Spermine colorimetric rapid detection method based on mimicking laccase activity of flower-like mn3o4 nanoparticles

Abstract

The application discloses a spermine colorimetric rapid detection method based on flower-like Mn3O4 nanoparticles simulating laccase activity, prepares a known target SPM concentration sample A1 to be measured; prepares a blank control sample A2; scans A1 and A2 respectively by using an enzyme marker, and measures the absorbance values; draws a standard curve of target SPM concentration and absorbance value change quantity according to the absorbance value difference between A2 and A1; establishes a regression equation of the mutual relationship between target SPM concentration and absorbance value according to the standard curve; prepares an unknown target SPM concentration sample C to be measured; takes the absorbance value of C, calculates the absorbance difference value between A2 and C, and substitutes the difference value into the linear regression equation, so that the concentration of target SPM in C can be obtained. The application applies the simulated laccase activity of flower-like Mn3O4 nanoparticles to SPM detection for the first time. In HEPES buffer solution, SPM can inhibit the laccase activity of flower-like Mn3O4 nanoparticles, so that the reaction solution appears light pink. The application can be widely applied to on-site rapid detection.

Description

Technical Field

[0001] This invention relates to a rapid colorimetric detection method for spermine, particularly a rapid colorimetric detection method for spermine based on flower-shaped Mn3O4 nanoparticles simulating laccase activity. Background Technology

[0002] Spermine (SPM) is a chemical substance that has been found to react with nitrosamines, commonly found in meat products and smoked foods, to produce carcinogens such as nitrosamines. Furthermore, spermine has been found to inhibit the activity of histamine and tyramine-related metabolic enzymes. Histamine and tyramine are present in some foods, such as fermented foods, dairy products, and certain vegetables. Under normal circumstances, the human body breaks them down through metabolic enzymes to prevent their accumulation. However, when spermine interferes with the activity of these metabolic enzymes, histamine and tyramine may accumulate in the body, leading to poisoning. Therefore, there is an urgent need to develop rapid methods for detecting SPM in food to prevent the consumption of foods with high SPM content. Currently, rapid detection methods for SPM mainly utilize noble metal nanoparticles (AuNPs) and AgNPs as colorimetric probes. However, the stability of noble metal nanoparticles is easily affected by high-salt particles and cationic polymers, thus affecting the accuracy of the detection results. In addition, some colorimetric methods use toxic dyes as sensing materials, which can easily threaten environmental quality and the safety of laboratory personnel. Since there are still challenges in the current rapid colorimetric detection methods for SPM, there is an urgent need to find a new sensing signal material to construct a rapid colorimetric detection method for SPM. Summary of the Invention

[0003] The purpose of this invention is to provide a rapid colorimetric detection method for spermine (SPM) based on flower-shaped Mn3O4 nanoparticles simulating laccase activity. This method offers advantages such as visual identification, ease of operation, rapid detection, low cost, good selectivity, and safety, and can be widely applied to the rapid detection of SPM in actual food products.

[0004] The technical solution of this invention: a rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity, comprising the following steps:

[0005] A: Plot a standard curve showing the change in spermine concentration versus absorbance;

[0006] B: Establish a linear regression equation for the relationship between spermine concentration and absorbance value based on the standard curve of spermine concentration and absorbance change;

[0007] C: Prepare a test solution with an unknown concentration of spermine to obtain product C;

[0008] D: Take product C and perform an absorption spectrum scan to measure its absorbance value at a wavelength of 510 nm. Calculate the absorbance difference between product C and the blank control solution A2. Substitute this absorbance difference into the linear regression equation to obtain the concentration of spermine in product C.

[0009] In the aforementioned rapid colorimetric detection method for spermine based on flower-shaped Mn3O4 nanoparticles simulating laccase activity, the specific method for plotting the standard curve of spermine concentration versus absorbance value change in step A is as follows:

[0010] A1: Take multiple graduated centrifuge tubes, add a certain concentration of Mn3O4 nanoparticles and a known concentration of spermine standard solution to each graduated centrifuge tube, mix well and incubate, then add substrate 2,4-DP and HEPES buffer, and finally add 4-AAP to prepare the test group, which is A1 product;

[0011] A2: Take one graduated centrifuge tube and prepare product A2 by replacing the known concentration of spermine standard solution with ultrapure water according to the method in A1.

[0012] A3: Take A1 and A2 products and measure their absorbance at 510 nm wavelength using an ELISA reader;

[0013] A4: Based on the absorbance values, plot the absorbance difference between A2 and different concentrations of A1 as the ordinate and the spermine concentration as the abscissa to obtain a standard curve of target concentration versus absorbance change.

[0014] In the aforementioned rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity, in step B, when the spermine concentration C is 0.3 μg·mL -1 ≤C≤2μg·mL -1 When the concentration and the change in absorbance value are, the regression equation is: y = 0.17916C - 0.10049; y is the absorbance difference (ΔA) = absorbance value of A2 sample - absorbance value of the sample containing target spermine.

[0015] In the aforementioned rapid colorimetric detection method for spermine based on flower-shaped Mn3O4 nanoparticles simulating laccase activity, the specific method for preparing the test solution C with an unknown target spermine concentration in step C is as follows: replace the target spermine standard solution of known concentration with the actual sample solution, and the test solution with an unknown target spermine concentration, namely C, can be prepared according to the method in A1.

[0016] In the aforementioned rapid colorimetric detection method for spermine based on laccase-mimicking activity using flower-shaped Mn3O4 nanoparticles, the concentration of the flower-shaped Mn3O4 nanoparticle solution is 1.25 mg·mL. -1 The concentration of the 2,4-DP solution was 25 mg / mL. -1The HEPES buffer was 15 mM, pH 7.5; the concentration of the 4-AAP solution was 1 mg / mL. -1 .

[0017] In the aforementioned rapid colorimetric detection method for spermine based on flower-shaped Mn3O4 nanoparticles simulating laccase activity, the preparation process of the A1, A2, or C product is as follows: add Mn3O4 solution and SPM to a centrifuge tube and mix thoroughly, incubate at 30°C for 10 min for later use, then add 2,4-DP solution, and then add HEPES buffer and 4-AAP mixture in sequence.

[0018] In the preparation of the A1, A2 or C products: the amount of flower-shaped Mn3O4 nanoparticles, spermine standard solution or actual sample solution, and 2,4-DP solution added is 10 μL; the amount of 4-AAP solution used is 30 μL; and the amount of HEPES buffer solution used is 440 μL.

[0019] In the aforementioned rapid detection method for spermine based on the simulated laccase activity of flower-shaped Mn3O4 nanoparticles, the preparation method of the flower-shaped Mn3O4 nanoparticle solution is as follows: 1.0 g of 6.3 mM KMnO4 is dissolved in 500 mL of ultrapure water and stirred vigorously for 30 min. Then, 10 mL of oleic acid is added to the solution and stirred continuously at 28 °C for 5 h. The resulting brown-black precipitate is washed with ultrapure water, and then the residual starting material in the precipitate is removed with ethanol. The treated precipitate is dried at 80 °C for 10 h to obtain particulate matter, which is then calcined in air at 200 °C for 5 h to obtain monodisperse nanoparticles.

[0020] The beneficial effects of this invention are as follows: Compared with the prior art, the detection principle of this method is as follows: This method utilizes flower-shaped Mn3O4 nanoparticles with laccase-mimicking activity as a colorimetric probe, and for the first time discovered that SPM can inhibit the laccase activity of the nanoparticles. Therefore, a rapid colorimetric detection method based on SPM simulating laccase activity using flower-shaped Mn3O4 nanoparticles has been developed. Flower-shaped Mn3O4 nanoparticles can catalyze the generation of superoxide anions (·O) from dissolved oxygen. 2- This process oxidizes the phenolic substrate 2,4-DP to produce a colorless quinone compound, which then reacts with 4-AAP to generate a red product. When SPM is added, it adsorbs onto the surface of the nanoparticles via electrostatic forces, occupying the active sites of the material. This reduces electron transfer between the flower-like Mn3O4 and 2,4-DP, thereby hindering the generation of dissolved oxygen (·O) in the nanoparticle catalytic reaction system. 2-This process inhibits the laccase activity of nanoparticles, resulting in a light pink color in the reaction solution. Furthermore, the degree of color change in the constructed colorimetric sensor is positively correlated with the SPM concentration, thus this method can be used for rapid detection of SPM in real samples. The developed colorimetric detection method has an average recovery rate within the range of 88.6-95.3%, with a relative standard deviation (RSD) ranging from 4.14-5.4%, and its detection performance is similar to that of high-performance liquid chromatography (HPLC). This method can specifically detect spermine despite interference from spermidine (SPD), histamine (HIS), cadaverine (CAD), putrescine (PUT), tryptamine (TRY), tyramine (TYR), arginine (ARG), proline (PRO), and lysine (LYS). The proposed rapid detection method has high sensitivity, strong specificity, and is simple, rapid, and visually perceptible, making it widely applicable for the rapid detection of SPM in real food samples. Attached Figure Description

[0021] Appendix Figure 1 A schematic diagram illustrating the feasibility of spermine detection;

[0022] Appendix Figure 2 A schematic diagram illustrating the principle of spermine detection;

[0023] Appendix Figure 3 The relationship between different concentrations of spermine and the absorbance difference (ΔA / A0);

[0024] Appendix Figure 4 A schematic diagram illustrating the effects of other interfering substances on spermine detection;

[0025] Appendix Figure 1 and attached Figure 2 The principle speculation and feasibility study of this invention show that the method of this invention can be used for spermine detection.

[0026] Appendix Figure 1 middle:

[0027] Sample 1: Flower-like Mn3O4 nanoparticles + 2,4-DP + 4-AAP;

[0028] Sample 2: Flower-like Mn3O4 nanoparticles + 2,4-DP + 4-AAP + 0.2 μg·mL -1 SPM;

[0029] Sample 3: Flower-like Mn3O4 nanoparticles + 2,4-DP + 4-AAP + 2 μg·mL -1 SPM;

[0030] Sample 4: Flower-like Mn3O4 nanoparticles + 2,4-DP + 4-AAP + 20 μg·mL -1 SPM;

[0031] Sample 5: Flower-like Mn3O4 nanoparticles + 4-AAP.

[0032] Appendix Figure 3 The linear range for detecting SPM demonstrated by this invention is 0.3-2 μg·mL. -1 It has a detection limit of 0.094 μg·mL. -1 This indicates that the invention has the characteristics of good stability and high sensitivity.

[0033] Appendix Figure 4 As can be seen, the method established in this invention can specifically detect SPM, while other competing targets have almost no interference with the detection of SPM. Detailed Implementation

[0034] 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.

[0035] Embodiments of the Invention: The inventors of this application have discovered for the first time that spermine (SPM) can inhibit the laccase-mimicking activity of flower-shaped Mn3O4 nanoparticles, preventing the reaction solution from turning red. Laccase is a green catalyst with a different catalytic mechanism than peroxidase, catalase, etc. Laccase catalyzes the oxidation of the substrate 2,4-DP, and its oxidation product combines with 4-AAP to produce a red product. This catalytic mechanism can avoid the influence of oxidase or peroxidase-active substances present in the sample on the detection results in actual detection. At the same time, the catalytic process of laccase does not require the participation of H2O2, avoiding problems such as self-decomposition and high toxicity in the catalytic reaction. Based on this, the inventors of this application have developed a rapid colorimetric detection method using SPM based on the laccase-mimicking activity of flower-shaped Mn3O4 nanoparticles.

[0036] The SPM colorimetric rapid detection method based on flower-like Mn3O4 nanoparticles mimicking laccase activity includes the following steps:

[0037] A: Plot a standard curve of target SPM concentration versus absorbance value change, as shown in the attached figure. Figure 3 As shown, it includes the following steps:

[0038] A1: Prepare the test solution with a known target SPM concentration to obtain product A1. Specifically, take 14 1.5 mL centrifuge tubes and add 10 μL of a solution with a concentration of 1.25 mg / mL to each graduated centrifuge tube. -1 The use of flower-shaped Mn3O4 solutions and SPM standard solutions of varying known concentrations maintained the SPM concentration in the entire detection system between 0.18 and 30 μg / mL. -1 After thoroughly mixing, incubate at 30°C for 10 min, then add 10 μL of a 25 mg / mL solution.-1 Add the substrate 2,4-DP solution, then add 440 μL of 15 mM HEPES buffer (pH 7.5), and finally add 30 μL of 1 mg / mL HEPES buffer. -1 The test group can be prepared by using a 4-AAP solution, and the final total volume is 500 μL, which is A1 grade.

[0039] A2: Take another 1.5mL centrifuge tube, replace the known concentration of SPM standard solution with 10μL of ultrapure water, and prepare the blank control group according to the method of A1, which is product A2;

[0040] A3: Take 200 μL of the standard solution and blank control solution prepared in steps A1 and A2 respectively and place them in a 96-well microplate. Use a microplate reader to scan and measure the absorbance value at 510 nm.

[0041] A4: Based on the absorbance values, plot the difference in absorbance values ​​between product A2 and product A1 at different concentrations on the ordinate and the spermine concentration on the abscissa to obtain a standard curve of target concentration versus absorbance change.

[0042] B: Based on the standard curve of target SPM concentration and absorbance change, establish a regression equation for the relationship between target SPM concentration and absorbance change.

[0043] When the spermine concentration is 0.3 μg / mL -1 ≤C≤2μg mL -1 When the concentration and the change in absorbance value are, the regression equation is: y = 0.17916C - 0.10049; y is the absorbance difference (ΔA) = absorbance value of A2 sample - absorbance value of the sample containing target spermine.

[0044] C: Prepare a test solution with unknown target SPM concentration to obtain product C;

[0045] D: Take product C and perform an absorption spectrum scan to measure its absorbance value at a wavelength of 510 nm. Calculate the absorbance difference between product A2 and product C. Substitute this absorbance difference into the linear regression equation in step B to obtain the concentration of the target SPM in product C.

[0046] The actual sample liquids were tap water, pork, and corn.

[0047] The preparation method of the flower-like Mn3O4 nanoparticle solution is as follows: 1.0 g of 6.3 mM KMnO4 is dissolved in 500 mL of ultrapure water and stirred vigorously for 30 min. Then, 10 mL of oleic acid is added to the solution and stirred continuously at 28 °C for 5 h. The resulting brown-black precipitate is washed with ultrapure water, and then the residual starting material in the precipitate is removed with ethanol. The treated precipitate is dried at 80 °C for 10 h to obtain particulate material. Then, it is calcined in air at 200 °C for 5 h to obtain monodisperse nanoparticles.

[0048] The basic principle of this invention is as follows: Flower-shaped Mn3O4 nanoparticles 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 510 nm. Upon addition of the target SPM, the electron transfer between the flower-shaped Mn3O4 nanoparticles and the substrate 2,4-DP is blocked by the SPM. The laccase-like activity of the nanoparticles is inhibited, preventing further oxidation of the substrate to produce the colorless quinone compound. Consequently, the nanoparticles cannot bind to 4-AAP to generate a colored product. Furthermore, the change in absorbance at 510 nm is directly proportional to the concentration of the target SPM. Therefore, this method can be used for SPM detection.

[0049] The detection method provided by this invention does not rely on large instruments and equipment, has high detection sensitivity, is simple and fast to operate, and is visible to the naked eye. It can be widely used for the rapid detection of SPM in actual biological or food samples.

Claims

1. A rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity, characterized in that: Includes the following steps: A: Plot a standard curve showing the change in spermine concentration versus absorbance; B: Establish a linear regression equation for the relationship between spermine concentration and absorbance value based on the standard curve of spermine concentration and absorbance change; C: Prepare a test solution with an unknown concentration of spermine to obtain product C; D: Take product C and perform an absorption spectrum scan to measure its absorbance value at a wavelength of 510 nm. Calculate the absorbance difference between product C and blank control solution A2. Substitute this absorbance difference into the linear regression equation to obtain the concentration of spermine in product C. In step A, the specific method for plotting the standard curve of spermine concentration versus absorbance value change is as follows: A1: Take multiple graduated centrifuge tubes, add a certain concentration of Mn3O4 nanoparticles and a known concentration of spermine standard solution to each graduated centrifuge tube, mix well and incubate, then add substrate 2,4-DP and HEPES buffer, and finally add 4-AAP to prepare the test group, which is A1 product; A2: Take one graduated centrifuge tube and prepare product A2 by replacing the known concentration of spermine standard solution with ultrapure water according to the method in A1. A3: Take A1 and A2 products and measure their absorbance at 510 nm wavelength using an ELISA reader; A4: Based on the absorbance values, plot the absorbance difference between A2 and different concentrations of A1 as the ordinate and the spermine concentration as the abscissa to obtain a standard curve of target concentration versus absorbance change.

2. The rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity according to claim 1, characterized in that: In step B, when the spermine concentration C is 0.3 μg·mL -1 ≤C≤2 μg·mL -1 When the concentration and the change in absorbance value are, the regression equation is: y = 0.17916C - 0.10049; y is the absorbance difference (ΔA) = absorbance value of A2 sample - absorbance value of the test sample containing target spermine.

3. The rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity according to claim 1, characterized in that: In step C, the specific method for preparing the test solution C with an unknown target spermine concentration is as follows: replace the target spermine standard solution with the actual sample solution, and prepare the test solution with an unknown target spermine concentration, which is product C, according to method A1.

4. The rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity according to claim 1, characterized in that: The concentration of the flower-like Mn3O4 nanoparticle solution was 1.25 mg·mL. -1 The concentration of the 2,4-DP solution was 25 mg / mL. -1 The HEPES buffer was 15 mM, pH 7.5; the concentration of the 4-AAP solution was 1 mg / mL. -1 .

5. The rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity according to claim 1, characterized in that: The preparation process of the A1, A2 or C products is as follows: add Mn3O4 solution and SPM to a centrifuge tube and mix thoroughly. Incubate at 30 °C for 10 min for later use. Then add 2,4-DP solution, followed by HEPES buffer and 4-AAP mixture in sequence. In the preparation of the A1, A2 or C products: the amount of flower-shaped Mn3O4 nanoparticles, spermine standard solution or actual sample solution, and 2,4-DP solution added is 10 μL; the amount of 4-AAP solution used is 30 μL; and the amount of HEPES buffer solution used is 440 μL.

6. The rapid colorimetric detection method for spermine based on flower-like Mn3O4 nanoparticles simulating laccase activity according to claim 1, characterized in that: The preparation method of the flower-like Mn3O4 nanoparticle solution is as follows: 1.0 g of 6.3 mM KMnO4 is dissolved in 500 mL of ultrapure water and stirred vigorously for 30 min. Then, 10 mL of oleic acid is added to the solution and stirred continuously at 28 °C for 5 h. The resulting brown-black precipitate is washed with ultrapure water, and then the residual starting material in the precipitate is removed with ethanol. The treated precipitate is dried at 80 °C for 10 h to obtain particulate material. Then, it is calcined in air at 200 °C for 5 h to obtain monodisperse nanoparticles.

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