White nano-enzyme with imitated alkaline phosphatase activity as well as preparation method and application of white nano-enzyme

Abstract

The invention discloses a white nano-enzyme with imitated alkaline phosphatase activity and a preparation method and application thereof, and relates to the field of pesticide detection.The white nano-enzyme with imitated alkaline phosphatase activity is synthesized from 1, 1, 2, 2-tetra (4-carboxyphenyl) ethylene, anhydrous zirconium tetrachloride, acetic acid, N, N-dimethylformamide and cetyltrimethylammonium bromide; the white nano-enzyme imitating alkaline phosphatase activity can be inhibited by the emamectin benzoate in a high-selectivity manner, so that when the white nano-enzyme is applied to paper-based detection of the emamectin benzoate, high-selectivity, visual and on-site rapid detection of the emamectin benzoate is realized, and the paper-based paper-based detection of the emamectin benzoate has the advantages that the paper-based paper-based paper-based detection of the emamectin benzoate is realized; and a promising way is provided for screening high-specificity pesticide recognition elements and developing high-selectivity enzyme-free colorimetric paper-based detection based on nano-enzyme.

Description

Technical Field

[0001] The present invention relates to the field of pesticide detection, and specifically to a white nanozyme with mimetic alkaline phosphatase activity, a preparation method thereof, and an application thereof. Background Art

[0002] Nanozymes are nanomaterials with mimetic enzyme activity. Due to their easy synthesis, high stability, and cost-effectiveness, they have become alternatives to biological enzymes in various application fields. In the prior art, applying nanozymes to colorimetric paper-based analytical devices can achieve pesticide detection intuitively, simply, and rapidly. Nanozymes known for use in paper-based analytical devices usually exhibit various colors, such as nanogold in wine red, Prussian blue in blue, iron oxide in black, cerium dioxide in yellow, and manganese dioxide in brown. In addition, most of the nanozymes used in paper-based analytical devices have mimetic oxidoreductase activity and are easily interfered by substances such as oxygen, reactive oxygen species, and antioxidants present in actual samples. Therefore, it is important to develop a nanozyme with non-interfering color and non-oxidoreductase mimetic enzyme activity for paper-based detection.

[0003] Currently, nanozymes have been widely used in visualization methods based on the inhibition of the activities of enzymes (such as acetylcholinesterase, butyrylcholinesterase, alkaline phosphatase, and acid phosphatase) due to their unique catalytic properties. However, due to the inherent limitations of enzyme inhibition, these enzymes are usually limited to the detection of organophosphorus, carbamate, and pyrethroid pesticides, resulting in a limited range of detectable pesticides and poor selectivity. Among them, emamectin benzoate is a biological pesticide. Due to its strong activity at low doses, long residual period, and relatively low toxicity to non-target organisms, it has been widely used in agriculture and aquaculture to control pests and parasites. However, long-term and large-scale use of emamectin benzoate has been found to have residues in water bodies, soil, and vegetables. Emamectin benzoate not only has cytotoxicity and genotoxicity but also remains toxic to non-target organisms such as bees, fish, silkworms, mice, and even humans. Since emamectin benzoate does not belong to common pesticide categories, traditional enzyme inhibition methods are not sufficient to detect emamectin benzoate. In addition, enzyme-based methods cannot completely avoid the participation of enzymes and cannot completely solve potential problems related to enzymes. Therefore, it is necessary to develop a nanozyme-based method for detecting emamectin benzoate to achieve specific detection without relying on enzymes.

[0004] Based on this, the present invention aims to provide a white nanozyme with mimetic alkaline phosphatase activity that can be used to detect emamectin benzoate, and to achieve paper-based detection by using the inhibitory effect of emamectin benzoate on the mimetic alkaline phosphatase activity of the white nanozyme. Summary of the Invention

[0005] The object of the present invention is to provide a white nanozyme with alkaline phosphatase mimicking activity and a preparation method thereof. The alkaline phosphatase mimicking activity of this nanozyme can be inhibited by emamectin benzoate in a highly selective manner.

[0006] According to the characteristics of the white nanozyme with alkaline phosphatase mimicking activity, another object of the present invention is to provide an application of the white nanozyme with alkaline phosphatase mimicking activity in paper-based detection, realizing highly selective, on-site, visual and rapid detection of emamectin benzoate.

[0007] To achieve the above object, the present invention provides the following technical solutions: A preparation method of a white nanozyme with alkaline phosphatase mimicking activity is as follows: Add 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, zirconium tetrachloride anhydrous, acetic acid, N,N-dimethylformamide and cetyltrimethylammonium bromide into a glass bottle, ultrasonically dissolve for a period of time, put the glass bottle containing the mixed solution into an oven, and heat at a certain temperature for a period of time; After cooling to room temperature, collect the product by centrifugation at a certain speed for a period of time, and wash it several times with N,N-dimethylformamide and methanol respectively; Finally, obtain the white nanozyme powder with alkaline phosphatase mimicking activity by vacuum drying.

[0008] In the present invention, for the preparation method of the white nanozyme with alkaline phosphatase mimicking activity, specifically: add 1.5−3.5 mg of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, 2−4 mg of zirconium tetrachloride anhydrous, 100−300 µL of acetic acid, 0.5−1.5 mL of N,N-dimethylformamide and 2−4 mg of cetyltrimethylammonium bromide into a glass bottle, ultrasonically dissolve for 2−10 min, put it into an oven at 80−100 °C and heat for 16−18 h; after cooling to room temperature, centrifuge at a speed of 5000−8000 rpm for 2−10 min, wash the product 2−3 times with N,N-dimethylformamide and methanol respectively, vacuum dry at 25−37 °C for 4−8 h to obtain the white nanozyme powder, and store it in a refrigerator at 4 °C for standby.

[0009] In the present invention, a white nanozyme with alkaline phosphatase mimicking activity obtained by the above preparation method.

[0010] An application of a white nanozyme with alkaline phosphatase mimicking activity in paper-based detection, fix the said white nanozyme on a test paper strip to prepare a white nanozyme test strip for detecting emamectin benzoate.

[0011] In the present invention, the preparation method of the white nanozyme detection test strip is specifically as follows: The white nanozyme with alkaline phosphatase-like activity is dissolved in water to form a solution with a concentration of 2−10 mg / mL. 2−50 μL of the white nanozyme solution is dropped onto the surface of the paper substrate, and then vacuum-dried at 25−37 °C and stored in a refrigerator at 4 °C for later use.

[0012] In the present invention, the paper substrate material of the test strip is qualitative filter paper, quantitative filter paper, absorbent paper, and nitrocellulose membrane, and the diameter of the paper substrate is 4−15 mm.

[0013] In the present invention, the process of detecting emamectin benzoate using the white nanozyme detection test strip is as follows: A certain amount of emamectin benzoate sample solution is dropped onto the white nanozyme detection test strip. After incubation at room temperature, a certain amount of 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES) buffer solution containing p-nitrophenyl phosphate and molybdate-malachite green solution are added in sequence. After reacting for a period of time, the color value of the white nanozyme detection test strip is identified by a smartphone to perform quantitative analysis of emamectin benzoate.

[0014] In the present invention, 2−50 μL of emamectin benzoate sample solution is dropped onto the white nanozyme detection test strip. After incubation at room temperature, 2−50 μL of HEPES buffer solution containing p-nitrophenyl phosphate and 2−50 μL of molybdate-malachite green solution are added in sequence. After reacting for 20 min, the color value of the white nanozyme detection test strip is identified by a smartphone to perform quantitative analysis of emamectin benzoate.

[0015] In the present invention, the concentration of p-nitrophenyl phosphate is 0.5−3 mmol / L. In the molybdate-malachite green solution, the concentration of molybdate is 1−2 mg / mL, and the concentration of malachite green is 0.5−1.5 mg / mL.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The nanozyme synthesized in the present invention exhibits significant alkaline phosphatase-like activity and does not have peroxidase-like activity. No reactive oxygen species are generated during the catalytic process, avoiding interference during pesticide detection.

[0017] 2. The nanozyme with alkaline phosphatase-like activity in the present invention can be inhibited by emamectin benzoate in a highly selective manner without the presence of an enzyme. Based on this property, qualitative and quantitative detection of emamectin benzoate can be achieved.

[0018] 3. The nanozyme prepared by the present invention is white itself. When it is immobilized on the test strip, it can achieve a whitening effect, reduce background interference, and thus enhance the visibility of the color development result. The developed white nanozyme-based test strip can realize the highly selective, visual, and on-site rapid detection of emamectin benzoate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a transmission electron microscope image of the white nanozyme (Zr-CPNP@CTAB nanozyme) with alkaline phosphatase-like activity provided by an example of the present invention.

[0020] Figure 2 It is an X-ray diffraction energy spectrum and X-ray photoelectron energy spectrum of the white nanozyme with alkaline phosphatase-like activity provided by an example of the present invention.

[0021] Figure 3 It is a verification diagram of the alkaline phosphatase-like activity of the white nanozyme (Zr-CPNP@CTAB nanozyme) provided by an example of the present invention.

[0022] Figure 4 It is a diagram of the inhibitory effect of emamectin benzoate (EMB) on alkaline phosphatase-like activity provided by an example of the present invention.

[0023] Figure 5 It is a diagram of the whitening effect of the white nanozyme with alkaline phosphatase-like activity (Zr-CPNP@CTAB nanozyme) on the test strip provided by an example of the present invention.

[0024] Figure 6 It is a schematic diagram of the detection of emamectin benzoate (EMB) based on the white nanozyme test strip provided by an example of the present invention.

[0025] Figure 7 It is a diagram of the quantitative and qualitative detection of emamectin benzoate (EMB) provided by an example of the present invention.

[0026] Figure 8 It is a diagram of the selectivity of the detection of emamectin benzoate (EMB) provided by an example of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0028] (1) A white nanozyme with alkaline phosphatase-like activity disclosed by the present invention Figure 1 is the morphology of the white nanozyme (Zr-CPNP@CTAB nanozyme) observed by transmission electron microscopy; Figure 2X-ray diffraction spectrum and X-ray photoelectron spectrum of the white nanozyme.

[0029] Example 1. The preparation method of the white nanozyme with alkaline phosphatase-like activity is as follows: Add 1.5 mg of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, 2 mg of anhydrous zirconium tetrachloride, 100 μL of acetic acid, 0.5 mL of N,N-dimethylformamide, and 2 mg of cetyltrimethylammonium bromide into a glass bottle, ultrasonically dissolve for 5 min, and place the mixed solution in an oven at 90 °C for 17 h. After cooling to room temperature, centrifuge to collect the product, and wash it several times with N,N-dimethylformamide and methanol respectively. Finally, obtain the powder of the white nanozyme with alkaline phosphatase-like activity (Zr-CPNP@CTAB nanozyme) by vacuum drying.

[0030] Example 2. The preparation method of the white nanozyme with alkaline phosphatase-like activity is as follows: Add 3.5 mg of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, 4 mg of anhydrous zirconium tetrachloride, 300 μL of acetic acid, 1.5 mL of N,N-dimethylformamide, and 4 mg of cetyltrimethylammonium bromide into a glass bottle, ultrasonically dissolve for 5 min, and place the mixed solution in an oven at 90 °C for 17 h. After cooling to room temperature, centrifuge to collect the product, and wash it several times with N,N-dimethylformamide and methanol respectively. Finally, obtain the powder of the white nanozyme with alkaline phosphatase-like activity (Zr-CPNP@CTAB nanozyme) by vacuum drying method.

[0031] (II) Analysis of the alkaline phosphatase-like activity of the white nanozyme: The alkaline phosphatase-like activity of the white nanozyme was studied using sodium p-nitrophenyl phosphate as the substrate. Usually, a 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution (0.1 mol / L, pH 8.0) containing sodium p-nitrophenyl phosphate (7 mmol / L) and the white nanozyme (Zr-CPNP@CTAB nanozyme) (0.5 mg / mL) was reacted at room temperature for 10 min, and the characteristic absorbance value of the hydrolysis product p-nitrophenol at 410 nm was monitored using a microplate reader.

[0032] As Figure 3 shown in a, the white Zr-CPNP@CTAB nanozyme can effectively catalyze the hydrolysis of sodium p-nitrophenyl phosphate and has obvious alkaline phosphatase-like activity; the other control groups have no obvious activity. In addition, the alkaline phosphatase-like activity of the white nanozyme (Zr-CPNP@CTAB nanozyme) depends on its concentration ( Figure 3 b), indicating that the white nanozyme (Zr-CPNP@CTAB nanozyme) has intrinsic alkaline phosphatase-like activity.

[0033] (III) Inhibitory effect of EMB on the mimetic alkaline phosphatase activity of white nanozyme: Experimental system a: The catalytic reaction system is 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution (pH 8.0, 0.1 mol / L) including the white nanozyme (Zr-CPNP@CTAB nanozyme) (0.5 mg / mL) obtained in the above example and the chromogenic agent p-nitrophenyl phosphate (7 mmol / L), and emamectin benzoate (EMB) with different concentrations. Another experimental system b is set up: The catalytic reaction system is 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution (pH 8.0, 0.1 mmol / L) of the white nanozyme (Zr-CPNP@CTAB nanozyme) (0.5 mg / mL) obtained in Example 1 above, the chromogenic agent p-nitrophenyl phosphate (7 mmol / L), and different kinds of pesticides (0.5 mM). React at room temperature for 10 min, and simultaneously monitor the absorbance value at 410 nm using an enzyme-linked immunosorbent assay reader.

[0034] As Figure 4 shown in a, as the concentration of EMB increases, the absorbance value at 410 nm decreases, indicating that EMB has an inhibitory effect on the mimetic alkaline phosphatase activity of the nanozyme, and the greater the concentration, the more obvious the inhibitory effect. As Figure 4 shown in b, only EMB shows a significant inhibitory effect on the mimetic alkaline phosphatase activity of the white nanozyme (Zr-CPNP@CTAB nanozyme), while other pesticides do not show obvious inhibitory effects, indicating that EMB has high selectivity for the mimetic alkaline phosphatase activity of the white nanozyme. Figure 4 c is the chemical structural formula of the pesticides selected during the experiment.

[0035] (IV) Application of a white nanozyme with mimetic alkaline phosphatase activity in paper-based detection. The white nanozyme is fixed on a test paper strip to prepare a white nanozyme detection test paper, and the paper-based detection of emamectin benzoate is realized by using the white nanozyme detection test paper.

[0036] Example 3. The preparation method of the white nanozyme detection test paper is specifically as follows: The white nanozyme with mimetic alkaline phosphatase activity is dissolved in water to form a solution with a concentration of 10 mg / mL. Take 2 μL of the white nanozyme solution and drop it on the surface of the paper substrate, then vacuum dry it at 37 °C and store it in a 4 °C refrigerator for later use.

[0037] Example 4. The preparation method of the white nanozyme detection test paper is specifically as follows: The white nanozyme with mimetic alkaline phosphatase activity is dissolved in water to form a solution with a concentration of 2 mg / mL. Take 50 μL of the white nanozyme solution and drop it on the surface of the paper substrate, then vacuum dry it at 37 °C and store it in a 4 °C refrigerator for later use.

[0038] Example 5, Whitening effect of white nanozyme on test strips: Experimental system a: The white nanozyme (Zr-CPNP@CTAB nanozyme) (5 mg / mL) obtained in the above example was added to the test strip; control system b: blank test strip. It can be Figure 5 seen that the gray value of the test strip added with the white nanozyme (Zr-CPNP@CTAB nanozyme) decreased significantly, and the test strip showed obvious white. It shows that the white nanozyme synthesized in the above example not only has no interference with the color development of this experiment, but also can make the color development of the test strip clearer.

[0039] Example 6, Quantitative and qualitative detection of emamectin benzoate: The white nanozyme (Zr-CPNP@CTAB nanozyme) has alkaline phosphatase mimicking activity, and emamectin benzoate can selectively inhibit the alkaline phosphatase mimicking activity of this nanozyme, so as to realize the quantitative and qualitative detection of emamectin benzoate. Figure 6 is a schematic diagram of the principle of using a test strip of white nanozyme (Zr-CPNP@CTAB nanozyme) to detect emamectin benzoate (EMB). The catalytic reaction system is incubated for 5 min with white nanozyme (Zr-CPNP@CTAB nanozyme) (5 mg / mL) and different concentrations of emamectin benzoate (EMB) (0−0.08 mmol / L), and then the 2-hydroxyethylpiperazine-N'-ethanesulfonic acid buffer solution (0.1 mol / L, pH 8.0) containing p-nitrophenyl phosphate ( p -NPP) (1.5 mmol / L) and molybdate (AM)-malachite green (MG) solution were added to the white nanozyme test strip in sequence. By using a smartphone to analyze the color development image of the white nanozyme test strip, and calculating the color (RGB) value of the color development image of the white nanozyme test strip by the following formula: color value = G / (R+G+B), where R, G, and B represent the red, green, and blue component values of each pixel point in the image respectively. Subsequently, the calculated color value was plotted against the emamectin benzoate concentration for subsequent emamectin benzoate detection and analysis. As Figure 7 shown, by observing the color change, it was found that the greater the concentration of emamectin benzoate, the lighter the green of the white nanozyme test strip, and its color (RGB) value conforms to the linear regression equation y = -0.00315x + 0.5374 (R 2 = 0.999), from which the qualitative and quantitative detection of emamectin benzoate can be realized.

[0040] A paper-based method for detecting emamectin benzoate using a white nanozyme with mimetic alkaline phosphatase activity is as follows: Solutions of emamectin benzoate at different concentrations (2−7 μL) were dropped onto the surface of the white nanozyme (Zr-CPNP@CTAB nanozyme) test strip and incubated at room temperature for 5 min. Then, 2−7 μL of 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer (0.1 mol / L, pH 8.0) containing 1.5 mmol / L of p-nitrophenyl phosphate and 2−7 μL of molybdate-malachite green solution were successively dropped onto the surface of the white nanozyme test strip. After reacting for 20 min, the color development image of the white nanozyme test strip was collected by a smartphone, and the color (RGB) value of the color development image of the white nanozyme test strip was calculated using the following formula: Color value = G / (R+G+B), where R, G, and B represent the component values of red, green, and blue of each pixel point in the image, respectively. Subsequently, the calculated color value was plotted against the concentration of emamectin benzoate EMB to be determined for subsequent detection and analysis of emamectin benzoate. Finally, qualitative detection was achieved by observing the color change of the white nanozyme test strip, and quantitative detection was achieved by collecting the color (RGB) value with a mobile phone.

[0041] Example 7, Selectivity analysis of emamectin benzoate detection: Solutions of different types of pesticides were dropped onto the white nanozyme test strip and incubated for 5 min. Then, 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer (0.1 mol / L, pH 8.0) containing 1.5 mmol / L of p-nitrophenyl phosphate and molybdate-malachite green solution were successively added to the white nanozyme test strip. After incubating at 25 °C for 20 min, the color development image of the white nanozyme test strip was collected by a mobile phone, and the color (RGB) value of the color development image of the white nanozyme test strip was calculated using the following formula: Color value = G / (R+G+B), where R, G, and B represent the component values of red, green, and blue of each pixel point in the image, respectively. Subsequently, the calculated color value was plotted against the concentration of emamectin benzoate EMB to be determined for subsequent selectivity analysis of emamectin benzoate detection. As Figure 8 can be seen, the color (RGB) value of the white nanozyme test strip with emamectin benzoate (EMB) added was the lowest and was significantly different from the control group and other experimental groups, indicating that emamectin benzoate has good inhibitory selectivity for the mimetic alkaline phosphatase activity of this white nanozyme.

[0042] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. Preparation method of white nanozyme with alkaline phosphatase-like activity, characterized in that, It includes the following steps: Add a certain amount of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, anhydrous zirconium tetrachloride, acetic acid, N,N-dimethylformamide, and cetyltrimethylammonium bromide into a glass bottle, ultrasonically dissolve at room temperature for a period of time, put the glass bottle containing the mixed solution into an oven, and heat at a certain temperature for a period of time; After cooling to room temperature, collect the product by centrifugation at a certain rotational speed for a period of time, and wash it several times with N,N-dimethylformamide and methanol respectively; Finally, obtain a white nanozyme powder with alkaline phosphatase mimicking activity by vacuum drying, and store it in a refrigerator for later use.

2. The white nanozyme with alkaline phosphatase-like activity according to claim 1, characterized in that, Add 1.5−3.5 mg of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, 2−4 mg of anhydrous zirconium tetrachloride, 100−300 μL of acetic acid, 0.5−1.5 mL of N,N-dimethylformamide, and 2−4 mg of cetyltrimethylammonium bromide into a glass bottle, ultrasonically dissolve for 2−10 min, put it into an oven at 80−100 °C and heat for 16−18 h; after cooling the product to room temperature, centrifuge at a rotational speed of 5000−8000 rpm for 2−10 min, then wash the product 2−3 times with N,N-dimethylformamide and methanol respectively, vacuum dry at 25−37 °C for 4−8 h, and finally store the obtained white nanozyme powder in a 4 °C refrigerator for later use.

3. A white nanozyme with alkaline phosphatase mimicking activity obtained by the preparation method according to claim 1 or 2.

4. Use of a white nanozyme with alkaline phosphatase-like activity as described in claim 3 in paper-based detection, characterized in that, The described white nanozyme is immobilized on a test paper strip to prepare a white nanozyme detection test paper, and the paper-based detection of emamectin benzoate is realized by using the white nanozyme detection test paper.

5. Use of the white nanozyme with alkaline phosphatase-like activity according to claim 4 in paper-based detection, characterized in that, The preparation method of the described white nanozyme detection test paper is specifically as follows: Prepare a solution of the white nanozyme with a concentration of 2−10 mg / mL by adding water, take 2−50 μL of the white nanozyme solution and drop it on the surface of the paper substrate, then vacuum dry at 25−37 °C, and store it in a 4 °C refrigerator for later use.

6. Use of the white nanozyme with alkaline phosphatase-like activity according to claim 5 in paper-based detection, characterized in that, The paper substrate material of the described test paper strip is qualitative filter paper, quantitative filter paper, blotting paper, and nitrocellulose membrane, and the diameter of the test paper strip is 4−15 mm.

7. Use of the white nanozyme with alkaline phosphatase-like activity according to any one of claims 4-6 in paper-based detection, characterized in that, The process of realizing the paper-based detection of emamectin benzoate by using the described white nanozyme detection test paper is as follows: Drop a certain amount of emamectin benzoate sample solution on the white nanozyme detection test paper, incubate at room temperature, then sequentially drop a certain amount of 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer solution containing p-nitrophenyl phosphate and molybdate-malachite green solution, after reacting for a period of time, identify the color value of the color development of the white nanozyme detection test paper by a smart phone, and perform quantitative analysis on emamectin benzoate.

8. Use of the white nanozyme with alkaline phosphatase-like activity according to claim 7 in paper-based detection, characterized in that, Add 2−50 μL of emamectin benzoate sample solution to the white nanozyme test paper. After incubation at room temperature, sequentially add 2−50 μL of 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer containing sodium p-nitrophenyl phosphate and 2−50 μL of molybdate-malachite green solution. After reacting for 20 min, identify the color value of the white nanozyme test paper color development through a smartphone to perform quantitative analysis of emamectin benzoate.

9. Use of the white nanozyme with alkaline phosphatase-like activity according to claim 8 in paper-based detection, characterized in that, The concentration of sodium p-nitrophenyl phosphate is 0.5−3 mmol / L. In the molybdate-malachite green solution, the concentration of molybdate is 1−2 mg / mL, and the concentration of malachite green is 0.5−1.5 mg / mL.

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