Kit and method for visual fluorescence detection of kasugamycin

Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to a kit and a method for detecting kasugamycin through visual fluorescence. The kit provided by the invention comprises a DT-Cu nano-enzyme dispersion liquid, a buffer solution, a hydrogen peroxide solution and a thiamine solution. The method comprises the following steps: mixing a to-be-detected solution with a DT-Cu nano-enzyme dispersion liquid to obtain a Y1 solution, mixing the Y1 solution with a buffer solution, a hydrogen peroxide solution and a thiamine solution, and carrying out a catalytic oxidation reaction to obtain a Y2 solution; and performing fluorescence detection or ultraviolet irradiation on the Y2 solution and a control solution, and realizing qualitative detection of kasugamycin in the to-be-detected solution according to a fluorescence spectrum obtained by comparing the Y2 solution and the control solution or fluorescence intensity of an ultraviolet irradiation picture. A fluorescence method is adopted for rapid detection, specificity is high, operation is easy, the result is reliable, the technical problem that kasugamycin cannot be rapidly detected is solved, and high practicability and wide applicability are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and specifically relates to a kit and method for visually fluorescently detecting kasugamycin. Background Art

[0002] Kasugamycin is an aminoglycoside antibiotic isolated from Streptomyces kasugaensis and is used to control various plant diseases. Although kasugamycin shows various advantages, including effective control of leaf spot disease, fire blight, and the ability to significantly inhibit microbial protein biosynthesis activity, etc., its antibiotic resistance and residue risks have raised concerns about its widespread use.

[0003] Kasugamycin is an amphoteric chemical with high hydrophilicity. Even at ambient temperature, it is unstable in alkaline media. Due to its aliphatic structure, it lacks a characteristic ultraviolet absorption spectrum, thus hindering the detection using common spectrophotometric methods or high-performance liquid chromatography based on ultraviolet detectors. Although high-performance liquid chromatography-tandem mass spectrometry can sensitively and reliably detect kasugamycin, due to the high polarity of kasugamycin, the pretreatment operation is complex. In addition, the dependence on professional technical personnel also limits its widespread application. Summary of the Invention

[0004] The purpose of the present invention is to provide a kit and method for visually fluorescently detecting kasugamycin. The method provided by the present invention is simple to operate and the results are reliable, realizing the rapid detection and visual response of kasugamycin, and having strong practicability and wide applicability.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a kit for visually fluorescently detecting kasugamycin, including a DT-Cu nanozyme dispersion, a buffer solution, a hydrogen peroxide solution, and a thiamine solution, wherein the DT-Cu nanozyme in the DT-Cu nanozyme dispersion is a coordination compound formed by 3,5-diamino-1,2,4-triazole and copper ions.

[0007] The present invention provides a method for visually fluorescently detecting kasugamycin, including the following steps:

[0008] Mix the test solution with the DT-Cu nanozyme dispersion and perform incubation treatment to obtain a Y1 solution;

[0009] Mix the Y1 solution, the buffer solution, the hydrogen peroxide solution, and the thiamine solution and perform a catalytic oxidation reaction to obtain a Y2 solution;

[0010] Perform fluorescence detection or ultraviolet irradiation on the Y2 solution to obtain the fluorescence spectrum or ultraviolet irradiation photo of the Y2 solution;

[0011] Qualitative detection of kasugamycin in the test solution is achieved based on the fluorescence spectrum or ultraviolet irradiation photograph of the Y2 solution and the fluorescence spectrum or ultraviolet irradiation photograph of the control solution.

[0012] The control solution is the blank control of the Y2 solution.

[0013] Preferably, the concentration of the DT-Cu nanozyme dispersion is 0.05 - 0.1 mg / mL; the volume ratio of the test solution to the DT-Cu nanozyme dispersion is 3:(1 - 2); the concentration range of kasugamycin in the test solution is 0.01 - 0.5 mg / mL.

[0014] Preferably, the temperature of the incubation treatment is 37 - 45 °C, and the time of the incubation treatment is 20 - 30 min.

[0015] Preferably, the concentration of the buffer solution is 0.1 - 0.2 M, the pH value is 5 - 6; the concentration of the thiamine solution is 0.01 - 0.05 M; the mass concentration of the hydrogen peroxide solution is 3 - 15%.

[0016] Preferably, the volume ratio of the Y1 solution to the buffer solution, hydrogen peroxide solution, and thiamine solution is 400:(280 - 350):(40 - 100):(80 - 200).

[0017] Preferably, the temperature of the catalytic oxidation reaction is 37 - 45 °C, and the time of the catalytic oxidation reaction is 30 - 60 min.

[0018] Preferably, the excitation wavelength of the fluorescence detection is 350 nm ± 10 nm; the emission wavelength of the fluorescence detection is 400 - 500 nm; the wavelength of the ultraviolet light is 365 nm.

[0019] Preferably, the preparation method of the DT-Cu nanozyme includes the following steps:

[0020] 3,5-Diamino-1,2,4-triazole and copper sulfate are mixed with water for a coordination reaction to obtain the DT-Cu nanozyme.

[0021] Preferably, the mass ratio of 3,5-diamino-1,2,4-triazole to copper sulfate is (15 - 18):(12.4 - 13); the temperature of the coordination reaction is 20 - 30 °C, and the time of the coordination reaction is 1 - 24 h.

[0022] The present invention provides a kit for visually fluorescent detection of kasugamycin, comprising a DT-Cu nanozyme dispersion, a buffer solution, a hydrogen peroxide solution and a thiamine solution. The DT-Cu nanozyme in the DT-Cu nanozyme dispersion is a coordination compound formed by 3,5-diamino-1,2,4-triazole and copper ions. The DT-Cu nanozyme has peroxidase-like catalytic activity at pH 5-6, and can catalyze the oxidation of thiamine to thiochrome, which produces blue fluorescence under the excitation of an excitation light with a wavelength of 350 nm and at the maximum emission wavelength of 440 nm. Hydrogen peroxide, as an electron acceptor, can accept the electrons provided by the substrate and be reduced itself, thereby promoting the progress of the redox reaction and oxidizing the substrate. Kasugamycin will inhibit the catalytic activity of the DT-Cu nanozyme, thereby inhibiting the catalytic oxidation of thiamine by the DT-Cu nanozyme and resulting in the hindrance of thiochrome formation.

[0023] The present invention provides a method for visually fluorescent detection of kasugamycin, which includes mixing a test solution with a DT-Cu nanozyme dispersion and performing an incubation treatment to obtain a Y1 solution; mixing the Y1 solution, a buffer solution, a hydrogen peroxide solution and a thiamine solution and performing a catalytic oxidation reaction to obtain a Y2 solution; performing fluorescence detection or ultraviolet irradiation on the Y2 solution to obtain the fluorescence spectrum or ultraviolet irradiation photo of the Y2 solution; and achieving qualitative detection of kasugamycin in the test solution according to the fluorescence spectrum or ultraviolet irradiation photo of the Y2 solution and the fluorescence spectrum or ultraviolet irradiation photo of a control solution; the control solution is a blank control of the Y2 solution. In the presence of kasugamycin, the blue fluorescence of the solution at 440 nm is weakened compared with the control solution, and the higher the content of kasugamycin in the solution, the weaker the blue fluorescence intensity of the solution, thus achieving rapid detection of kasugamycin.

[0024] Furthermore, compared with the prior art in which high performance liquid chromatography tandem mass spectrometry is used to detect kasugamycin, the present invention uses a fluorescence method for rapid detection, which is simple to operate, reliable in results and low in cost, solves the technical problem of rapid detection of kasugamycin, and has strong practicability and wide applicability. Description of the Drawings

[0025] Figure 1 Fluorescence spectra of Solution I, Solution II, Solution III and Solution IV in the examples and pictures under ultraviolet irradiation at 365 nm. Detailed Embodiments

[0026] The present invention provides a kit for visually fluorescent detection of kasugamycin, comprising a DT-Cu nanozyme dispersion, a buffer solution, a hydrogen peroxide solution and a thiamine solution. The DT-Cu nanozyme in the DT-Cu nanozyme dispersion is a coordination compound formed by 3,5-diamino-1,2,4-triazole and copper ions.

[0027] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well-known to those skilled in the art.

[0028] The present invention provides a method for visually fluorescently detecting kasugamycin, comprising the following steps:

[0029] Mix the test solution with the DT-Cu nanozyme dispersion, and perform incubation treatment to obtain solution Y1;

[0030] Mix the solution Y1, buffer solution, hydrogen peroxide solution, and thiamine solution, and perform a catalytic oxidation reaction to obtain solution Y2;

[0031] Perform fluorescence detection or ultraviolet irradiation on the solution Y2 to obtain the fluorescence spectrum or ultraviolet irradiation photo of the solution Y2;

[0032] Based on the fluorescence spectrum or ultraviolet irradiation photo of the solution Y2 and the fluorescence spectrum or ultraviolet irradiation photo of the control solution, qualitative detection of kasugamycin in the test solution is achieved;

[0033] The control solution is the blank control of the solution Y2.

[0034] In the present invention, the test solution is mixed with the DT-Cu nanozyme dispersion, and incubation treatment is performed to obtain solution Y1.

[0035] As an embodiment of the present invention, the concentration of the DT-Cu nanozyme dispersion can be 0.05 - 0.1 mg / mL, and further can be 0.05 - 0.08 mg / mL; the volume ratio of the test solution to the DT-Cu nanozyme solution can be 3:(1 - 2), specifically can be 3:1, 3:1.5, and 3:2; the concentration range of kasugamycin in the test solution is 0.01 - 0.5 mg / mL, and further can be 0.05 - 0.3 mg / mL.

[0036] As an embodiment of the present invention, the solvent of the DT-Cu nanozyme dispersion can be water; the mixing of the test solution and the DT-Cu nanozyme dispersion in the present invention is denoted as the first mixing; the first mixing can be adding the test solution to the DT-Cu nanozyme dispersion and shaking well; there are no special limitations on the time and temperature of the first mixing in the present invention, and it is sufficient to mix evenly. In the embodiments of the present invention, the test solution is specifically a kasugamycin standard solution with a concentration of 1 mg / mL, and the solvent of the kasugamycin standard solution is water.

[0037] As an embodiment of the present invention, the temperature of the incubation treatment can be 37 - 45 °C, specifically can be 37 - 40 °C; the time of the incubation treatment can be 20 - 30 min, specifically can be 20 - 25 min.

[0038] As an embodiment of the present invention, the preparation method of the DT-Cu nanozyme comprises the following steps:

[0039] Mix 3,5-diamino-1,2,4-triazole, copper sulfate and water for a coordination reaction to obtain the DT-Cu nanozyme.

[0040] As an embodiment of the present invention, the mass ratio of 3,5-diamino-1,2,4-triazole to copper sulfate is (15-18):(12.4-13).

[0041] As an embodiment of the present invention, specifically, an aqueous solution of 3,5-diamino-1,2,4-triazole can be mixed with an aqueous solution of copper sulfate for a coordination reaction; the concentration of the aqueous solution of 3,5-diamino-1,2,4-triazole can be 15-18 mg / mL, specifically 16 mg / mL; the concentration of the aqueous solution of copper sulfate can be 6.2-6.5 mg / mL, specifically 6.4 mg / mL; the volume ratio of the aqueous solution of 3,5-diamino-1,2,4-triazole to the aqueous solution of copper sulfate is based on ensuring that the mass ratio of 3,5-diamino-1,2,4-triazole to copper sulfate is satisfied.

[0042] As an embodiment of the present invention, the coordination reaction is carried out under stirring, the time of the coordination reaction can be 1-24 h, further 1-12 h, specifically 1 h; the present invention has no special limitation on the temperature of the coordination reaction, and it can be carried out at room temperature, specifically 20-30 °C.

[0043] As an embodiment of the present invention, after the coordination reaction, the present invention may further include solid-liquid separation and drying treatment of the obtained product liquid to finally obtain the DT-Cu nanozyme.

[0044] As an embodiment of the present invention, the solid-liquid separation can be centrifugation, the rotation speed of the solid-liquid separation can be 8000 rpm, the time of the solid-liquid separation can be 10 min, and the present invention has no special limitation on the temperature of the solid-liquid separation, and it can be carried out at room temperature.

[0045] As an embodiment of the present invention, before the drying treatment, the precipitate can be rinsed, and the rinsing can be carried out by rinsing with ultrapure water and ethanol 3-5 times in sequence, specifically 3 times.

[0046] As an embodiment of the present invention, the drying treatment can be vacuum drying, the temperature of the drying treatment can be 100 °C, and the present invention has no special limitation on the time of the drying treatment, and the material is completely dried.

[0047] After obtaining the Y1 solution, the present invention mixes the Y1 solution, a buffer solution, a hydrogen peroxide solution, and a thiamine solution, and performs a catalytic oxidation reaction to obtain a Y2 solution.

[0048] As an embodiment of the present invention, the buffer solution may include a MES buffer solution; the concentration of the buffer solution may be 0.1 - 0.2 M, specifically 0.1 M; the pH value of the buffer solution may be 5 - 6, specifically pH = 6.

[0049] As an embodiment of the present invention, the concentration of the thiamine solution may be 0.01 - 0.05 M, specifically 0.01 M; the solvent of the thiamine solution may be water.

[0050] As an embodiment of the present invention, the mass concentration of the hydrogen peroxide solution may be 3 - 15%, specifically 3%.

[0051] The hydrogen peroxide solution of the present invention serves as an electron acceptor, capable of accepting the electrons provided by the thiamine solution, reducing itself, thereby promoting the progress of the oxidation-reduction reaction and oxidizing the thiamine solution.

[0052] As an embodiment of the present invention, the volume ratio of the Y1 solution, the buffer solution, the hydrogen peroxide solution, and the thiamine solution may be 400:(280 - 350):(40 - 100):(80 - 200), specifically 400:350:50:200, 400:300:100:200, 400:400:100:100, and 500:350:50:100.

[0053] As an embodiment of the present invention, the mixing of the Y1 solution, the buffer solution, the hydrogen peroxide solution, and the thiamine solution by the present invention is denoted as the second mixing; the second mixing may be adding the buffer solution, the hydrogen peroxide solution, and the thiamine solution to the Y1 solution, and the present invention has no special limitation on the second mixing method.

[0054] As an embodiment of the present invention, the temperature of the catalytic oxidation reaction may be 37 - 45 °C, specifically 37 - 40 °C, and the time of the catalytic oxidation reaction may be 30 - 60 min, specifically 30 - 40 min.

[0055] After obtaining the Y2 solution, the present invention performs fluorescence detection or ultraviolet irradiation on the Y2 solution to obtain the fluorescence spectrum or ultraviolet irradiation photo of the Y2 solution; according to the fluorescence spectrum or ultraviolet irradiation photo of the Y2 solution and the fluorescence spectrum or ultraviolet irradiation photo of the control solution, qualitative detection of kasugamycin in the test solution is achieved; the control solution is the blank control of the Y2 solution.

[0056] As an embodiment of the present invention, the excitation wavelength for fluorescence detection may be 350 nm ± 10 nm; the emission wavelength for fluorescence detection may be 400 - 500 nm, and the maximum emission wavelength for fluorescence detection may be 440 nm; the wavelength of the ultraviolet light may be 365 nm.

[0057] As an embodiment of the present invention, the present invention determines whether kasugamycin or the content of kasugamycin is contained in the Y2 solution according to the change in fluorescence intensity between the Y2 solution and the control solution:

[0058] If the fluorescence intensity of the Y2 solution is the same as that of the control solution, then the Y2 solution does not contain kasugamycin;

[0059] If the Y2 solution has no fluorescence or the fluorescence intensity decreases compared with the control solution, then the Y2 solution contains kasugamycin, and the lower the fluorescence intensity of the Y2 solution, the higher the content of kasugamycin.

[0060] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0061] Example 1

[0062] Dissolve 2.00 g of 3,5-diamino-1,2,4-triazole in 125 mL of ultrapure water to obtain a 3,5-diamino-1,2,4-triazole aqueous solution with a concentration of 16 mg / mL; dissolve 1.60 g of copper sulfate in 250 mL of ultrapure water to obtain a copper sulfate aqueous solution with a concentration of 6.4 mg / mL; mix the 3,5-diamino-1,2,4-triazole aqueous solution and the copper sulfate aqueous solution, stir and react at room temperature for 1 h, centrifuge the obtained product liquid at a rotation speed of 8000 rpm at room temperature for 10 min to obtain a green precipitate; rinse the green precipitate with ultrapure water and ethanol three times respectively, and vacuum dry at 100 °C for 6 h to obtain DT-Cu nanozyme.

[0063] Example 2

[0064] Using water as the solvent, the DT-Cu nanozyme obtained in Example 1 was formulated into a DT-Cu nanozyme dispersion with a concentration of 0.05 mg / mL. 300 μL of a kasugamycin standard solution with a concentration of 0.1 mg / mL (solvent is water) was added to 100 μL of the DT-Cu nanozyme dispersion with a concentration of 0.05 mg / mL, shaken and mixed evenly, and incubated at 37 °C for 20 min to obtain Solution Y1; 350 μL of a MES buffer with a concentration of 0.1 M and pH = 6.0, 50 μL of a hydrogen peroxide solution with a mass concentration of 3%, and 200 μL of a thiamine solution with a concentration of 0.01 M were added to Solution Y1, and the reaction was carried out at 37 °C for 30 min to obtain Solution II.

[0065] Test Example

[0066] Basically the same as Example 2, except that 300 μL of the kasugamycin standard solution with a concentration of 0.1 mg / mL was replaced with 300 μL of water (without kasugamycin), and finally Solution I was obtained as a control solution.

[0067] Directly use the DT-Cu nanozyme dispersion with a concentration of 0.05 mg / mL prepared in Example 2 as Solution III.

[0068] Directly use the kasugamycin standard solution with a concentration of 0.1 mg / mL as Solution IV.

[0069] Solutions I, II (from Example 2), III, and IV were respectively excited by an excitation light with a wavelength of 350 nm, and the fluorescence intensity at 440 nm was measured, where the slits of the excitation wavelength and the emission wavelength were both 5 nm, to obtain Figure 1 A in; and photos of Solutions I, II, III, and IV were respectively taken and recorded under ultraviolet light at 365 nm to obtain Figure 1 B in.

[0070] Figure 1 A in is a fluorescence spectrogram of the relationship between the fluorescence intensity and wavelength of the four solutions. Figure 1 B in is a diagram of the four solutions under ultraviolet light at 365 nm. From Figure 1As can be seen from A and B in [the context], since the DT-Cu nanozyme can catalyze the oxidation of thiamine to thiochrome, Solution I has the highest fluorescence intensity at a wavelength of 440 nm and exhibits strong blue fluorescence under ultraviolet irradiation at 365 nm. In the presence of kasugamycin, the fluorescence intensity of Solution II at 440 nm decreases. Correspondingly, the blue fluorescence of the solution under ultraviolet light at 365 nm weakens. Solutions III and IV indicate that the DT-Cu nanozyme solution and the kasugamycin standard solution themselves have no fluorescence intensity at 440 nm and do not exhibit blue fluorescence under ultraviolet light at 365 nm, that is, the possibility that the DT-Cu nanozyme solution and the kasugamycin standard solution themselves have fluorescence and thus affect the accuracy of the method of the present invention is excluded.

[0071] Therefore, the method provided by the present invention can qualitatively determine the content of kasugamycin in the test solution. The present invention can judge whether the test solution contains kasugamycin or the content of kasugamycin according to the change of the fluorescence intensity of the test solution:

[0072] If the fluorescence intensity of the test solution is the same as that of the control solution, the test solution does not contain kasugamycin;

[0073] If the test solution has no fluorescence or the fluorescence intensity decreases compared with the control solution, the test solution contains kasugamycin, and the lower the fluorescence intensity of the test solution, the higher the content of kasugamycin.

[0074] Therefore, the visual fluorescence detection method provided by the present invention has excellent application potential in the field of detecting kasugamycin.

[0075] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A kit for visual fluorescence detection of kasugamycin, comprising a DT-Cu nanozyme dispersion, a buffer solution, a hydrogen peroxide solution and a thiamine solution, wherein the DT-Cu nanozyme in the DT-Cu nanozyme dispersion is a coordination compound formed by 3,5-diamino-1,2,4-triazole and copper ions.

2. A method for visual fluorescence detection of kasugamycin, comprising the following steps: The test solution is mixed with the DT-Cu nanozyme dispersion and incubated to obtain a Y1 solution; The Y1 solution, buffer solution, hydrogen peroxide solution and thiamine solution are mixed to perform a catalytic oxidation reaction to obtain a Y2 solution; The Y2 solution is subjected to fluorescence detection or ultraviolet irradiation to obtain a fluorescence spectrum or an ultraviolet irradiation photograph of the Y2 solution; According to the fluorescence spectrum or ultraviolet irradiation photograph of the Y2 solution and the fluorescence spectrum or ultraviolet irradiation photograph of the control solution, qualitative detection of kasugamycin in the test solution is achieved; The control solution is a blank control of the Y2 solution.

3. The method for visual fluorescence detection of kasugamycin according to claim 2, characterized in that: The concentration of the DT-Cu nanozyme dispersion is 0.05-0.1 mg / mL; the volume ratio of the test solution to the DT-Cu nanozyme dispersion is 3:(1-2); the concentration range of kasugamycin in the test solution is 0.01-0.5 mg / mL.

4. The method for visual fluorescence detection of kasugamycin according to claim 2, characterized in that: The incubation temperature is 37-45° C., and the incubation time is 20-30 min.

5. The method for visual fluorescence detection of kasugamycin according to claim 2 or 3, characterized in that: The concentration of the buffer solution is 0.1-0.2M, and the pH value is 5-6; the concentration of the thiamine solution is 0.01-0.05M; and the mass concentration of the hydrogen peroxide solution is 3-15%.

6. The method for visual fluorescence detection of kasugamycin according to claim 5, characterized in that: The volume ratio of the Y1 solution to the buffer solution, the hydrogen peroxide solution, and the thiamine solution is 400:(280-350):(40-100):(80-200).

7. The method for visual fluorescence detection of kasugamycin according to claim 2, characterized in that: The temperature of the catalytic oxidation reaction is 37-45° C., and the time of the catalytic oxidation reaction is 30-60 minutes.

8. The method for visual fluorescence detection of kasugamycin according to claim 2, characterized in that: The excitation light wavelength of the fluorescence detection is 350nm±10nm; the emission wavelength of the fluorescence detection is 400-500nm; and the wavelength of the ultraviolet light is 365nm.

9. The method for visual fluorescence detection of kasugamycin according to claim 2, characterized in that: The preparation method of the DT-Cu nanozyme comprises the following steps: 3,5-Diamino-1,2,4-triazole and copper sulfate were mixed with water for coordination reaction to obtain DT-Cu nanozyme.

10. The method for visual fluorescence detection of kasugamycin according to claim 9, characterized in that: The mass ratio of the 3,5-diamino-1,2,4-triazole to copper sulfate is (15-18):(12.4-13); the temperature of the coordination reaction is 20-30° C., and the time of the coordination reaction is 1-24 hours.

Images