL. -1 After mixing, incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM) to each centrifuge tube, followed by 390-410 μL of HEPES buffer (9-11 mM) to each centrifuge tube. Finally, add 48-52 μL of 4-aminoantipyrine solution (4.5-5.5 mM) to each centrifuge tube to obtain 8 groups of histamine standard solutions of different concentrations. (4) Preparation of spermine standard solution: Take 6 portions of spermine reference standard and add ultrapure water to prepare solutions of 98-102 mg / L. -1 Spermine standard solution, 248-252 mg / L -1 Spermine standard solution, 498-502 mg / L -1 Spermine standard solution, 748-752 mg / L -1 Spermine standard solution, 998-1002 mg·L -1 Spermine standard solution and 1248-1252 mg·L -1 Take 8-12 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 28-32 μL of Pro-Ag nanozyme solution (concentration of 0.9-1.1 mg·mL⁻¹). -1 After mixing, incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM) to each centrifuge tube, followed by 390-410 μL of HEPES buffer (10 mM) to each centrifuge tube. Finally, add 48-52 μL of 4-aminoantipyrine solution (4.5-5.5 mM) to each centrifuge tube to obtain 6 sets of spermine standard solutions with different concentrations. (5) Construction of histamine absorbance standard curve: Take 200 μL of the 8 groups of histamine standard solutions of different concentrations prepared in step (3) and the blank control solution prepared in step (6) respectively and place them in a 96-well microplate. Use a microplate reader to scan and measure the absorbance value at 506 nm. Based on the absorbance value, use the difference in absorbance value between the 8 groups of histamine standard solutions of different concentrations and the blank control solution as the ordinate and the histamine concentration as the abscissa to draw a standard curve of target concentration versus absorbance change, which is the histamine absorbance standard curve. (6) Construction of spermine absorbance standard curve: Take 200 μL of the six groups of spermine standard solutions of different concentrations prepared in step (5) and the blank control solution prepared in step (6) into a 96-well microplate, and use a microplate reader to scan and measure the absorbance value at 506 nm. Based on the absorbance value, use the difference in absorbance value between the six groups of spermine standard solutions of different concentrations and the blank control solution as the ordinate and the spermine concentration as the abscissa to draw a standard curve of target concentration versus absorbance change, which is the spermine absorbance standard curve. (7) Constructing a linear regression equation: Based on the standard curves of histamine absorbance and spermine absorbance, establish a regression equation for the relationship between the concentrations of histamine and spermine and the changes in absorbance.

[0005] (8) Preparation of the test sample: After ultrasonic degassing of beer, dilute it with double-distilled water to prepare the test solution. Take 8-12 μL of the diluted test solution and add it to a container containing 28-32 μL of a solution with a concentration of 0.9-1.1 mg·mL. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM), followed by 390-410 μL of HEPES buffer (8-12 mM), and finally 48-52 μL of 4-aminoantipyrine solution (4.5-5.5 mM) to obtain the sample to be tested. (9) Detection of histamine and spermine content in the sample to be tested: Take the sample to be tested and perform absorption spectral scanning to measure its absorbance value at a wavelength of 506 nm. Calculate the absorbance difference between the sample to be tested and the blank control solution. Substitute the absorbance difference into the linear regression equation to obtain the content of histamine and spermine in the sample to be tested.

[0006] In step (1) above, the Pro-Ag nanozyme solution was prepared as follows: 10 mL of 5 mM silver nitrate aqueous solution was slowly added to 10 mL of 10 mM proline alkaline solution, and the reaction was carried out at 25°C for 12 hours. The solution was centrifuged at 10000 rpm for 10 min, the precipitate was collected, washed 4 times with ultrapure water, and finally dried at 60°C to obtain monodisperse nanozyme. Ultrapure water was added to prepare a solution of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution is ready for use.

[0007] In step (2) above, the blank control solution is prepared by taking one centrifuge tube and adding 1 mg·mL⁻¹. -1 Add 29-31 μL of Pro-Ag nanozyme, then add 9-11 μL of ultrapure water, mix well, and incubate at room temperature for 9-11 min. Then add 9-11 μL of 2,4-dichlorophenol solution with a concentration of 29-31 mM, then add 395-405 μL of HEPES buffer with a concentration of 9-11 mM, and finally add 49-51 μL of 4-aminoantipyrine solution with a concentration of 5 mM to obtain the blank control solution.

[0008] Specifically, in step (2) above, the blank control solution is prepared by taking one centrifuge tube and adding 1 mg·mL⁻¹. -1Add 30 μL of Pro-Ag nanozyme and 10 μL of ultrapure water, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine solution to obtain the blank control solution.

[0009] In step (3) above, the preparation of the histamine standard solution to be tested is as follows: Take 8 portions of histamine reference standard and add ultrapure water to prepare solutions of 24-26 mg·L⁻¹. -1 Histamine standard solution, 49-51 mg / L -1 Histamine standard solution, 74-76 mg / L -1 Histamine standard solution, 99-101 mg / L -1 Histamine standard solution, 199-201 mg / L -1 Histamine standard solution, 299-301 mg / L -1 Histamine standard solution, 399-401 mg / L -1 Histamine standard solution and 499-501 mg·L -1 Take 9-11 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 28-32 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 0.95-1.05 mg·mL. -1 After mixing, incubate at room temperature for 9-11 min. Then, add 9-11 μL of 2,4-dichlorophenol solution (29-31 mM) to each centrifuge tube, followed by 395-405 μL of HEPES buffer (9.5-10.5 mM) to each centrifuge tube. Finally, add 49-51 μL of 4-aminoantipyrine solution (5 mM) to each centrifuge tube to obtain 8 sets of histamine standard solutions of different concentrations.

[0010] Specifically, in step (3) above, the preparation of the histamine standard solution to be tested is as follows: Take 8 portions of histamine reference standard and add ultrapure water to prepare 25 mg·L⁻¹ solutions. -1 Histamine standard solution, 50 mg / L -1 Histamine standard solution, 75 mg / L -1 Histamine standard solution, 100 mg / L -1 Histamine standard solution, 200 mg / L -1 Histamine standard solution, 300 mg / L -1 Histamine standard solution, 400 mg / L -1 Histamine standard solution and 500 mg·L -1Take 10 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 30 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 1 mg·mL⁻¹. -1 After mixing, incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution to each centrifuge tube, followed by 400 μL of 10 mM HEPES buffer to each centrifuge tube. Finally, add 50 μL of 5 mM 4-aminoantipyrine solution to each centrifuge tube to obtain 8 sets of histamine standard solutions of different concentrations.

[0011] In step (4) above, the preparation of the spermine standard solution to be tested is as follows: Take 6 portions of spermine reference standard and add ultrapure water to prepare solutions of 99-101 mg·L⁻¹. -1 Spermine standard solution, 249-251 mg / L -1 Spermine standard solution, 499-501 mg / L -1 Spermine standard solution, 749-751 mg / L -1 Spermine standard solution, 999-1001 mg·L -1 Spermine standard solution and 1249-1251 mg·L -1 Take 9-11 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 29-31 μL of Pro-Ag nanozyme solution (concentration of 0.95-1.05 mg·mL). -1 After mixing, incubate at room temperature for 9-11 min. Then add 9-11 μL of 2,4-dichlorophenol solution (29-31 mM) to each centrifuge tube, followed by 395-405 μL of HEPES buffer (10 mM) to each centrifuge tube. Finally, add 49-51 μL of 4-aminoantipyrine solution (5 mM) to each centrifuge tube to obtain 6 sets of spermine standard solutions with different concentrations.

[0012] Specifically, in step (4) above, the preparation of the spermine standard solution to be tested is as follows: take 6 portions of spermine reference standard and add ultrapure water to prepare 100 mg·L⁻¹ solutions. -1 Spermine standard solution, 250 mg / L -1 Spermine standard solution, 500 mg / L -1 Spermine standard solution, 750 mg·L -1 Spermine standard solution, 1000 mg·L -1 Spermine standard solution and 1250 mg·L -1Take 10 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 30 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 1 mg·mL⁻¹. -1 After mixing, incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution to each centrifuge tube, followed by 400 μL of 10 mM HEPES buffer to each centrifuge tube. Finally, add 50 μL of 5 mM 4-aminoantipyrine solution to each centrifuge tube to obtain 6 sets of spermine standard solutions of different concentrations.

[0013] In step (8) above, the preparation of the test sample is as follows: Take 4-6 mL of beer, degas it by sonication for 15-25 min, dilute it with 4-6 mL of double-distilled water, take 10 μL of the diluted test solution, and add 28-32 μL of a solution with a concentration of 0.9-1.1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 9-11 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM), followed by 395-405 μL of HEPES buffer (9-11 mM), and finally 49-51 μL of 4-aminoantipyrine solution (5 mM) to obtain the sample to be tested.

[0014] Specifically, in step (8) above, the preparation of the test sample is as follows: take 5 mL of beer, degas it by sonication for 20 min, add 5 mL of double-distilled water for dilution, take 10 μL of the diluted test solution, and add 30 μL of a solution with a concentration of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution was placed in a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer, and finally 50 μL of 5 mM 4-aminoantipyrine solution to obtain the sample to be tested.

[0015] The basic principle of this invention is as follows: Pro-Ag nanozymes catalyze the oxidation of the substrate 2,4-DP to a colorless quinone compound under alkaline HEPES buffer. This colorless quinone compound further binds to 4-AAP to generate a red product, causing the sensing solution to appear red, with a characteristic peak at 506 nm. Upon addition of target histamine or spermine, the electron transfer between the Pro-Ag nanozyme and the substrate 2,4-DP is blocked by histamine or spermine. The nanozyme's laccase-like activity is inhibited, preventing further oxidation of the substrate to produce the colorless quinone compound. Consequently, it cannot bind to 4-AAP to generate a colorimetric product. Furthermore, the change in absorbance at 506 nm is directly proportional to the concentrations of the target histamine and spermine. Therefore, this method can be used for the detection of histamine and spermine (see image). Figure 1 ).

[0016] Beneficial effects of this invention: The detection method provided by this invention does not rely on large-scale instruments and equipment, has high detection sensitivity, is simple and rapid to operate, and is visible to the naked eye. It can be applied to the rapid detection of histamine and spermine. Furthermore, it has strong anti-interference effects and is unaffected by spermidine, tryptamine, putrescine, cadaverine, phenylethylamine, and tyramine. Attached Figure Description

[0017] Figure 1 A schematic diagram illustrating the principle of histamine and spermine detection; Figure 2 The relationship between different concentrations of histamine and the absorbance difference (ΔA / A0) is shown. Figure 3 The relationship between different concentrations of spermine and the absorbance difference (ΔA / A0); Figure 4 A schematic diagram illustrating the feasibility of histamine detection (1: blank control solution; 1: 2 mg·L⁻¹). -1 Histamine sample; 2: 10 mg·L -1 Histamine sample; 3:30 mg·L -1 Histamine sample; 4: 70 mg·L -1 Histamine sample); Figure 5 A schematic diagram illustrating the feasibility of spermine detection (1: blank control solution; 1: 10 mg·L⁻¹). -1 Spermine sample; 2: 20 mg·L -1 Spermine sample; 3: 50 mg·L -1 Spermine sample; 4: 200 mg·L -1 (spermine sample); Figure 6 This diagram illustrates the effect of other interfering substances on the detection of histamine and spermine. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention. Example 1: Reagent Preparation

[0019] Preparation of 5 mM silver nitrate aqueous solution: Add silver nitrate to ultrapure water to prepare a 5 mM silver nitrate aqueous solution for later use; Preparation of 10 mM proline alkaline solution: Add proline to sodium hydroxide aqueous solution to prepare a 10 mM proline alkaline solution for later use; Preparation of 30 mM 2,4-dichlorophenol solution: Add 2,4-dichlorophenol to N,N-dimethylformamide (DMF) to prepare a 30 mM 2,4-dichlorophenol solution (2,4-DP) for later use; Preparation of 10 mM HEPES buffer: Add HEPES to ultrapure water to prepare a 10 mM HEPES buffer for later use. Preparation of 5 mM 4-aminoantipyrine solution: Add 4-aminoantipyrine to ultrapure water to prepare a 5 mM 4-aminoantipyrine solution (4-AAP) for later use.

[0020] Example 2: Constructing a linear regression equation (1) Preparation of Pro-Ag nanozyme solution: 10 mL of 5 mM silver nitrate aqueous solution was slowly added to 10 mL of 10 mM proline alkaline solution. The reaction was carried out at 25°C for 12 hours. The solution was centrifuged at 10000 rpm for 10 min, the precipitate was collected, and washed 4 times with 30 mL of ultrapure water each time. Finally, the solution was dried at 60°C to obtain monodisperse nanozyme, which was then diluted with ultrapure water to prepare a solution of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution is ready for use.

[0021] (2) Preparation of blank control solution: Take one centrifuge tube and add 1 mg·mL -1 Add 30 μL of Pro-Ag nanozyme and 10 μL of ultrapure water, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine solution to obtain the blank control solution.

[0022] (3) Preparation of the histamine standard solution to be tested: Take 8 portions of histamine reference standard and add ultrapure water to prepare 25 mg·mL solutions respectively. -1 Histamine standard solution, 50 mg / mL -1 Histamine standard solution, 75 mg / mL-1 Histamine standard solution, 100 mg / mL -1 Histamine standard solution, 200 mg / mL -1 Histamine standard solution, 300 mg / mL -1 Histamine standard solution, 400 mg / mL -1 Histamine standard solution and 500 mg / mL -1 Take 10 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 30 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 1 mg·mL⁻¹. -1 After mixing, incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution to each centrifuge tube, followed by 400 μL of 10 mM HEPES buffer to each centrifuge tube. Finally, add 50 μL of 5 mM 4-aminoantipyrine to each centrifuge tube to obtain 8 groups of histamine standard solutions of different concentrations.

[0023] (4) Preparation of the standard solution of spermine to be tested: Take 6 portions of spermine reference standard and add ultrapure water to prepare 100 mg·mL. -1 Spermine standard solution, 250 mg / mL -1 Spermine standard solution, 500 mg / mL -1 Spermine standard solution, 750 mg / mL -1 Spermine standard solution, 1000 mg / mL -1 Spermine standard solution and 1250 mg / mL -1 Take 10 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 30 μL of Pro-Ag nanozyme solution (the concentration of Pro-Ag nanozyme solution is 1 mg·mL⁻¹). -1 After mixing, incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution to each centrifuge tube, followed by 400 μL of 10 mM HEPES buffer to each centrifuge tube. Finally, add 50 μL of 5 mM 4-aminoantipyrine to each centrifuge tube to obtain 6 sets of spermine standard solutions of different concentrations.

[0024] (5) Construction of histamine absorbance standard curve: Take 200 μL of the 8 groups of histamine standard solutions of different concentrations prepared in step (3) and the blank control solution prepared in step (6) respectively and place them in a 96-well microplate. Use a microplate reader to scan and measure the absorbance value at 506 nm. Based on the absorbance value, use the difference in absorbance value between the 8 groups of histamine standard solutions of different concentrations and the blank control solution as the ordinate and the histamine concentration as the abscissa to draw a standard curve of target concentration versus absorbance change, which is the histamine absorbance standard curve. (6) Construction of spermine absorbance standard curve: Take 200 μL of the six groups of spermine standard solutions of different concentrations prepared in step (5) and the blank control solution prepared in step (6) into a 96-well microplate, and use a microplate reader to scan and measure the absorbance value at 506 nm. Based on the absorbance value, use the difference in absorbance value between the six groups of spermine standard solutions of different concentrations and the blank control solution as the ordinate and the spermine concentration as the abscissa to draw a standard curve of target concentration versus absorbance change, which is the spermine absorbance standard curve. (7) Constructing a linear regression equation: Based on the standard curves of histamine absorbance and spermine absorbance, establish a regression equation for the relationship between the concentrations of histamine and spermine and the changes in absorbance.

[0025] Following the steps above, the histamine concentration C was found to be 0.5 mg·L⁻¹. -1 ≤C≤10 mg·L -1 When the concentration and the change in absorbance value are, the regression equation is: y = 0.07021C + 0.13397; The spermine concentration C is 2 mg·L -1 ≤C≤25 mg·L -1 When the concentration and the change in absorbance value are, the regression equation is: y = 0.02344C + 0.12978; y is the absorbance difference (ΔA) = absorbance value of A2 sample - absorbance value of the test sample containing target histamine.

[0026] Depend on Figure 2 and Figure 3 The correlation between different concentrations of histamine and the absorbance difference (ΔA / A0) and the correlation between different concentrations of spermine and the absorbance difference (ΔA / A0) demonstrate that the detection method has the characteristics of good stability and high sensitivity.

[0027] Example 3: Detection of the sample to be tested (1) Preparation of the test sample: Take 5 mL of beer, degas it by sonication for 20 min, dilute it with 5 mL of double-distilled water, take 10 μL of the diluted test solution, and add it to a 30 μL container containing a concentration of 1 mg·mL⁻¹-1 The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer, and finally 50 μL of 5 mM 4-aminoantipyrine solution to obtain the sample to be tested.

[0028] (2) Detection of the sample to be tested: Take the sample to be tested and perform absorption spectral scanning to measure its absorbance value at a wavelength of 506 nm. Calculate the absorbance difference between the sample to be tested and the blank control solution. Substitute the absorbance difference into the linear regression equation to obtain the content of histamine and spermine contained in the sample to be tested.

[0029] Example 4: Feasibility Experiment for Histamine Detection (1) 2 mg·L -1 Preparation of histamine sample: Dissolve histamine in ultrapure water to prepare a solution of 100 mg·L⁻¹ -1 To determine the concentration, take 10 μL of histamine and add it to a 30 μL container containing a solution of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer. Finally, 50 μL of 5 mM 4-aminoantipyrine was added to obtain a final concentration of 2 mg / L. -1 Histamine sample to be tested.

[0030] (2) 10 mg·L -1 Preparation of histamine sample: Dissolve histamine in ultrapure water to prepare a solution of 500 mg·L⁻¹. -1 The concentration was increased by adding 30 μL of a solution containing 1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, followed by 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine to obtain a final concentration of 10 mg·L⁻¹. -1 Histamine sample.

[0031] (3) 30 mg·L -1 Preparation of histamine sample: Dissolve histamine in ultrapure water to prepare a solution of 1500 mg·L⁻¹ -1 The concentration was increased by adding 30 μL of a solution containing 1 mg·mL⁻¹. -1The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer. Finally, 50 μL of 5 mM 4-aminoantipyrine was added to obtain a final concentration of 30 mg·L⁻¹. -1 Histamine sample.

[0032] (4) 70 mg·L -1 Preparation of histamine sample: Dissolve histamine in ultrapure water to prepare a solution of 3500 mg·L⁻¹. -1 The concentration was increased by adding 30 μL of a solution containing 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer. Finally, 50 μL of 5 mM 4-aminoantipyrine was added to obtain a final concentration of 70 mg·L⁻¹. -1 Histamine sample.

[0033] (5) Absorbance detection: Take the blank control solution prepared in Example 2 and the 2 mg·L⁻¹ solution prepared in the above steps. -1 Histamine sample, 10 mg·L -1 Histamine sample, 30 mg·L -1 Histamine sample and 70 mg·L -1 Histamine samples were subjected to absorption spectral scanning, and their absorbance values ​​at a wavelength of 506 nm were measured. The results are shown below. Figure 4 .

[0034] Example 5: Feasibility Experiment for Spermine Detection (1) 10 mg·L -1 Preparation of spermine sample: Dissolve spermine in ultrapure water to prepare a solution of 500 mg·L⁻¹ -1 To determine the concentration, take 10 μL and add it to a 30 μL container containing a concentration of 1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, followed by 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine to obtain a final concentration of 10 mg·L⁻¹. -1 Spermine sample.

[0035] (2) 20 mg·L -1 Preparation of spermine sample: Dissolve spermine in ultrapure water to prepare a solution of 1000 mg·L⁻¹-1 To determine the concentration, take 10 μL and add it to a 30 μL container containing a concentration of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer. Finally, 50 μL of 5 mM 4-aminoantipyrine was added to obtain a final concentration of 20 mg·L⁻¹. -1 Spermine sample.

[0036] (3) 50 mg·L -1 Preparation of spermine sample: Dissolve spermine in ultrapure water to prepare a solution of 2500 mg·L⁻¹ -1 To determine the concentration, take 10 μL and add it to a 30 μL container containing a concentration of 1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, followed by 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine to obtain a final concentration of 50 mg·L⁻¹. -1 Spermine sample.

[0037] (4) 200 mg·L -1 Preparation of spermine sample: Dissolve spermine in ultrapure water to prepare a solution of 4000 mg·L⁻¹ -1 To determine the concentration, take 10 μL and add it to a 30 μL container containing a concentration of 1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, followed by 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine to obtain a final concentration of 200 mg·L⁻¹. -1 Spermine sample.

[0038] (5) Absorbance determination: Take the blank control solution prepared in Example 2 and the 10 mg·L⁻¹ solution prepared in the above steps. -1 Spermine sample, 20 mg·L -1 Spermine sample, 50 mg·L -1 Spermine sample and 200 mg·L -1 Spermine samples were subjected to absorption spectroscopy scans, and their absorbance values ​​at a wavelength of 506 nm were measured. The results are shown below. Figure 5 .

[0039] Example 6: Anti-interference test To verify the specificity of this colorimetric sensing method for the target biogenic amines, eight common food biogenic amines (histamine, spermine, spermidine, tryptamine, putrescine, cadaverine, phenylethylamine, and tyramine) were used as interfering agents. Each of these biogenic amines was added to the Pro-Ag-catalyzed 2,4-DP sensing solution, with the final concentration controlled at 10 mg·L⁻¹. -1 The experimental method for examining the changes in the response signal of the sensing system is as follows: Preparation of test samples: Histamine, spermine, spermidine, tryptamine, putrescine, cadaverine, phenylethylamine, and tyramine were dissolved in ultrapure water to obtain a concentration of 500 mg·L⁻¹ for each sample. -1 The solutions were each added to 30 μL containers containing a concentration of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer, and finally 50 μL of 5 mM 4-aminoantipyrine solution was added to obtain the test samples of histamine, spermine, spermidine, tryptamine, putrescine, cadaverine, phenethylamine, and tyramine.

[0040] Detection of the test samples: Take the prepared histamine, spermine, spermidine, tryptamine, putrescine, cadaverine, phenylethylamine and tyramine test samples respectively and perform absorption spectral scanning to measure their absorbance value at a wavelength of 506 nm. Calculate the absorbance difference between the test sample and the blank control solution. Substitute the absorbance difference into the linear regression equation to obtain the content of histamine and spermine in the test sample.

[0041] See results Figure 6 This demonstrates that the detection method of the present invention can specifically detect histamine and spermine, while other competing targets have almost no interference with the detection of histamine and spermine.

Claims

1. A colorimetric detection method for histamine and spermine based on Pro-Ag-mimicked laccase activity, characterized in that: First, standard curves for histamine and spermine absorbance are plotted separately. Then, linear regression equations are constructed based on these standard curves to determine the relationship between the concentrations of histamine and spermine and their absorbance values. The concentrations of histamine and spermine in the test samples are then determined using these linear regression equations. The specific steps are as follows: (1) Preparation of Pro-Ag nanozyme solution: 9-11 mL of 5 mM silver nitrate aqueous solution was slowly added to 9-11 mL of 10 mM proline alkaline solution. The reaction was carried out at 22-28°C for 11-13 hours, centrifuged for 8-12 min, the precipitate was collected, washed 4 times with ultrapure water, and finally dried at 55-65°C to obtain monodisperse nanozyme. Ultrapure water was added to prepare a solution of 0.9-1.1 mg·mL -1 Pro-Ag nanozyme solution, for later use; (2) Preparation of blank control solution: Take one centrifuge tube and add 1 mg·mL -1 Add 28-32 μL of Pro-Ag nanozyme, then add 8-12 μL of ultrapure water, mix well, and incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution with a concentration of 28-32 mM, then add 390-410 μL of HEPES buffer with a concentration of 8-12 mM, and finally add 48-52 μL of 4-aminoantipyrine solution with a concentration of 4.5-5.5 mM to obtain the blank control solution. (3) Preparation of the histamine standard solution to be tested: Take 8 portions of histamine reference standard and add ultrapure water to prepare solutions of 23-27 mg·L⁻¹. -1 Histamine standard solution, 48-52 mg / L -1 Histamine standard solution, 73-77 mg / L -1 Histamine standard solution, 98-102 mg / L -1 Histamine standard solution, 198-202 mg / L -1 Histamine standard solution, 298-302 mg·L -1 Histamine standard solution, 398-402 mg·L -1 Histamine standard solution and 498-502 mg·L -1 Take 8-12 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 25-35 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 0.9-1.1 mg·mL. -1 After mixing, incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM) to each centrifuge tube, followed by 390-410 μL of HEPES buffer (9-11 mM) to each centrifuge tube. Finally, add 48-52 μL of 4-aminoantipyrine solution (4.5-5.5 mM) to each centrifuge tube to obtain 8 groups of histamine standard solutions of different concentrations. (4) Preparation of spermine standard solution: Take 6 portions of spermine reference standard and add ultrapure water to prepare solutions of 98-102 mg / L. -1 Spermine standard solution, 248-252 mg / L -1 Spermine standard solution, 498-502 mg / L -1 Spermine standard solution, 748-752 mg / L -1 Spermine standard solution, 998-1002 mg·L -1 Spermine standard solution and 1248-1252 mg·L -1 Take 8-12 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 28-32 μL of Pro-Ag nanozyme solution (concentration of 0.9-1.1 mg·mL⁻¹). -1 After mixing, incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM) to each centrifuge tube, followed by 390-410 μL of HEPES buffer (10 mM) to each centrifuge tube. Finally, add 48-52 μL of 4-aminoantipyrine solution (4.5-5.5 mM) to each centrifuge tube to obtain 6 sets of spermine standard solutions with different concentrations. (5) Construction of histamine absorbance standard curve: Take 200 μL of the 8 groups of histamine standard solutions of different concentrations prepared in step (3) and the blank control solution prepared in step (6) respectively and place them in a 96-well microplate. Use a microplate reader to scan and measure the absorbance value at 506 nm. Based on the absorbance value, use the difference in absorbance value between the 8 groups of histamine standard solutions of different concentrations and the blank control solution as the ordinate and the histamine concentration as the abscissa to draw a standard curve of target concentration versus absorbance change, which is the histamine absorbance standard curve. (6) Construction of spermine absorbance standard curve: Take 200 μL of the six groups of spermine standard solutions of different concentrations prepared in step (5) and the blank control solution prepared in step (6) into a 96-well microplate, and use a microplate reader to scan and measure the absorbance value at 506 nm. Based on the absorbance value, use the difference in absorbance value between the six groups of spermine standard solutions of different concentrations and the blank control solution as the ordinate and the spermine concentration as the abscissa to draw a standard curve of target concentration versus absorbance change, which is the spermine absorbance standard curve. (7) Constructing a linear regression equation: Based on the standard curves of histamine absorbance and spermine absorbance, establish a regression equation for the relationship between the concentrations of histamine and spermine and the changes in absorbance.

2. (8) Preparation of the test sample: After ultrasonic degassing of beer, dilute it with double-distilled water to prepare the test solution. Take 8-12 μL of the diluted test solution and add it to a container containing 28-32 μL of a solution with a concentration of 0.9-1.1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 8-12 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM), followed by 390-410 μL of HEPES buffer (8-12 mM), and finally 48-52 μL of 4-aminoantipyrine solution (4.5-5.5 mM) to obtain the sample to be tested. (9) Detection of histamine and spermine content in the sample to be tested: Take the sample to be tested and perform absorption spectral scanning to measure its absorbance value at a wavelength of 506 nm. Calculate the absorbance difference between the sample to be tested and the blank control solution. Substitute the absorbance difference into the linear regression equation to obtain the content of histamine and spermine in the sample to be tested.

3. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 1, characterized in that: In step (1), the Pro-Ag nanozyme solution was prepared as follows: 10 mL of 5 mM silver nitrate aqueous solution was slowly added to 10 mL of 10 mM proline alkaline solution, and the reaction was carried out at 25°C for 12 hours. The solution was then centrifuged at 10000 rpm for 10 min, the precipitate was collected, washed four times with ultrapure water, and finally dried at 60°C to obtain monodisperse nanozyme. Ultrapure water was added to prepare a solution of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution is ready for use.

4. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 1, characterized in that: In step (2), the blank control solution is prepared by taking one centrifuge tube and adding 1 mg·mL⁻¹. -1 Add 29-31 μL of Pro-Ag nanozyme, then add 9-11 μL of ultrapure water, mix well, and incubate at room temperature for 9-11 min. Then add 9-11 μL of 2,4-dichlorophenol solution with a concentration of 29-31 mM, then add 395-405 μL of HEPES buffer with a concentration of 9-11 mM, and finally add 49-51 μL of 4-aminoantipyrine solution with a concentration of 5 mM to obtain the blank control solution.

5. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 3, characterized in that: In step (2), the blank control solution is prepared by taking one centrifuge tube and adding 1 mg·mL⁻¹. -1 Add 30 μL of Pro-Ag nanozyme and 10 μL of ultrapure water, mix well, and incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution, 400 μL of 10 mM HEPES buffer, and finally add 50 μL of 5 mM 4-aminoantipyrine solution to obtain the blank control solution.

6. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 1, characterized in that: In step (3), the preparation of the histamine standard solution to be tested is as follows: Take 8 portions of histamine reference standard and add ultrapure water to prepare solutions of 24-26 mg·L⁻¹. -1 Histamine standard solution, 49-51 mg / L -1 Histamine standard solution, 74-76 mg / L -1 Histamine standard solution, 99-101 mg / L -1 Histamine standard solution, 199-201 mg / L -1 Histamine standard solution, 299-301 mg / L -1 Histamine standard solution, 399-401 mg / L -1 Histamine standard solution and 499-501 mg·L -1 Take 9-11 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 28-32 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 0.95-1.05 mg·mL. -1 After mixing, incubate at room temperature for 9-11 min. Then, add 9-11 μL of 2,4-dichlorophenol solution (29-31 mM) to each centrifuge tube, followed by 395-405 μL of HEPES buffer (9.5-10.5 mM) to each centrifuge tube. Finally, add 49-51 μL of 4-aminoantipyrine solution (5 mM) to each centrifuge tube to obtain 8 sets of histamine standard solutions of different concentrations.

7. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 5, characterized in that: In step (3), the preparation of the histamine standard solution to be tested is as follows: Take 8 portions of histamine reference standard and add ultrapure water to prepare 25 mg·L⁻¹ solutions. -1 Histamine standard solution, 50 mg / L -1 Histamine standard solution, 75 mg / L -1 Histamine standard solution, 100 mg / L -1 Histamine standard solution, 200 mg / L -1 Histamine standard solution, 300 mg / L -1 Histamine standard solution, 400 mg / L -1 Histamine standard solution and 500 mg·L -1 Take 10 μL of each of the eight different concentrations of histamine standard solution and add them separately to eight centrifuge tubes containing 30 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 1 mg·mL⁻¹. -1 After mixing, incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution to each centrifuge tube, followed by 400 μL of 10 mM HEPES buffer to each centrifuge tube. Finally, add 50 μL of 5 mM 4-aminoantipyrine solution to each centrifuge tube to obtain 8 groups of histamine standard solutions of different concentrations.

8. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 1, characterized in that: In step (4), the preparation of the spermine standard solution to be tested is as follows: Take 6 portions of spermine reference standard and add ultrapure water to prepare solutions of 99-101 mg·L⁻¹. -1 Spermine standard solution, 249-251 mg / L -1 Spermine standard solution, 499-501 mg / L -1 Spermine standard solution, 749-751 mg / L -1 Spermine standard solution, 999-1001 mg·L -1 Spermine standard solution and 1249-1251 mg·L -1 Take 9-11 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 29-31 μL of Pro-Ag nanozyme solution (concentration of 0.95-1.05 mg·mL). -1 After mixing, incubate at room temperature for 9-11 min. Then add 9-11 μL of 2,4-dichlorophenol solution (29-31 mM) to each centrifuge tube, followed by 395-405 μL of HEPES buffer (10 mM) to each centrifuge tube. Finally, add 49-51 μL of 4-aminoantipyrine solution (5 mM) to each centrifuge tube to obtain 6 sets of spermine standard solutions with different concentrations.

9. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 7, characterized in that: In step (4), the preparation of the spermine standard solution to be tested is as follows: Take 6 portions of spermine reference standard and add ultrapure water to prepare 100 mg·L⁻¹ solutions. -1 Spermine standard solution, 250 mg / L -1 Spermine standard solution, 500 mg / L -1 Spermine standard solution, 750 mg·L -1 Spermine standard solution, 1000 mg·L -1 Spermine standard solution and 1250 mg·L -1 Take 10 μL of each of the six different concentrations of spermine standard solution and add them separately to six centrifuge tubes containing 30 μL of Pro-Ag nanozyme solution. The concentration of the Pro-Ag nanozyme solution is 1 mg·mL⁻¹. -1 After mixing, incubate at room temperature for 10 min. Then add 10 μL of 30 mM 2,4-dichlorophenol solution to each centrifuge tube, followed by 400 μL of 10 mM HEPES buffer to each centrifuge tube. Finally, add 50 μL of 5 mM 4-aminoantipyrine solution to each centrifuge tube to obtain 6 sets of spermine standard solutions of different concentrations.

10. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 1, characterized in that: In step (8), the sample to be tested is prepared as follows: Take 4-6 mL of beer, degas it by sonication for 15-25 min, dilute it with 4-6 mL of double-distilled water, take 10 μL of the diluted test solution, and add 28-32 μL of a solution with a concentration of 0.9-1.1 mg·mL⁻¹. -1 Add the Pro-Ag nanozyme solution to a centrifuge tube, mix well, and incubate at room temperature for 9-11 min. Then add 8-12 μL of 2,4-dichlorophenol solution (28-32 mM), followed by 395-405 μL of HEPES buffer (9-11 mM), and finally 49-51 μL of 4-aminoantipyrine solution (5 mM) to obtain the sample to be tested.

11. The colorimetric detection method for histamine and spermine based on Pro-Ag simulated laccase activity according to claim 9, characterized in that: In step (8), the sample to be tested is prepared as follows: 5 mL of beer is taken, degassed by sonication for 20 min, and then diluted with 5 mL of double-distilled water. 10 μL of the diluted sample is taken and added to a 30 μL container containing a solution of 1 mg·mL⁻¹. -1 The Pro-Ag nanozyme solution was added to a centrifuge tube, mixed well, and incubated at room temperature for 10 min. Then, 10 μL of 30 mM 2,4-dichlorophenol solution was added, followed by 400 μL of 10 mM HEPES buffer, and finally 50 μL of 5 mM 4-aminoantipyrine solution to obtain the sample to be tested.

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