A rapid detection method for latent malachite green based on nanozyme oxidation and a method for preparing the nanozyme.

Abstract

This invention discloses a rapid detection method for leucomall green based on nanozyme oxidation and a method for preparing nanozymes. The specific steps are as follows: (1) Solution preparation: Prepare an acetate-sodium acetate buffer solution, a leucomall green solution, and a nanozyme stock solution for rapid detection of leucomall green; (2) Add the nanozyme stock solution to the acetate-sodium acetate buffer solution and shake well, then add the leucomall green solution, and measure the absorbance of the solution before and after adding leucomall green at 618 nm; (3) Adjust the final concentration of the malachite green solution, the leucomall green solution, and the mixed solution after nanozyme catalysis of leucomall green to 10 μM, and determine the sample under liquid chromatography-tandem mass spectrometry conditions to obtain an ion chromatogram, confirming that the product of nanozyme catalysis of leucomall green is malachite green. The nanozyme catalysis of leucomall green using the rapid oxidation of leucomall green of this invention is simple to operate, rapid and sensitive, low in cost, and highly efficient.

Description

Technical Field

[0001] This invention relates to the fields of rapid food safety detection and nanomaterial applications, specifically to a PSS / ZIF-67 nanozyme for rapid detection of leuco malachite green and its detection method. Background Technology

[0002] Malachite green (MG) is widely used in aquaculture as a cost-effective treatment for fungal and parasitic infections. However, malachite green has carcinogenic, teratogenic, and mutagenic effects, and many countries have banned its use in aquaculture. Establishing detection methods for malachite green in aquatic products is of great significance for ensuring the safety of aquatic food products.

[0003] Malachite green is rapidly absorbed and metabolized by tissues after entering the body of aquatic animals, with approximately 90% existing in muscle tissue as leucomall green. Leucomall green is lipophilic, more toxic than regular malachite green, and remains in tissues for a longer period. The content of leucomall green is a better indicator of malachite green residues in aquatic products. Leucomall green is a colorless and non-absorbent substance, making its detection extremely difficult. The state has established national standard methods for malachite green and leucomall green, GB / T19857-2005 and GB / T20361-2006, for high-performance liquid chromatography-derivatization and high-performance liquid chromatography-mass spectrometry, respectively, to monitor and regulate the market. These methods not only require large instruments and cumbersome sample pretreatment, resulting in high detection costs and long detection cycles, but also necessitate the oxidation of leucomall green to malachite green using a PbO2 or iodine column for detection. This method is highly unsuitable for on-site monitoring and supervision of large batches of market samples. Currently, the main method for detecting malachite green is the national standard method.

[0004] Nanozymes possess characteristics such as high catalytic efficiency and environmental friendliness. This study synthesized a PSS / ZIF-67 nanozyme, an oxidase-like enzyme that selectively oxidizes cryptic malachite green. This enzyme not only instantly oxidizes cryptic malachite green to malachite green but also prevents further oxidation of the generated malachite green. Therefore, by monitoring the absorption of the oxidized malachite green, rapid, simple, sensitive, and selective detection of cryptic malachite green can be achieved, providing a practical strategy and method for the immediate detection of cryptic malachite green. Summary of the Invention

[0005] Compared to reported methods, this patent proposes for the first time an analytical detection strategy for latent malachite green based on nanozyme oxidation; a novel method for detecting latent malachite green is constructed based on this strategy. This method is highly selective, requires no cumbersome sample pretreatment, is simple and rapid, and is highly suitable for real-time monitoring of water quality in aquaculture as well as on-site monitoring and supervision of aquatic products in the market.

[0006] This invention provides a rapid detection method for leucomall green based on nanozyme oxidation and a method for preparing the nanozyme. The nanozyme for rapidly oxidizing leucomall green, namely the PSS / ZIF-67 nanozyme, exhibits a UV-Vis absorption peak at 618 nm when catalyzing the oxidation of leucomall green. Using this method to oxidize leucomall green enables real-time visual detection; the method is simple, rapid, sensitive, low-cost, and highly efficient.

[0007] The object of this invention is achieved in the following manner:

[0008] A method for preparing a nanozyme for rapid oxidation of cryptic malachite green.

[0009] The specific steps are as follows:

[0010] Step 1: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water, and stirred to obtain a mixed solution, which is called solution A;

[0011] Step 2: Dissolve 2-methylimidazole in a mixed solution of N,N-dimethylformamide and deionized water, and stir to obtain a mixed solution, which is called solution B.

[0012] Step 3: Quickly pour solution A from step 1 into solution B from step 2, stir and let stand. Centrifuge the resulting product, wash and dry it to obtain a solid product, which is the nanozyme that can rapidly oxidize leucomalle green, namely PSS / ZIF-67.

[0013] In the above-mentioned method for preparing nanozymes for rapid oxidation of malachite green, in step 1, the concentration of Co(NO3)2·6H2O in solution A is 0.029M-0.035M, the concentration of sodium polystyrene sulfonate is 0.90mg / mL-1.10mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1, and the mixture is stirred for 25-35 min; in step 2, the concentration of 2-methylimidazole in solution B is 0.09M-0.035M. The volume ratio of .17M, N,N-dimethylformamide and deionized water is 1:1, and the mixture is stirred for 25-35 min. In step 3, the ratio of A solution to B solution is 0.9-1.1:0.9-1.1. After stirring for 0.5-1.5 min, the mixture is allowed to stand for 20-28 h. Then, the mixture is centrifuged at 7500-8500 rpm / min for 3-7 min, and then dried in a vacuum drying oven at 55-65℃ for 3.5-4.5 h.

[0014] In the above-mentioned method for preparing nanozymes for rapid oxidation of cryptic malachite green, in step 1, the concentration of Co(NO3)2·6H2O is 0.033M, the concentration of sodium polystyrene sulfonate is 1mg / mL, and the mixture is stirred for 30min.

[0015] In the above-mentioned method for preparing nanozymes for rapid oxidation of cryptic malachite green, in step 2, the concentration of 2-methylimidazole in solution B is 0.13M, and the mixture is stirred for 30 minutes.

[0016] In the above-mentioned method for preparing nanozymes for rapid oxidation of malachite green, in step 3, the ratio of solution A to solution B is 1:1. After stirring for 1 min, the mixture is allowed to stand for 24 h. Then, the mixture is centrifuged at 8000 rpm / min for 5 min. The precipitate is washed with a mixed solution of N,N-dimethylformamide and deionized water, and then dried in a vacuum drying oven at 60 °C for 4 h.

[0017] A rapid detection method for latent malachite green based on nanozyme oxidation, comprising the following specific steps:

[0018] (1) Solution preparation: Prepare an acetate-sodium acetate buffer solution with a concentration of 90mM-105mM, a leucomalle green solution with a concentration of 9mM-12mM, and a nanoenzyme stock solution with a concentration of 0.9mg / mL-1.1mg / mL; wherein the pH of the acetate-sodium acetate buffer solution is 2.8-3.2, the solvent of the nanoenzyme stock solution is acetonitrile, and the solvent of the leucomalle green solution is deionized water;

[0019] (2) Add 99-110 μL of nanozyme stock solution with a concentration of 0.9-1.1 mg / mL to 890-901 μL of 90 mM-105 mM acetate-sodium acetate buffer solution and shake well to obtain a reaction system with a total volume of 1 ml. At this time, the reaction system has no absorption peak at 618 nm. Then add 0.9-1.1 μL of leucomall green solution with a concentration of 9-12 mM. At this time, the mixed solution has an absorption peak at 618 nm. Measure the absorbance of the solution at 618 nm before and after adding leucomall green.

[0020] (3) The final concentration of the malachite green solution, the leucomalle green solution and the mixed solution after oxidation of leucomalle green by PSS / ZIF-67 nanozyme were all adjusted to 10 μM. The samples were measured according to the liquid chromatography-tandem mass spectrometry conditions. During the measurement, the quantitative ion m / z of malachite green was monitored separately: m / z 329 / 313; the quantitative ion m / z of leucomalle green was monitored separately. The ion chromatogram was obtained, and the product of nanozyme catalytic oxidation of leucomalle green was determined to be malachite green.

[0021] The above-mentioned rapid detection method for cryptic malachite green based on nanozyme oxidation,

[0022] It also includes step (4), a rapid detection method for the content of leuco malachite green in actual samples:

[0023] (4.1) Plotting a standard working curve: Add a certain amount of nanozyme stock solution to different volumes of acetate-sodium acetate buffer solution, and then add different amounts of leucomalle green solution. Use a UV-Vis spectrophotometer to measure the absorbance of each solution at 618 nm, record the UV-Vis absorption spectrum, obtain the relationship between absorbance and the concentration of leucomalle green solution, and plot a standard working curve.

[0024] The acetate-sodium acetate buffer solution had a pH of 3 and a concentration of 0.1 M. The concentrations of the leucomalle green solution were 2, 3, 5, 7, 8, 10, 13, and 15 mM. The concentration of the nanozyme stock solution for rapid detection of leucomalle green was 100 mg / mL. The volumes of the acetate-sodium acetate buffer solution were 898, 897, 895, 893, 892, 890, 887, and 885 μL. The volume of the nanozyme stock solution was 100 μL. 2, 3, 5, 7, 8, 10, 13, and 15 μL of leucomalle green solution were added.

[0025] (4.2) Content of leucomall green in actual sample: The extract of actual sample for preparing leucomall green was added to a mixture of acetic acid-sodium acetate buffer solution and nanozyme solution. The absorbance at 618 nm was measured by UV-Vis spectrophotometer. The content of leucomall green in the product was calculated based on the standard working curve obtained in step (4.1).

[0026] The above-mentioned rapid detection method for leucomall green based on nanozyme oxidation, in step (1), the pH of the acetate-sodium acetate buffer solution is 3 and the concentration is 100mM. A leucomall green solution with a concentration of 10mM is prepared using acetonitrile as the solution, and a rapid detection stock solution with a concentration of 1mg / mL is prepared using deionized water as the solvent. In step (2), 101μL of a nanozyme stock solution with a concentration of 1mg / mL for rapid oxidation of leucomall green is added to 899μL of acetate-sodium acetate buffer solution with a concentration of 100nM. At this time, the mixed solution has no absorption peak at 618nm. Then, 1μL of a leucomall green solution with a concentration of 10mM is added. At this time, the mixed solution has an absorption peak at 618nm. The absorbance of the solution before and after adding leucomall green is measured at 618nm.

[0027] The above-mentioned rapid detection method for latent malachite green based on nanozyme oxidation is used in seafood products selected from any one of fish, shrimp, crab, clam, and oyster.

[0028] Compared with the prior art, the present invention has the following technical effects:

[0029] (1) This invention is the first to propose a method for rapid detection of leucomalle green based on nanozymes;

[0030] (2) This invention can achieve simple, fast, and visual detection of leucomalle green in 1 minute;

[0031] (3) The present invention has extremely high selectivity in detecting leucomalle green in actual aquatic products.

[0032] The nanoenzyme prepared by this invention oxidizes leucomall green, thereby realizing a method for the determination of leucomall green that is simple to operate, rapid, sensitive, low in cost, and highly efficient. Attached Figure Description

[0033] Figure 1 This diagram illustrates the synthesis steps of the nanozyme for the rapid oxidation of leuco malachite green according to the present invention.

[0034] Figure 2 TEM and XPS images of the nanozyme for the rapid oxidation of leuco malachite green in Example 2 of this invention;

[0035] Figure 3 The images show the UV-Vis absorption spectra of different solutions in Example 9 of this invention between 400-800 nm. The insets are photographs of the corresponding solutions under natural light. ① represents a 100 μg / mL nanozyme solution (PSS / ZIF-67 solution is a mixture of PSS / ZIF-67 stock solution and acetate-sodium acetate buffer solution); ② represents a mixture of leucomalleite green solution and acetate-sodium acetate buffer solution (leucomalleite green final concentration is 10 μM); ③ represents a mixture of PSS / ZIF-67 solution and leucomalleite green solution (PSS / ZIF-67 solution final concentration is 100 μg / mL, leucomalleite green final concentration is 10 μM).

[0036] Figure 4 The images show the ion chromatograms of leucomalle green and malachite green standard solutions from Example 9 of this invention, as well as the ion chromatogram of the leucomalle green solution after catalytic oxidation with PSS / ZIF-67.

[0037] Figure 5 Example 9 of the present invention shows the UV-Vis absorption spectra after adding 100 μL LPSS / ZIF-67 stock solution and different amounts of leucomalle green solution (final concentration of leucomalle green 2-15 μM, final volume 1 mL) to an acetate-sodium acetate buffer solution (100 mM, pH=3), and the standard working curve of absorbance at 618 nm for leucomalle green concentration in the range of 2-15 μM.

[0038] Figure 6 The bar chart shows the absorbance at 618 nm of the PSS / ZIF-67 solution in Example 9 of this invention after reacting with leucomalle green and an interfering substance with a concentration 10 times higher than that of leucomalle green. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0040] This invention relates to a rapid detection method for leucomall green based on nanozyme oxidation and a method for preparing the nanozyme. Examples 1-7 describe in detail a method for preparing a nanozyme for rapidly oxidizing leucomall green. Examples 8-12 describe a rapid detection method for leucomall green based on nanozyme oxidation.

[0041] Example 1:

[0042] A method for preparing a nanozyme for rapid detection of cryptic malachite green.

[0043] The specific steps are as follows:

[0044] Step 1: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water, and stirred to obtain a mixed solution, which is called solution A;

[0045] Step 2: Dissolve 2-methylimidazole in a mixed solution of N,N-dimethylformamide and deionized water, and stir to obtain a mixed solution, which is called solution B.

[0046] Step 3: Quickly pour solution A from step 1 into solution B from step 2, stir and let stand. Centrifuge the resulting product, wash and dry it to obtain a solid product, which is the nanozyme that can rapidly oxidize leucomalle green, namely PSS / ZIF-67.

[0047] The method for preparing the nanozyme for rapid oxidation of malachite green according to the present invention comprises the following steps: In step 1, the concentration of Co(NO3)2·6H2O in solution A is 0.029M-0.035M, the concentration of sodium polystyrene sulfonate is 0.90mg / mL-1.10mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1, with stirring for 25-35 min; In step 2, the concentration of 2-methylimidazole in solution B is 0.09M. The volume ratio of 0.17M N,N-dimethylformamide and deionized water is 1:1, and the mixture is stirred for 25-35 min. In step 3, the ratio of A solution to B solution is 0.9-1.1:0.9-1.1. After stirring for 0.5-1.5 min, the mixture is allowed to stand for 20-28 h. Then, the mixture is centrifuged at 7500-8500 rpm / min for 3-7 min, and then dried in a vacuum drying oven at 55-65℃ for 3.5-4.5 h.

[0048] Example 2:

[0049] A method for preparing a nanozyme for the rapid oxidation of leucomalle green comprises the following steps: 0.291 g of Co(NO3)2·6H2O and 0.03 g of sodium polystyrene sulfonate are dissolved in a mixed solution of 15 mL of N,N-dimethylformamide and 15 mL of deionized water, referred to as solution A. 0.328 g of 2-methylimidazole is then dissolved in a mixed solution of 15 mL of N,N-dimethylformamide and 15 mL of deionized water, referred to as solution B. Equal masses of solutions A and B are stirred separately for 0.5 h. Solution A is then rapidly poured into solution B, stirred for 1 min, and allowed to stand at room temperature for 24 h. The mixture is then centrifuged at 8000 rpm / min for 5 min. The resulting precipitate is washed with a mixed solution of N,N-dimethylformamide and deionized water, and then dried in a vacuum drying oven at 60 °C for 4 h. The resulting product is a nanozyme for the rapid oxidation of leucomalle green, named PSS / ZIF-67.

[0050] like Figure 2 As shown, transmission electron microscopy and X-ray photoelectron spectroscopy both indicate the successful preparation of PSS / ZIF-67 nanozymes.

[0051] Transmission electron microscopy results showed that the PSS / ZIF-67 nanozyme exhibited an ultrathin two-dimensional morphology, displaying a wrinkled nanohydrangea structure.

[0052] The X-ray photoelectron spectroscopy results show that the two main peaks at 780.0 eV and 795.6 eV correspond to Co2p, respectively. 3 / 2 and Co2p 1 / 2 The peaks at 284.4 eV correspond to C1s, 529.0 eV and 530.5 eV correspond to O1s, and 164.9 eV and 165.7 eV correspond to S2p.

[0053] Example 3:

[0054] A method for preparing a nanozyme for rapid oxidation of cryptic malachite green comprises the following steps: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 25 min, which is called solution A. The concentration of Co(NO3)2·6H2O in solution A is 0.029 M, the concentration of sodium polystyrene sulfonate is 0.90 mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1.

[0055] Then, 2-methylimidazole was dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 25 minutes. This solution was called solution B. The concentration of 2-methylimidazole in solution B was 0.09 M, and the volume ratio of N,N-dimethylformamide to deionized water was 1:1.

[0056] After stirring solutions A and B separately for 24 min, solution A was quickly poured into solution B at a ratio of 0.9:0.95. The mixture was stirred for 0.5 min and then allowed to stand at room temperature for 20 h. The mixture was then centrifuged at 7500 rpm for 3 min. The resulting precipitate was washed with a mixture of N,N-dimethylformamide and deionized water, and then dried in a vacuum oven at 55 °C for 3.5 h. The resulting product was a nanozyme for the rapid oxidation of leuco malachite green, named PSS / ZIF-67.

[0057] Example 4:

[0058] A method for preparing a nanozyme for rapid oxidation of cryptic malachite green comprises the following steps: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 28 min, which is called solution A. The concentration of Co(NO3)2·6H2O in solution A is 0.030 M, the concentration of sodium polystyrene sulfonate is 0.95 mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1.

[0059] Then, 2-methylimidazole was dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 27 minutes. This solution was called solution B. The concentration of 2-methylimidazole in solution B was 0.10 M, and the volume ratio of N,N-dimethylformamide to deionized water was 1:1.

[0060] After stirring solutions A and B separately for 28 min, solution A was quickly poured into solution B at a ratio of 1:1.02. The mixture was stirred for 0.6 min and then allowed to stand at room temperature for 23 h. The mixture was then centrifuged at 7800 rpm for 4 min. The resulting precipitate was washed with a mixture of N,N-dimethylformamide and deionized water, and then dried in a vacuum oven at 58 °C for 3.8 h. The resulting product was a nanozyme for the rapid oxidation of leuco malachite green, named PSS / ZIF-67.

[0061] Example 5:

[0062] A method for preparing a nanozyme for rapid oxidation of cryptic malachite green comprises the following steps: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 32 min, which is called solution A. The concentration of Co(NO3)2·6H2O in solution A is 0.031 M, the concentration of sodium polystyrene sulfonate is 1.02 mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1.

[0063] Then, 2-methylimidazole was dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 31 minutes. This solution was called solution B. The concentration of 2-methylimidazole in solution B was 0.14 M, and the volume ratio of N,N-dimethylformamide to deionized water was 1:1.

[0064] After stirring solutions A and B separately for 35 min, solution A was quickly poured into solution B at a ratio of 1.02:1. The mixture was stirred for 0.8 min and then allowed to stand at room temperature for 27 h. The mixture was then centrifuged at 8100 rpm for 6 min. The resulting precipitate was washed with a mixture of N,N-dimethylformamide and deionized water, and then dried in a vacuum oven at 62 °C for 4.2 h. The resulting product was a nanozyme for the rapid oxidation of leuco malachite green, named PSS / ZIF-67.

[0065] Example 6:

[0066] A method for preparing a nanozyme for rapid oxidation of cryptic malachite green comprises the following steps: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 35 min, which is called solution A. The concentration of Co(NO3)2·6H2O in solution A is 0.034 M, the concentration of sodium polystyrene sulfonate is 1.08 mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1.

[0067] Then, 2-methylimidazole was dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 34 minutes. This solution was called solution B. The concentration of 2-methylimidazole in solution B was 0.17M, and the volume ratio of N,N-dimethylformamide to deionized water was 1:1.

[0068] After stirring solutions A and B separately for 36 min, solution A was quickly poured into solution B at a ratio of 1.1:1. The mixture was stirred for 1.5 min and then allowed to stand at room temperature for 28 h. The mixture was then centrifuged at 8400 rpm for 6 min. The resulting precipitate was washed with a mixture of N,N-dimethylformamide and deionized water, and then dried in a vacuum oven at 65 °C for 4.4 h. The resulting product was a nanozyme for the rapid oxidation of leuco malachite green, named PSS / ZIF-67.

[0069] Example 7:

[0070] A method for preparing a nanozyme for rapid oxidation of cryptic malachite green comprises the following steps: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 35 min, which is called solution A. The concentration of Co(NO3)2·6H2O in solution A is 0.035 M, the concentration of sodium polystyrene sulfonate is 1.1 mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1.

[0071] Then, 2-methylimidazole was dissolved in a mixed solution of N,N-dimethylformamide and deionized water and stirred for 35 minutes. This solution was called solution B. The concentration of 2-methylimidazole in solution B was 0.17M, and the volume ratio of N,N-dimethylformamide to deionized water was 1:1.

[0072] After stirring solutions A and B separately for 36 min, solution A was quickly poured into solution B at a ratio of 1:1. The mixture was stirred for 1.5 min and then allowed to stand at room temperature for 27 h. The mixture was then centrifuged at 8500 rpm for 7 min. The resulting precipitate was washed with a mixture of N,N-dimethylformamide and deionized water, and then dried in a vacuum oven at 65 °C for 4.5 h. The resulting product was a nanozyme for the rapid oxidation of leuco malachite green, named PSS / ZIF-67.

[0073] Example 8

[0074] A rapid detection method for latent malachite green based on nanozyme oxidation, the specific steps of which are as follows:

[0075] (1) Solution preparation: Prepare an acetate-sodium acetate buffer solution with a concentration of 90mM-105mM, a leucomalle green solution with a concentration of 9mM-12mM, and a nano-enzyme stock solution for rapid detection of leucomalle green with a concentration of 0.9mg / mL-1.1mg / mL; wherein the pH of the acetate-sodium acetate buffer solution is 2.8-3.2, the solvent of the nano-enzyme stock solution for rapid oxidation of leucomalle green is acetonitrile, and the solvent of the leucomalle green solution is deionized water;

[0076] (2) Add 99-110 μL of nanozyme stock solution with a concentration of 0.9-1.1 mg / mL to 890-901 μL of 90 mM-105 mM acetate-sodium acetate buffer solution and shake well to obtain a reaction system with a total volume of 1 ml. At this time, the reaction system has no absorption peak at 618 nm. Then add 0.9-1.1 μL of leucomalle green solution with a concentration of 9-12 mM. At this time, the mixed solution has an absorption peak at 618 nm. Measure the absorbance of the solution at 618 nm before and after adding leucomalle green.

[0077] (3) The final concentration of the malachite green solution, the leucomalle green solution and the mixed solution of leucomalle green catalyzed by PSS / ZIF-67 nanozyme were all adjusted to 10 μM. The samples were measured according to the liquid chromatography-tandem mass spectrometry conditions. During the measurement, the quantitative ion m / z of malachite green was monitored separately: m / z 329 / 313; the quantitative ion m / z of leucomalle green was monitored separately. The ion chromatogram was obtained, and the product of nanozyme catalyzing the oxidation of leucomalle green was determined to be malachite green.

[0078] Example 9

[0079] A rapid detection method for latent malachite green based on nanozyme oxidation, the specific steps of which are as follows:

[0080] (1) Solution preparation: Prepare an acetate-sodium acetate buffer solution with pH=3 and a final concentration of 100mM. Prepare a leucomalle green solution with acetonitrile as solvent. Dissolve the solid PSS / ZIF-67 prepared in Example 1 in deionized water to prepare a 1mg / mL PSS / ZIF-67 stock solution.

[0081] (2) Add 10 μM leucomalle green to a reaction system containing 100 μL LPSS / ZIF-67 stock solution and 0.1 M acetic acid-sodium acetate buffer solution (pH=3.0) to a final volume of 1 mL. Shake well and react at room temperature for 1 min. Then measure the absorbance of the reaction system at 618 nm.

[0082] (3) The malachite green solution, the leucomalle green solution and the mixed solution after PSS / ZIF-67 nanozyme catalytic oxidation of leucomalle green were detected by liquid chromatography-tandem mass spectrometry, so that the final concentration of the malachite green solution, the leucomalle green solution and the mixed solution after PSS / ZIF-67 nanozyme catalytic oxidation of leucomalle green was 10 μM.

[0083] Figure 3 The results show that neither the PSS / ZIF-67 nanozyme nor leucomalle green has a UV-Vis absorption peak at 618 nm. However, the solution after the PSS / ZIF-67 nanozyme catalyzes the oxidation of leucomalle green has a significant UV-Vis absorption peak at 618 nm, and the solution color changes from colorless to blue.

[0084] Figure 4 The results show that, under the same experimental conditions, the reaction system of PSS / ZIF-67 and leucomalle green mixed solution was determined by liquid chromatography-tandem mass spectrometry. The results were compared with the ion chromatograms of leucomalle green and malachite green standard solutions. It can be seen that PSS / ZIF-67 catalyzes the oxidation of leucomalle green to produce a large amount of malachite green, but a small amount of leucomalle green still did not react completely.

[0085] (4) The method for detecting the content of leuco malachite green in fish tissue is as follows:

[0086] (4.1) Investigate the relationship between absorbance at 618 nm and concentration of leuco malachite green, and plot a standard working curve.

[0087] 100 μL of LPSS / ZIF-67 stock solution was added to different volumes of acetate-sodium acetate buffer solution, followed by different amounts of leucomalle green solution, to achieve final concentrations of 2, 3, 5, 7, 8, 10, 13, and 15 μM, respectively, with a final volume of 1 mL. The absorbance of each solution at 618 nm was measured using a UV-Vis spectrophotometer, and the UV-Vis absorption spectra were recorded.

[0088] Figure 5 The results show that the absorption peak at 618 nm gradually increases with increasing concentration of leucomall green solution (2-15 μM). Leucomall green exhibits a good linear relationship with absorbance at 618 nm within the 2-15 μM concentration range, with a linear regression equation of A618 = 0.07205[LMG] - 0.15298 (R² = 0.9961). The detection limit (LOD = 3σ) of the PSS / ZIF-67 nanozyme sensor for LMG is 18.55 nM. Here, A618 represents the absorbance at 618 nm, and [LMG] represents the concentration of leucomall green. This indicates that the PSS / ZIF-67 nanozyme sensor has good application prospects in the detection of leucomall green.

[0089] (4.2) Investigate the selectivity of PSS / ZIF-67 for leucomall green and determine the influence of interfering substances on the results.

[0090] 100 μL PSS / ZIF-67 stock solution was added to different volumes of acetate-sodium acetate buffer solution, followed by the addition of 10 mM leucomalle green solution and potentially interfering substances from fish tissue at concentrations 10 times higher than the leucomalle green solution. These interfering substances included metal ions (Na+). + K + Fe 3+ Mg 2+ Ca 2+ Al 3+ Fe 2+ and Cu 2+ The absorbance at 618 nm was observed after the interaction of L-glutamic acid (L-Glu), L-cysteine ​​(L-Cys), L-tryptophan (L-Try), L-phenylalanine (L-Phe), and L-methionine (L-Met).

[0091] Figure 6This indicates that the interfering agent at a concentration ten times that of leucomall green has almost no effect on the detection results of leucomall green. Only after the addition of leucomall green can the reaction system show a UV-Vis absorption peak at 618 nm, indicating that the PSS / ZIF-67 nanoenzyme sensor has good selectivity for leucomall green.

[0092] (4.3) Detection of the content of leucomalle green in fish tissue

[0093] Preparation of the extract: Remove the skin from fresh crucian carp and perch, then cut a portion of muscle from the back of the fish and mince it using a food processor. Accurately weigh 4g of the minced fish back muscle tissue and pour it into a 50mL centrifuge tube. Add 20mL of acetonitrile to the centrifuge tube while stirring to disperse the fish meat. Sonicate for 5 minutes, centrifuge at 12000rpm / min for 5 minutes, and then collect the supernatant as the sample solution to be tested.

[0094] The content of leucomall green in the test sample solution was detected by a spiked recovery experiment. 3 μL of the test sample solution and leucomall green standard solutions of different concentrations (0, 3.0, 8.0, 13.0 μM) were added to 100 μL LSS / ZIF-67 and acetate-sodium acetate buffer solution, with a final volume of 1 mL. After the mixed solution was left to stand at room temperature for 2 min, the absorbance of the reaction system was measured at 618 nm. The content of leucomall green in the fish tissue was calculated according to the standard working curve. The recovery rate and relative standard deviation were calculated, and the results are shown in Table 1.

[0095] Table 1. Detection results, recovery rate, and relative standard deviation of leucomalle green content in crucian carp and perch.

[0096]

[0097] In addition to the aforementioned ability to rapidly detect the content of leucomall green in fish tissue, this invention can also rapidly detect the content of leucomall green in other seafood products, such as shrimp, crab, clams, and oysters. The specific detection method is the same as the detection procedure for leucomall green in fish tissue. This invention can also detect leucomall green in other products besides seafood.

[0098] Example 10

[0099] A rapid detection method for leucomall green based on nanozyme oxidation, the specific steps of which are as follows:

[0100] (1) Solution preparation: Prepare a 95mM acetate-sodium acetate buffer solution, a 9.5mM leucomalle green solution, and a 0.98mg / mL nanozyme stock solution for rapid detection of leucomalle green; wherein the pH of the acetate-sodium acetate buffer solution is 2.9, the solvent of the nanozyme stock solution for rapid oxidation of leucomalle green is acetonitrile, and the solvent of the leucomalle green solution is deionized water;

[0101] (2) 110 μL of a nano-enzyme stock solution for rapid oxidation of leucomall green with a concentration of 1 mg / mL was added to 890 μL of 95 mM acetate-sodium acetate buffer solution and shaken thoroughly to obtain a reaction system with a total volume of 1 ml. At this time, the reaction system did not have an absorption peak at 618 nm. Then, 0.98 μL of 10 mM leucomall green solution was added. At this time, the mixed solution showed an absorption peak at 618 nm. The absorbance of the solution before and after adding leucomall green was measured at 618 nm.

[0102] (3) The final concentration of the malachite green solution, the leucomalle green solution and the mixed solution of leucomalle green catalyzed by PSS / ZIF-67 nanozyme were all adjusted to 10 μM. The samples were measured according to the liquid chromatography-tandem mass spectrometry conditions. During the measurement, the quantitative ion m / z of malachite green was monitored separately: m / z 329 / 313; the quantitative ion m / z of leucomalle green was monitored separately. The ion chromatogram was obtained, and the product of leucomalle green catalyzed by nanozyme for rapid oxidation of leucomalle green was determined to be malachite green.

[0103] (4) Rapid detection method for leuco malachite green content in actual samples:

[0104] (4.1) Plotting a standard working curve: Add a certain amount of nanozyme stock solution to different volumes of acetate-sodium acetate buffer solution, and then add different amounts of leucomalle green solution. Use a UV-Vis spectrophotometer to measure the absorbance of each solution at 618 nm, record the UV-Vis absorption spectrum, obtain the relationship between absorbance and the concentration of leucomalle green solution, and plot a standard working curve.

[0105] The acetate-sodium acetate buffer solution had a pH of 3 and a concentration of 0.1 M. The concentrations of the leucomalle green solutions were 2, 3, 5, 7, 8, 10, 13, and 15 mM. The concentration of the nanozyme stock solution for rapid detection of leucomalle green was 100 mg / mL. The volumes of the acetate-sodium acetate buffer solution were 898, 897, 895, 893, 892, 890, 887, and 885 μL. The volume of the nanozyme stock solution for rapid detection of leucomalle green was 100 μL. 2, 3, 5, 7, 8, 10, 13, and 15 μL of leucomalle green solution were added.

[0106] (4.2) Analysis of leucomall green content in actual samples: The actual sample extract of leucomall green was prepared and added to the mixture of acetic acid-sodium acetate buffer solution and nanozyme solution. The absorbance at 618 nm was measured by UV-Vis spectrophotometer. The content of leucomall green in the actual sample was calculated according to the standard working curve obtained in step (4.1).

[0107] Example 11

[0108] A rapid detection method for latent malachite green based on nanozyme oxidation, the specific steps of which are as follows:

[0109] (1) Solution preparation: Prepare a 102 mM acetate-sodium acetate buffer solution, an 11 mM leucomalle green solution, and a 1 mg / mL nanozyme stock solution for rapid detection of leucomalle green; wherein the pH of the acetate-sodium acetate buffer solution is 3.1, the solvent of the nanozyme stock solution for rapid oxidation of leucomalle green is acetonitrile, and the solvent of the leucomalle green solution is deionized water;

[0110] (2) Add 100 μL of 1.1 mg / mL nanozyme stock solution to 900 μL of 105 mM acetate-sodium acetate buffer solution and shake well to obtain a reaction system. The total volume of the reaction system is 1 ml. At this time, the reaction system has no absorption peak at 618 nm. Then add 1.1 μL of 12 mM leucomalle green solution. At this time, the mixed solution has an absorption peak at 618 nm. Measure the absorbance of the solution at 618 nm before and after adding leucomalle green.

[0111] (3) The final concentration of the malachite green solution, the leucomalle green solution and the mixed solution of leucomalle green catalyzed by PSS / ZIF-67 nanozyme were all adjusted to 10 μM. The samples were measured according to the liquid chromatography-tandem mass spectrometry conditions. During the measurement, the quantitative ion m / z of malachite green was monitored separately: m / z 329 / 313; the quantitative ion m / z of leucomalle green was monitored separately. The ion chromatogram was obtained, and the product of the rapid oxidation of leucomalle green by nanozyme catalyzed oxidation of leucomalle green was determined to be malachite green.

[0112] Example 12

[0113] A rapid detection method for latent malachite green based on nanozyme oxidation, the specific steps of which are as follows:

[0114] (1) Solution preparation: Prepare a 90 mM acetate-sodium acetate buffer solution, an 11 mM leucomalle green solution, and a 0.98 mg / mL nanozyme stock solution for rapid detection of leucomalle green; wherein the pH of the acetate-sodium acetate buffer solution is 3.0, the solvent of the nanozyme stock solution for rapid oxidation of leucomalle green is acetonitrile, and the solvent of the leucomalle green solution is deionized water;

[0115] (2) Add 99 μL of 0.9 / mL nanozyme stock solution to 901 μL of 105 mM acetate-sodium acetate buffer solution and shake well to obtain a reaction system. The total volume of the reaction system is 1 ml. At this time, the reaction system has no absorption peak at 618 nm. Then add 1.1 μL of 9 mM leucomalle green solution. At this time, the mixed solution has an absorption peak at 618 nm. Measure the absorbance of the solution at 618 nm before and after adding leucomalle green.

[0116] (3) The final concentration of the malachite green solution, the leucomalle green solution and the mixed solution of leucomalle green catalyzed by PSS / ZIF-67 nanozyme were all adjusted to 10 μM. The samples were measured according to the liquid chromatography-tandem mass spectrometry conditions. During the measurement, the quantitative ion m / z of malachite green was monitored separately: m / z 329 / 313; the quantitative ion m / z of leucomalle green was monitored separately. The ion chromatogram was obtained, and the product of the rapid oxidation of leucomalle green by nanozyme catalyzed oxidation of leucomalle green was determined to be malachite green.

[0117] Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A rapid detection method for latent malachite green based on nanozyme oxidation, characterized in that: The specific steps are as follows: (1) Solution preparation: Prepare an acetate-sodium acetate buffer solution with a concentration of 90mM-105mM, a leucomalle green solution with a concentration of 9mM-12mM, and a nanozyme stock solution for rapid detection of leucomalle green with a concentration of 0.9mg / mL-1.1mg / mL; wherein the pH of the acetate-sodium acetate buffer solution is 2.8-3.2, the solvent of the nanozyme stock solution for rapid detection of leucomalle green is acetonitrile, and the solvent of the leucomalle green solution is deionized water; The preparation method of the nanozyme for rapid detection of leuco malachite green is as follows: Step 1: Co(NO3)2·6H2O and sodium polystyrene sulfonate are dissolved in a mixed solution of N,N-dimethylformamide and deionized water, and stirred to obtain a mixed solution, which is called solution A; Step 2: Dissolve 2-methylimidazole in a mixed solution of N,N-dimethylformamide and deionized water, and stir to obtain a mixed solution, which is called solution B. Step 3: Quickly pour solution A from step 1 into solution B from step 2, stir and let stand. Centrifuge the resulting product, wash and dry it to obtain a solid product, which is the nanozyme that can rapidly oxidize leucomalle green, namely PSS / ZIF-67. (2) Add 99-110 μL of nanozyme stock solution with a concentration of 0.9-1.1 mg / mL to 890-901 μL of 90 mM-105 mM acetate-sodium acetate buffer solution, shake well to obtain the reaction system, the total volume of the reaction system is 1 ml. At this time, the reaction system has no absorption peak at 618 nm. Then add 0.9-1.1 μL of leucomall green solution with a concentration of 9-12 mM. At this time, the mixed solution has an absorption peak at 618 nm. Measure the absorbance of the solution at 618 nm before and after adding leucomall green. (3) The final concentration of the malachite green solution, the leucomalle green solution and the mixed solution of leucomalle green catalyzed by PSS / ZIF-67 nanozyme were all adjusted to 10 μM. The samples were measured according to the liquid chromatography-tandem mass spectrometry conditions. During the measurement, the quantitative ion m / z of malachite green was monitored separately: m / z 329 / 313; the quantitative ion m / z of leucomalle green was monitored separately. The ion chromatogram was obtained, and the product of nanozyme catalyzing the oxidation of leucomalle green was determined to be malachite green.

2. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 1, characterized in that: In step (1), the preparation method of the nanozyme for rapid detection of leucomall green, step 1, the concentration of Co(NO3)2·6H2O in solution A is 0.029M-0.035M, the concentration of sodium polystyrene sulfonate is 0.90mg / mL-1.10mg / mL, and the volume ratio of N,N-dimethylformamide to deionized water is 1:1, stirred for 25-35min; in step 2, the concentration of 2-methylimidazole in solution B is 0.09M- The volume ratio of 0.17M, N,N-dimethylformamide and deionized water is 1:1, and the mixture is stirred for 25-35 min. In step 3, the ratio of A solution to B solution is 0.9-1.1:0.9-1.

1. After stirring for 0.5-1.5 min, the mixture is allowed to stand for 20-28 h. Then, the mixture is centrifuged at 7500-8500 rpm / min for 3-7 min, and then dried in a vacuum drying oven at 55-65℃ for 3.5-4.5 h.

3. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 2, characterized in that: In step 1 of the method for preparing nanozymes for rapid detection of leucomall green, the concentration of Co(NO3)2·6H2O is 0.033M, the concentration of sodium polystyrene sulfonate is 1mg / mL, and the mixture is stirred for 30min.

4. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 2, characterized in that: In step 2 of the method for preparing nanozymes for rapid detection of leucomalle green in step (1), the concentration of 2-methylimidazole in solution B is 0.13M, and the mixture is stirred for 30 minutes.

5. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 2, characterized in that: In step 3 of the method for preparing nanozymes for rapid detection of leucomalle green in step (1), the ratio of A solution to B solution is 1:

1. After stirring for 1 min, the mixture is allowed to stand for 24 h. Then, the mixture is centrifuged at 8000 rpm / min for 5 min. The precipitate is washed with a mixed solution of N,N-dimethylformamide and deionized water, and then dried in a vacuum drying oven at 60℃ for 4 h.

6. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 1, characterized in that: It also includes step (4), a rapid detection method for the content of leuco malachite green in actual samples: (4.1) Plotting a standard working curve: Add a certain amount of nanozyme stock solution to different volumes of acetate-sodium acetate buffer solution, and then add different amounts of leucomalle green solution. Use a UV-Vis spectrophotometer to measure the absorbance of each solution at 618 nm, record the UV-Vis absorption spectrum, obtain the relationship between absorbance and the concentration of leucomalle green solution, and plot a standard working curve. The acetate-sodium acetate buffer solution had a pH of 3 and a concentration of 0.1 M. The concentrations of the leucomalle green solutions were 2, 3, 5, 7, 8, 10, 13, and 15 mM. The concentration of the nanozyme stock solution for rapid detection of leucomalle green was 100 mg / mL. The volumes of the acetate-sodium acetate buffer solution were 898, 897, 895, 893, 892, 890, 887, and 885 μL. The volume of the nanozyme stock solution for rapid detection of leucomalle green was 100 μL. 2, 3, 5, 7, 8, 10, 13, and 15 μL of leucomalle green solution were added. (4.2) Analysis of leucomall green content in actual samples: The actual sample extract of leucomall green was prepared and added to the mixture of acetic acid-sodium acetate buffer solution and nanozyme solution. The absorbance at 618 nm was measured by UV-Vis spectrophotometer. The content of leucomall green in the actual sample was calculated according to the standard working curve obtained in step (4.1).

7. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 1, characterized in that: In step (1), the pH of the acetate-sodium acetate buffer solution is 3 and the concentration is 100mM. A leucomalle green solution with a concentration of 10mM is prepared using acetonitrile as the solution, and a rapid detection stock solution with a concentration of 1mg / mL is prepared using deionized water as the solvent. In step (2), 101μL of a rapid oxidation leucomalle green nanoenzyme stock solution with a concentration of 1mg / mL is added to 899μL of acetate-sodium acetate buffer solution with a concentration of 100nM. At this time, the mixed solution has no absorption peak at 618nm. Then, 1μL of a leucomalle green solution with a concentration of 10mM is added. At this time, the mixed solution has an absorption peak at 618nm. The absorbance of the solution at 618nm before and after the addition of leucomalle green is measured.

8. The rapid detection method for latent malachite green based on nanozyme oxidation according to claim 6, characterized in that: The actual sample was a seafood product.

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