A spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme and its preparation method and application

Abstract

The present invention relates to the field of nano-enzyme-mimicking catalytic materials, and specifically provides a spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme and a preparation method and application thereof. The material has good nano-mimetic oxidase activity and simulated peroxidase activity, and can play a bifunctional nano-enzyme role. The material is guided by a positive hexadecyltrimethylammonium bromide (CTAB) template, and a copper-ruthenium bimetallic-based nano-mimetic enzyme (CTAB@Cu-Ru) is prepared by a "one-pot reduction method". The copper-ruthenium metals interact strongly with each other, further promoting charge transfer between each other, and synergistically enhancing the catalytic activity of the CTAB@Cu-Ru simulated oxidase / peroxidase. In detection applications, the CTAB@Cu-Ru nano-mimetic enzyme has excellent specificity and selectivity for o-aminophenol and m-aminophenol, good anti-interference, and is convenient and fast, highly sensitive, accurate in detection results, and highly visible, and can effectively detect ultra-trace o-AP and m-AP in environmental samples.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials, and specifically relates to a spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme and a preparation method and application thereof. Background Art

[0002] o-Aminophenol and m-aminophenol are two important pollutants. This compound degrades very slowly in the environment and is easily absorbed by the skin or mucous tissue, which can cause methemoglobinemia and asthma. At present, common methods for detecting aminophenol isomers (o-aminophenol, m-aminophenol, p-aminophenol) include high performance liquid chromatography, fluorescence spectroscopy, capillary electrophoresis, gas chromatography, electrochemistry, microfluidic devices, etc. Although effective detection effects can be achieved, the required instruments are expensive, sample pretreatment is relatively cumbersome, and it is easily interfered by the coexistence of aminophenol isomers; traditional colorimetric analysis is convenient and fast, but the detection limit is low and the selectivity is poor, which cannot meet the high sensitivity and selective colorimetric detection of o-aminophenol, m-aminophenol, and p-aminophenol. Therefore, it is of great significance to develop a convenient, efficient and sensitive colorimetric detection method for catalytic signal amplification of o-aminophenol and m-aminophenol.

[0003] As a highly efficient bionic catalyst, nano-mimetic enzymes are low-cost, highly stable, and can catalyze various reactions under mild conditions. They have high substrate specificity, selectivity, and catalytic efficiency. They have been widely used in anti-cancer, photodynamic therapy, bionic catalysis, biosensing, and environmental analysis and detection. Summary of the invention

[0004] In order to deal with the environmental pollution caused by o-aminophenol, m-aminophenol and p-aminophenol, and solve the defects of expensive conventional instrument analysis, complex sample processing, low sensitivity and poor selectivity of traditional colorimetric detection, the purpose of the present invention is to provide a spindle-shaped Cu-Ru bimetallic mesoporous nanomimetic enzyme and its preparation method, as well as the application of the nanomimetic enzyme in catalytic visual detection of o-aminophenol and m-aminophenol. Based on the good synergistic enhancement between copper and ruthenium bimetallic, a spindle-shaped Cu-Ru bimetallic nanomimetic enzyme (CTAB@Cu-Ru) was successfully prepared by "one-pot reduction" assisted by surfactant hexadecyltrimethylammonium bromide (CTAB); through research, CTAB@Cu-Ru mimetic enzyme was applied to the simultaneous colorimetric detection of o-aminophenol (o-AP) and m-aminophenol (m-AP), with strong visibility, simple operation, good selectivity and high sensitivity.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a spindle-shaped Cu-Ru bimetallic mesoporous nanomimetic enzyme (CTAB@Cu-Ru), comprising Cu nanoparticles and Ru nanoparticles, wherein the nano Cu and Ru are connected via metal bonds.

[0007] Furthermore, during the formation of the nano-mimetic enzyme, the CTAB solution easily forms a double atomic layer micelle, the surface of which reacts with the hydrolysis product OH of the reducing agent. - After ion exchange, the OH - Based on the strong electrostatic interaction and Cu 2+ 、Ru 3 + The nanostructured materials are combined, rapidly reduced by a reducing agent, and assembled on CTAB to finally obtain a spin-cone-shaped nanomaterial.

[0008] Furthermore, the particle size of the nano-mimetic enzyme is between 150 and 250 nm; the particle size of the nano-mimetic enzyme is uniform, the dispersibility is good, and the stability is good.

[0009] In a second aspect of the present invention, a method for preparing a CTAB@Cu-Ru nano-mimetic enzyme is provided, comprising the following steps:

[0010] (1) Containing Cu 2+ Ion, Ru 3+ The raw material of the ion is added into the dispersion of hexadecyltrimethylammonium bromide and mixed to obtain a precursor mixed solution;

[0011] (2) adding a reducing agent to the precursor mixture, stirring at room temperature for 0.5 to 3 hours, and centrifuging to obtain a solid product, namely, the nanomimetic enzyme CTAB@Cu-Ru.

[0012] Furthermore, the Cu 2+ The ion source is selected from at least one of Cu(NO3)2, CuSO4, and CuCl2.

[0013] Furthermore, the Ru 3+ The ion source is selected from at least one of RuCl3 hydrate, ruthenium acetate, and anhydrous RuCl3.

[0014] Furthermore, the Cu 2+ Ions and Ru 3+ The molar ratio of ions is 0.1-0.4:0.02-0.06.

[0015] Furthermore, the Cu 2+ The feed ratio of Cu to hexadecyltrimethylammonium bromide is 0.1-0.4:100-300; 2+ The amount is in mol and the amount of hexadecyltrimethylammonium bromide is in mg.

[0016] Furthermore, in step (1), Cu 2+ Ion source, Ru 3+ The ion source is dispersed in a cationic hexadecyltrimethylammonium bromide solution.

[0017] Furthermore, the reducing agent in step (2) is selected from at least one of NaBH4, LiAlH4, hydrazine hydrate, and vitamin C; - Based on the strong electrostatic interaction and Cu 2+ 、Ru 3+ The nanostructured materials are combined, rapidly reduced by a reducing agent, and assembled on CTAB to finally obtain a spin-cone-shaped nanomaterial.

[0018] Furthermore, after the solid product is separated by centrifugation in step (2), it is washed by centrifugation with ethanol and water, and then the solid product is vacuum dried to obtain the target product CTAB@Cu-Ru nano-mimetic enzyme.

[0019] The third aspect of the present invention provides an application of the CTAB@Cu-Ru nano-mimetic enzyme in the fields of environmental water, biology, medicine, etc.

[0020] Furthermore, the application is to detect o-aminophenol using CTAB@Cu-Ru nano-mimetic enzyme, comprising the following steps:

[0021] S1. Add the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB), disodium hydrogen phosphate-citric acid buffer solution, and the sample solution to be tested to the CTAB@Cu-Ru standard solution to obtain a test solution;

[0022] S2. Observe the color change of the liquid to be tested.

[0023] Furthermore, the standard solution described in step S1 is a CTAB@Cu-Ru solution with a concentration of 0.1 mg / mL.

[0024] Furthermore, the amounts of 3,3',5,5'-tetramethylbenzidine (TMB) and sodium hydrogen phosphate-citric acid buffer solution added in step S1 are: 20-200 μL 1.5 mM TMB, 50-200 μL pH 3.5 sodium hydrogen phosphate-citric acid buffer solution.

[0025] Furthermore, the color change in step S2 is a visible color change from blue to colorless, which means that the sample liquid to be tested contains o-aminophenol.

[0026] Furthermore, the application is to use the CTAB@Cu-Ru nano-mimetic enzyme to detect m-aminophenol, comprising the following steps:

[0027] S3, adding the colorimetric substrate ABTS, disodium hydrogen phosphate-citrate buffer solution, H2O2, and the sample solution to be tested to the CTAB@Cu-Ru standard solution to obtain a test solution;

[0028] S4. Observe the color change of the test liquid.

[0029] Furthermore, in step S3, the standard solution is a CTAB@Cu-Ru solution with a concentration of 0.1 mg / mL.

[0030] Furthermore, in step S3, the added amounts of ABTS, sodium hydrogen phosphate-citric acid buffer solution, and H2O2 are respectively: 20-280 μL 1.5 mM ABTS, 50-500 μL 5.0 mM H2O2, and 50-200 μL pH 3.0 sodium hydrogen phosphate-citric acid buffer solution.

[0031] Furthermore, in step S4, the color change is a visible color change from blue-green to pink, which means that the sample liquid to be tested contains m-aminophenol.

[0032] Furthermore, the application is to use CTAB@Cu-Ru nano-mimetic enzyme to perform dual-mode colorimetric detection of o-aminophenol and m-aminophenol, comprising the following steps:

[0033] S1. Add the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB), disodium hydrogen phosphate-citric acid buffer solution, and the sample solution to be tested to the CTAB@Cu-Ru standard solution to obtain a test solution;

[0034] S2, observing the color change of the test liquid;

[0035] S3, adding the colorimetric substrate ABTS, disodium hydrogen phosphate-citrate buffer solution, H2O2, and the sample solution to be tested to the CTAB@Cu-Ru standard solution to obtain a test solution;

[0036] S4. Observe the color change of the test liquid.

[0037] Furthermore, the standard solution described in step S1 is a CTAB@Cu-Ru solution with a concentration of 0.1 mg / mL.

[0038] Furthermore, the amount of 3,3',5,5'-tetramethylbenzidine (TMB) and disodium hydrogen phosphate-citric acid buffer solution added in step S1 is:

[0039] Furthermore, the color change in step S2 is a visible color change from blue to colorless, which means that the sample liquid to be tested contains o-aminophenol.

[0040] Furthermore, in step S3, the standard solution is a CTAB@Cu-Ru solution with a concentration of 0.1 mg / mL.

[0041] Furthermore, in step S3, the added amounts of ABTS, disodium hydrogen phosphate-citric acid buffer solution, and H2O2 are:

[0042] Furthermore, in step S4, the color change is a visible color change from blue-green to pink, which means that the sample liquid to be tested contains m-aminophenol.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The present invention provides a spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme (CTAB@Cu-Ru), which has good nano-mimetic oxidase activity and simulated peroxidase activity, and can play a bifunctional nano-enzyme role. At room temperature and air, 3,3',5,5'-tetramethylbenzidine (TMB) can be rapidly catalyzed and oxidized to generate blue oxTMB, with a characteristic absorption peak at 653nm. CTAB@Cu-Ru-TMB has a strong visual colorimetric recognition ability for o-AP, and the characteristic absorption intensity at 653nm gradually decreases, accompanied by a visual "blue-light blue-colorless" change. At room temperature and H2O2, ABTS can be rapidly catalyzed and oxidized to generate blue-green oxABTS, with a characteristic absorption peak at 727nm. CTAB@Cu-Ru-TMB-H2O2 has a strong visual colorimetric recognition ability for m-AP, and the characteristic absorption intensity at 727nm gradually decreases, a new characteristic absorption peak appears at 513nm, and the absorption intensity gradually increases, accompanied by a visual "blue-pink purple-pink" change. It is convenient and efficient to visually detect ultra-trace o-AP and m-AP in environmental water, commercial fruit juice drinks, etc., with high sensitivity and accurate detection results.

[0045] (2) The present invention provides a method for preparing the CTAB@Cu-Ru nanomimetic enzyme, specifically, under the guidance of a positive hexadecyltrimethylammonium bromide (CTAB) template, a copper-ruthenium bimetallic nanomimetic enzyme (CTAB@Cu-Ru) is prepared by a one-pot "reduction method", wherein: the empty d orbitals on the Cu and Ru atoms and the electron-rich coordination bonds of CTAB are loaded on the CTAB template, and since the CTAB template is soluble, it can be easily removed during the water centrifugal washing process after synthesis, and the CTAB template can form a bi-micelle surface, which is bonded to Cu by strong electrostatic interaction. 2+ 、Ru 3+Combined to avoid the formation of irregular nanoparticles. At the same time, the interaction between copper and ruthenium metals further promotes the charge transfer between them and synergistically enhances the catalytic activity of CTAB@Cu-Ru simulated oxidase / peroxidase.

[0046] (3) The present application provides an application of the CTAB@Cu-Ru nanomimetic enzyme, which exhibits excellent specificity and selectivity for o-aminophenol and m-aminophenol in detection samples such as environmental water and commercial fruit juice beverages; the CTAB@Cu-Ru nanomimetic enzyme has good anti-interference ability, and is convenient and fast in detection applications, has high sensitivity, and accurate detection results, and can effectively detect ultra-trace o-AP and m-AP in samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 .TEM image of CTAB@Cu-Ru nano-mimetic enzyme prepared in Example 1.

[0048] Figure 2 .Elemental analysis EDS (Figure a) and HR-TEM (Figure b) of the CTAB@Cu-Ru nano-mimetic enzyme prepared in Example 1.

[0049] Figure 3 .XPS spectrum (Figure a) and XRD spectrum (Figure b) of the CTAB@Cu-Ru nano-mimetic enzyme prepared in Example 1.

[0050] Figure 4 . The CTAB@Cu-Ru nano-mimetic enzyme prepared in Example 1 optimizes the amount of surfactant (Figure a), the ratio of Cu and Ru (Figure b); the enzyme activity curve of the CTAB@Cu-Ru nano-mimetic enzyme under different pH systems (Figure c), TMB concentrations (Figure d), and reaction times (Figure e).

[0051] Figure 5 .The enzymatic activity curve of the CTAB@Cu-Ru nano-mimetic enzyme prepared in Example 1 under different system pH (Figure a), ABTS concentration (Figure b), H2O2 concentration (Figure c), and reaction time (Figure d).

[0052] Figure 6 .The kinetic curve of the CTAB@Cu-Ru nanomimetic enzyme prepared in Example 1 when the substrate is TMB (Figure a); Figure b is a double reciprocal curve graph.

[0053] Figure 7 .The kinetic curves of the CTAB@Cu-Ru nanomimetic enzyme prepared in Example 1 as the concentration of ABTS and H2O2 change (Figures a, c); Figure b is a double reciprocal curve diagram of the change of ABTS concentration, and Figure d is a double reciprocal curve diagram of the change of H2O2 concentration.

[0054] Figure 8 .Figure a shows the effect of different interfering ions on the spectral performance of the CTAB@Cu-Ru nanomimetic enzyme prepared in Example 1 (illustration: color comparison chart); Figure b shows the effect of the coexistence of interfering ions and o-AP on the spectral performance of the CTAB@Cu-Ru nanomimetic enzyme prepared in Example 1.

[0055] Fig. 9 .Figure a shows the effect of different interfering ions on the spectral properties of the CTAB@Cu-Ru nano-enzyme prepared in Example 1 (illustration: color comparison chart); Figure b shows the effect of the coexistence of interfering ions and m-AP on the spectral properties of the CTAB@Cu-Ru nano-enzyme prepared in Example 1.

[0056] Fig.10 Figure a is the spectral titration curve of colorimetric sensor o-AP (inset: color change picture; Figure b is the absorbance intensity (A 653 ) and o-aminophenol concentration (c o-AP ) between them.

[0057] Fig.11 Figure a is the spectral titration curve of colorimetric sensor m-AP (inset: color change picture); Figure b is lg (A 513 / A 727 ) or lg(A 513 / A 415 ) and m-aminophenol concentration (c m-AP ) between them. DETAILED DESCRIPTION

[0058] The present application will be further described in detail below by way of example so that those skilled in the art can practice the present application. It should be understood that other embodiments may be adopted, and appropriate changes may be made without departing from the spirit or scope of the present application. In order to avoid unnecessary details for enabling those skilled in the art to practice the present application, the description may omit certain information known to those skilled in the art. Therefore, the following detailed description should not be understood in a restrictive sense, and the scope of the present invention is defined only by the appended claims. The following examples are convenient for a better understanding of the present application, but are not intended to limit the scope of the present application.

[0059] Embodiment 1

[0060] (1) Accurately weigh 0.1180 g (0.4 mmol) Cu(NO3)2·3H2O and 0.0108 g (0.05 mmol) anhydrous RuCl3 and dissolve them in 25 mL 10 mg / mL CTAB dispersion and stir to obtain a precursor mixture;

[0061] (2) Add 5.0 mL (0.2 M) of freshly prepared NaBH4 solution to the precursor mixture obtained in step (1), stir for 1 h, centrifuge, and then wash three times with ethanol and water by centrifugation (speed of 7000 rpm) to remove the template CTAB, and then vacuum dry at 60°C to obtain CTAB@Cu-Ru nanoenzyme.

[0062] Embodiment 2

[0063] (1) Accurately weigh 0.0635 g (0.4 mmol) CuSO4 and 0.0139 g (0.05 mmol) ruthenium acetate and dissolve them in 25 mL 10 mg / mL CTAB dispersion, and stir to obtain a precursor mixture;

[0064] (2) Add 5.0 mL of freshly prepared hydrazine hydrate solution (0.2 M) to the precursor mixture obtained in step (1), stir for 1 h, wash with ethanol and water three times by centrifugation (speed of 7000 rpm) to remove the template CTAB, and then vacuum dry the solid product obtained by centrifugation at 60°C to obtain CTAB@Cu-Ru nano-enzyme.

[0065] Embodiment 3

[0066] (1) Accurately weigh 0.0682 g (0.4 mmol) of CuCl2·2H2O and 0.0139 g (0.05 mmol) of ruthenium acetate and dissolve them in 25 mL of 10 mg / mL CTAB dispersion and stir to obtain a precursor mixture;

[0067] (2) Add 5.0 mL of 0.2 M LiAlH4 to the precursor mixture obtained in step (1) and stir for 1 h. Wash with ethanol and water by centrifugation (speed of 7000 rpm) for 3 times to remove the template CTAB. Then, vacuum dry the solid product obtained by centrifugation at 60°C to obtain CTAB@Cu-Ru nano-enzyme.

[0068] Performance characterization and testing:

[0069] 1. The spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme prepared in Example 1 was observed and tested by transmission electron microscopy. Figures 1 to 3 shown. Figure 1 The microscopic image of the CTAB@Cu-Ru under a transmission electron microscope shows that the prepared CTAB@Cu-Ru nano-mimetic enzyme has a multi-conical structure and is evenly dispersed with a particle size of about 150 to 250 nm. It can provide a larger surface area and more active sites, thereby improving the detection capability of organic aminophenols. Figure 2b shows three distinct lattice spacings of 0.234nm, 0.255nm and 0.263nm, which belong to the (111) and (002) crystal planes of Cu and the lattice of the copper-ruthenium alloy, respectively. Figure 3 In a, it can be seen that the XPS spectrum contains all the elements of Cu, Ru, and O; Figure 3 b It can be seen that the characteristic crystal diffraction signals of Cu (2θ=32.7°, 35.9°, 38.7°, 48.8°, 54.1°, 58.2°, 61.5°, 66.7° and 68.1°) and the characteristic crystal diffraction signals of Ru (2θ=26.7°, 35.4°, 42.5°, 53.4° and 57.5°).

[0070] 2. Test of the detection ability of the spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme of Example 1 for o-AP and m-AP:

[0071] (1) Preparation of 0.1 mg / mL CTAB@Cu-Ru standard solution: Weigh 0.01 g of the CTAB@Cu-Ru sample and disperse it into 100 mL of pure water under ultrasonic conditions to prepare a 0.1 mg / mL CTAB@Cu-Ru standard solution, which is then stored at room temperature away from light for later use.

[0072] (2) Preparation of o-AP standard solution: Add 0.01091 g of o-aminophenol to a 100 mL volumetric flask. After it is completely dissolved, dilute to 100 mL with ethanol to prepare a solution with a concentration of 1.0 × 10 -3 mol / L, store at room temperature for future use.

[0073] Preparation of m-AP standard solution: Add 0.01091 g of o-aminophenol to a 100 mL volumetric flask. After it is completely dissolved, dilute to 100 mL with ethanol to prepare a solution with a concentration of 1.0 × 10 -3 mol / L, store at room temperature for future use.

[0074] (3) Preparation of actual samples to be tested:

[0075] (i) Randomly measure 50.0 mL of environmental water samples (such as water from the Yi River, Liao River, and apartment tap water, etc.), filter them three times through a 4 μm microporous filter membrane, and distill and concentrate them to 10.0 mL. Store the samples in a refrigerator (4°C) for later use.

[0076] (ii) Weigh 50.0 mg of Gankang tablets, dissolve in 50.0 mL of pH 3.5 solution, filter through a 4 μm microporous filter membrane three times, and store the sample in a refrigerator (4° C.) for later use.

[0077] (4) Test of CTAB@Cu-Ru simulated oxidase catalytic activity: 500 μL (0.1 mg / mL) CTAB@Cu-Ru standard solution and 160 μL (1.5 mM) TMB were placed in a 5 mL biological centrifuge tube, mixed thoroughly, and diluted to 3.0 mL with sodium hydrogen phosphate-citric acid buffer solution (pH = 3.5). The mixture was aged at room temperature for 45 min and the UV-visible absorption spectrum was measured in the range of 300 to 800 nm. The system had a strong absorption peak at 653 nm (A 653 =0.532), the color of the solution changed from colorless to blue.

[0078] (5) Test of CTAB@Cu-Ru simulated peroxidase catalytic activity: 200 μL of the CTAB@Cu-Ru standard solution, 220 μL (1.5 mM) ABTS and 400 μL (5.0 mM) H2O2 were measured and placed in a 5 mL biological centrifuge tube. The mixture was thoroughly mixed and made up to 3.0 mL with disodium hydrogen phosphate-citric acid buffer solution (pH = 3.0). The mixture was aged at room temperature for 75 min and the UV-visible absorption spectrum in the range of 300 to 800 nm was measured. The system had two strong absorption peaks at 415 nm and 727 nm (A 415 =2.01, A 727 =0.83), accompanied by a color change from “colorless to blue-green”.

[0079] (6) Optimization of enzyme catalysis experimental conditions: In order to obtain the best catalytic activity of CTAB@Cu-Ru nanoenzyme, we first took TMB colorimetric substrate as an example to explore the effects of surfactant dosage and molar ratio of material components; on this basis, we explored the effects of system pH, reaction temperature, TMB concentration, ABTS concentration, H2O2 concentration and reaction time. That is, according to the methods of (4) and (5) above, we studied the effects of pH (2.5-7.0), Cu:Ru molar ratio (20:1-7:1), reaction temperature (20-90°C), TMB content (0.01-0.15mM), ABTS content (0.01-0.14mM) or H2O2 (0.083-0.83mM) on the catalytic activity of CTAB@Cu-Ru nanoenzyme.

[0080] Depend on Figure 4 It can be seen that the optimized conditions of the CTAB@Cu-Ru simulated oxidase are: pH = 3.5, 0.08 mM TMB, aging at 30 ° C for 45 min; Figure 5The optimized conditions for the simulated peroxidase are: pH = 3.0, 0.11 mM ABTS, 0.67 mM H2O2, and aging at 30°C for 75 min. That is, the CTAB@Cu-Ru nano-mimetic enzyme has good nano-simulated oxidase activity or simulated peroxidase activity.

[0081] from Figure 6 and Figure 7 It can be seen that the Michaelis constant Km of the CTAB@Cu-Ru nanoenzyme catalyzing TMB is 0.0072mM, and the maximum starting rate Vmax is 6.33×10 -8 M.s -1 The Michaelis constants (Km) of CTAB@Cu-Ru nanoenzyme catalyzing ABTS and H2O2 were 0.017mM and 3.81mM, respectively, and the maximum initial rate (Vmax) was 6.47×10 -8 M.s -1 and 8.23×10 -8 M.s -1 ; This means that the CTAB@Cu-Ru nanoenzyme has good enzyme-like catalytic activity and catalytic rate, and can quickly catalyze the reaction of substrates such as TMB, ABTS and H2O2 to completion, thereby achieving the detection of the target analyte.

[0082] (7) Figure 8 and Fig. 9 The activity selectivity of CTAB@Cu-Ru nanoenzyme was studied by using common interfering ions. Figure 8 and Fig. 9 It can be seen that the common coexisting ions in the test samples are: Ba 2+ Mg 2+ , K + , Hg 2+ When o-AP coexists with phenylalanine, glycine, threonine, alanine, glucose, aniline, urea, phenol, acetone, m-aminophenol and p-aminophenol, the absorption intensity change value of CTAB@Cu-Ru-TMB system at 653nm ( Figure 8 a), and the absorption intensity change value of CTAB@Cu-Ru-TMB-H2O2 system at 727nm when coexisting with m-AP ( Fig. 9a), all within the analytical error range of 5%. The CTAB@Cu-Ru-TMB system has a specific and selective colorimetric response to o-AP, accompanied by a color change from blue to colorless; the CTAB@Cu-Ru-TMB-H2O2 system has a colorimetric response with good visibility to m-AP, accompanied by a visible "blue-green-pink-purple" change, so that the CTAB@Cu-Ru nano-mimetic enzyme can be used to detect o-AP and m-AP in the sample quickly and efficiently with the naked eye without the aid of other observation instruments.

[0083] (8) Enzyme-catalyzed colorimetric detection of o-AP content: 500 μL (0.1 mg / mL) of the CTAB@Cu-Ru standard solution was placed in a 5 mL biological centrifuge tube, 160 μL (1.5 mM) TMB and 200 μL of sodium dihydrogen phosphate-citric acid buffer solution at pH = 3.5 were added, mixed evenly, and the volume was adjusted to 2.8 mL with three times distilled water. The solution was placed at room temperature for 45 min, 200 μL of o-AP standard solution of different concentrations was added, and the solution was placed for 3 min. The UV-vis absorption spectrum and color change in the range of 300 to 800 nm were measured, and A was found. 653 The linear relationship between the concentration of o-AP (co-AP) and Fig.10 As shown in Figure 2, as the o-AP concentration gradually increases (the curves from top to bottom represent the o-AP concentrations of 0, 0.33, 0.67, 1.00, 1.67, 2.67, 4.3, 5.00, 5.67, 6.67, 8.00, 9.33, 10.67, and 11.67 μM), the absorption intensity (A) of the CTAB@Cu-Ru-TMB system at 653 nm increases. 653 ) gradually decreases, and its color gradually changes from blue to colorless, ranging from 0 to 11.67×10 -6 There is a good linear relationship in the range of mol / L, and the linear regression equation is: 653 =0.52776-0.02955c o-Ap (R 2 =0.9981), the detection limit is 1.60×10 -8 mol / L(S / N=3).

[0084] (9) Enzyme-catalyzed colorimetric detection of m-AP content: Take 200 μL (0.1 mg / mL) of the CTAB@Cu-Ru standard solution in a 5 mL biological centrifuge tube, add 220 μL (1.5 mM) ABTS, 200 μL of sodium dihydrogen phosphate-citric acid buffer solution with pH = 3.5 and 400 μL H2O2 (5.0 mM) and mix well, and dilute to 2.8 mL with three times distilled water, let stand at room temperature for 75 min, add 200 μL of m-AP standard solution of different concentrations, let stand for 30 min, and measure the UV-vis absorption spectrum and color change in the range of 300 to 800 nm. Fig.11 As shown in Figure 2, a new characteristic peak appears in the CTAB@Cu-Ru-ABTS-H2O2 system at 513 nm. As the concentration of m-AP increases gradually (the curves from top to bottom represent the concentrations of o-AP: 0, 0.667, 1.33, 2, 2.67, 3.33, 5, 6.67, 8.33, 10, 11.67, 13.33, 15, 16.67, 18.33, 21.67 μM), the absorption intensity at 513 nm (A 513 ) gradually increased, accompanied by a color change of "blue-green-pink-purple", and lg(A 513 / A 727 ) or lg(A 513 / A 415 ) and m-aminophenol concentration (c m-AP ) in the range of 0 to 21.67×10 -6 There is a good multi-ratio linear relationship in the mol / L range. The linear regression equation is lg(A 513 / A 727 )=0.07187c m-AP -0.60598(R 2 =0.9979) or lg(A 513 / A 415 )=0.06315c m-AP -0.97034(R 2 =0.9987). The detection limit is 3.25×10 -8 mol / L(S / N=3).

[0085] (10) According to the above methods (8) and (9) for enzyme-catalyzed colorimetric detection of o-AP and m-AP content, colorimetric detection of trace amounts of o-AP and m-AP in environmental water, commercial fruit juice beverages and pharmaceutical samples was performed. The results are shown in Tables 1 and 2.

[0086] Table 1 Detection results of trace o-AP in samples (n=5) a

[0087]

[0088] a PB, pH = 3.5, c CTAB@Cu-Ru-TMB =0.1mg / mL

[0089] Table 2 Detection results of trace m-AP in samples (n=5) a

[0090]

[0091]

[0092] a PB, pH = 3.5, c CTAB@Cu-Ru-TMB =0.1mg / mL

[0093] As can be seen from Table 1, the recovery rate of o-AP in the sample is between 97.2% and 103.5%, and the relative error (RSD) is less than 4.58%; as can be seen from Table 2, the recovery rate of m-AP in the sample is between 96.6% and 102.27%, and the relative error (RSD) is less than 3.34%. This shows that the CTAB@Cu-Ru nanomimetic enzyme described above is convenient, fast, highly sensitive, and has accurate detection results when applied to trace o-AP and m-AP in environmental water, commercial fruit juice beverages, and pharmaceutical samples, and can effectively detect ultra-trace o-AP and m-AP in samples.

[0094] The above description only illustrates several embodiments of the present invention, and it cannot be understood as limiting the scope of the present invention. It should be pointed out that other persons in the art may make modifications, substitutions, improvements, etc. without departing from the concept and scope of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of the present invention shall be based on the described claims.

Claims

1. A spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme, characterized in that: The invention comprises Cu nanoparticles and Ru nanoparticles, wherein the nano Cu and Ru are connected through metal bonds.

2. The spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme according to claim 1, characterized in that: The particle size of the nano-mimetic enzyme is between 150 and 250 nm.

3. A method for preparing a spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme, characterized in that: The steps include: (1) Containing Cu 2+ 、Ru 3+ The raw materials are added into the dispersion of hexadecyltrimethylammonium bromide to obtain a precursor mixed solution; (2) Adding a reducing agent to the precursor mixture, washing and separating the solid product after the reaction is completed, thereby obtaining the nano-mimetic enzyme.

4. The preparation method according to claim 3, characterized in that: The Cu 2+ The raw material is selected from at least one of Cu(NO3)2, CuSO4, and CuCl2.

5. The preparation method according to claim 3, characterized in that: The Ru 3+ The raw material is selected from at least one of RuCl3 hydrate, ruthenium acetate, and anhydrous RuCl3.

6. The preparation method according to claim 3, characterized in that: The Cu in step (1) 2+ With Ru 3+ The molar ratio is 0.1~0.4:0.02~0.

06.

7. The preparation method according to claim 3, characterized in that: The Cu in step (1) 2+ The feed ratio of Cu to hexadecyltrimethylammonium bromide is 0.1~0.4:100~300; 2+ The amount is in mol and the amount of hexadecyltrimethylammonium bromide is in mg.

8. The preparation method according to claim 3, characterized in that: The reducing agent described in step (2) is at least one selected from NaBH4, LiAlH4, hydrazine hydrate, and vitamin C.

9. An application of the spindle-shaped Cu-Ru bimetallic mesoporous nanomimetic enzyme according to any one of claims 1 to 2 or the spindle-shaped Cu-Ru bimetallic mesoporous nanomimetic enzyme prepared by the method according to any one of claims 3 to 8 in the fields of environmental water, biology, and medicine, characterized in that: The nano-mimetic enzyme is used to detect o-aminophenol and / or m-aminophenol.

10. The use according to claim 9, characterized in that: The detection process of described o-aminophenol is: S1, adding the colorimetric substrate 3,3',5,5'-tetramethylbenzidine, disodium hydrogen phosphate-citric acid buffer solution, and the sample solution to be tested to the spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme standard solution to obtain a test solution; S2, observing the color change of the test liquid; The detection process of the meta-aminophenol is: S3, adding a colorimetric substrate ABTS, a disodium hydrogen phosphate-citric acid buffer solution, H2O2, and a sample solution to be tested to the spindle-shaped Cu-Ru bimetallic mesoporous nano-mimetic enzyme standard solution to obtain a test solution; S4. Observe the color change of the test liquid.

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