Microminiature trimetal nano-enzyme sensor, preparation thereof and application of microminiature trimetal nano-enzyme sensor in colorimetric detection

Abstract

The invention discloses a subminiature three-metal nano-enzyme sensor, preparation thereof and application of the subminiature three-metal nano-enzyme sensor in colorimetric detection. The nano enzyme comprises multi-metal oxide nano particles, and the multi-metal oxide nano particles comprise monovalent copper, bivalent copper, divalent iron, ferric iron and divalent manganese. A partial area of the surface of the multi-metal oxide nano particle is provided with a coating layer, and the coating layer comprises a nitrogen-containing polymer. The particle size range is 5-10 nm; under the condition that the pH value is 6.5-7.5 (namely a neutral condition), the enzyme has peroxidase-like and oxidase-like double-enzyme activity at the same time. A colorimetric sensor constructed on the basis of the nano-enzyme can realize quantitative detection of biomarkers such as glutathione, glucose, glucose-6-phosphate dehydrogenase and the like. Besides, further, the colorimetric sensor is combined with the gold nanomaterial, and a new thought is provided for visual detection of the indexes by naked eyes through integration of equipment such as a smart phone and an image analysis technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a super-small three-metal nanosensor and its preparation and application in colorimetric detection. BACKGROUND

[0002] Living organisms regulate a series of key life activities such as energy transduction, redox homeostasis and development process through specific biomolecules. Glucose (Glu) as the most important metabolic substrate is the core energy source for ATP synthesis in cells. In clinical practice, chronic hyperglycemia is closely related to the functional impairment of multiple systems and organs, therefore, the management of metabolic diseases such as diabetes highly depends on the dynamic monitoring of glucose levels. Glucose-6-phosphate (G6P), as a metabolic hub, determines the flow direction of carbon sources into glycolysis, oxidative phosphorylation, pentose phosphate shunt and glycogen synthesis. The level change of G6P in body fluid can reflect the functional status of the enzyme system related to it, especially the evaluation of the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme for NADPH synthesis, is of great significance. G6PD deficiency is currently the most common enzyme deficiency disease in humans, which maintains the reduced state of glutathione (GSH) in red blood cells to resist oxidative stress hemolysis. Recent studies have shown that G6PD dysfunction is also closely related to various cardiometabolic diseases and tumor-related signaling pathways. Glutathione (GSH) is an important redox-regulating molecule in cells, which plays a variety of biological functions, including: ① scavenging reactive oxygen free radicals; ② participating in the detoxification reaction of phase II metabolism; ③ regulating the redox state of protein thiol / disulfide bond; ④ affecting the calcium signaling pathway. Abnormal levels of GSH not only affect redox homeostasis, but also mediate apoptosis and immune response. Therefore, it is of great significance to realize sensitive, stable and low-cost detection of key indicators such as Glu, G6PD and GSH for early diagnosis and large-scale screening of diseases.

[0003] In recent years, artificially synthesized nanosensors have become a research hotspot in the field of biosensing due to their superior catalytic activity, structural stability and environmental adaptability compared to natural enzymes. The current mainstream types of nanosensors include nanomaterials with peroxidase (POD), oxidase (OXD) and superoxide dismutase (SOD) like activities. Among them, POD-like and OXD-like nanosensors are widely used in colorimetric detection due to their adaptation to a variety of substrate reactions.

[0004] However, most of the existing POD-like and OXD-like nanozymes exhibit optimal catalytic performance in acidic environment, and their activity significantly decreases under neutral or physiological pH conditions, which greatly limits their practicality in biomedical detection. Although some studies have developed nanozymes with residual activity under neutral conditions, their catalytic efficiency is still far inferior to that under acidic conditions. So far, there is no literature report on nanozymes with high-efficiency POD-like and OXD-like dual enzyme activities under neutral conditions. Therefore, developing multifunctional nanozymes that can work under physiological pH conditions has become a major technical challenge in the field of biological sensing. SUMMARY

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a super-small three-metal nanozyme sensor and its preparation and application in colorimetric detection. The nanozyme is a super-small copper-iron-manganese three-metal nanozyme (CuFeMn Nanozyme), abbreviated as CF4M. By regulating the multi-valence of copper and iron elements, the nanozyme realizes high-efficiency enzyme catalytic performance under neutral pH conditions. Based on the peroxidase (POD) and oxidase (OXD) dual enzyme activities of CF4M, the reduction reaction of oxidized substrates and the tandem catalytic reaction are used to construct a multi-index colorimetric biosensing platform, realizing the quantitative detection of biomarkers such as glutathione (GSH), glucose (Glu) and glucose-6-phosphate dehydrogenase (G6PD). In addition, the platform is combined with gold nanomaterials, and through the integration of devices such as smartphones and image analysis technology, a new idea is provided for the naked-eye visual detection of Glu, GSH and G6PD indicators.

[0006] To achieve the above-mentioned purposes and other related purposes, the present application provides a super-small three-metal nanozyme, which comprises a multi-metal oxide nanoparticle, wherein the multi-metal oxide nanoparticle comprises monovalent copper (Cu + ), divalent copper (Cu 2+ ), divalent iron (Fe 2+ ), trivalent iron (Fe 3+ ) and divalent manganese (Mn 2+ );

[0007] Part of the surface area of the multi-metal oxide nanoparticle has a coating layer, and the coating layer comprises a nitrogen-containing polymer;

[0008] The particle size of the super-small three-metal nanozyme ranges from 5 nm to 10 nm;

[0009] The super-small three-metal nanozyme has peroxidase-like and oxidase-like dual enzyme activities under neutral conditions (i.e. under conditions with pH ranging from 6.5 to 7.5).

[0010] In an embodiment of the present application, in the multi-metal oxide nanoparticles, the mass fraction of copper is 0.76% to 46.28%, the mass fraction of iron is 53.65% to 98.32%, and the mass fraction of manganese is 0.07% to 1.04%, based on the total amount of metal elements being 100%. Preferably, in the multi-metal oxide nanoparticles, the mass fractions of copper, iron and manganese are 43.97%, 55.87% and 0.16%, respectively, based on the total amount of metal elements being 100%.

[0011] In an embodiment of the present application, the nitrogen-containing polymer is polyvinylpyrrolidone.

[0012] The present application also provides a preparation method of the ultra-small three-metal nanoscale enzyme as described above, comprising the following steps:

[0013] The copper salt, the iron salt and the manganese salt are added into a polyvinylpyrrolidone solution, mixed, and then the sodium hydroxide and hydrogen peroxide solution are added and stirred for reaction. After the reaction, the nanoparticles are purified by ultracentrifugation, washed with water, and the obtained precipitate is collected to obtain the ultra-small three-metal nanoscale enzyme.

[0014] In an embodiment of the present application, the molar ratio of the copper salt, the iron salt and the manganese salt is 1-4:1-4:1-4, for example, 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:2:1, 1:3:1, 1:4:1, 2:1:1, 3:1:1 and 4:1:1, etc.

[0015] In an embodiment of the present application, the copper salt includes but is not limited to copper chloride, copper nitrate, copper acetate, copper sulfate and corresponding metal salt hydrates, etc., the iron salt includes but is not limited to ferric chloride, ferrous chloride, ferric nitrate, ferrous nitrate, ferric sulfate, ferrous sulfate and corresponding metal salt hydrates, etc., and the manganese salt includes but is not limited to manganese chloride, manganese acetate, manganese acetylacetone, manganese sulfate and corresponding metal salt hydrates, etc.

[0016] In an embodiment of the present application, the polyvinylpyrrolidone solution is an aqueous solution of polyvinylpyrrolidone, and the concentration is 0.1-0.5 g / mL, for example, 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL and 0.5 g / mL, etc. The polyvinylpyrrolidone can be used as a stabilizer for nanoparticles, which has the effect of preventing agglomeration and maintaining dispersibility.

[0017] In an embodiment of the present application, the molar ratio of the copper salt, the iron salt, the manganese salt and the sodium hydroxide is 1-4:1-4:1-4:0.02-0.04, for example, 1:1:4:0.02, 1:1:4:0.025, 1:1:4:0.03, 1:1:4:0.035 and 1:1:4:0.04, etc.

[0018] In an embodiment of the present application, the hydrogen peroxide solution is an aqueous solution of hydrogen peroxide with a concentration of 10-40%, such as 10%, 20%, 30% and 40%, etc.

[0019] In an embodiment of the present application, the stirring rate of the stirring reaction is 800-1600 rpm / min, such as 800 rpm / min, 1200 rpm / min and 1600 rpm / min, etc.

[0020] In an embodiment of the present application, the reaction time of the stirring reaction is 20-40 minutes, such as 20 minutes, 30 minutes, 40 minutes, etc.

[0021] It should be noted that the above preparation method involves chemical reactions at room temperature.

[0022] The present application also provides a colorimetric detection kit for detecting glutathione, glucose and / or glucose-6-phosphate dehydrogenase, which comprises the ultra-small tri-metal nanoscale enzyme as described above, and further comprises a color developing agent.

[0023] In an embodiment of the present application, the color developing agent is 3,3',5,5'-tetramethylbenzidine (TMB).

[0024] In an embodiment of the present application, when used for detecting glucose, the kit further comprises glucose oxidase (Gox).

[0025] In an embodiment of the present application, when used for detecting glutathione and / or glucose-6-phosphate dehydrogenase, the kit further comprises hydrogen peroxide.

[0026] In an embodiment of the present application, when used for detecting glucose-6-phosphate dehydrogenase, the kit further comprises nicotinamide adenine dinucleotide (NAD) and glucose-6-phosphate (G6P).

[0027] In an embodiment of the present application, the kit further comprises a diluent, and the pH of the diluent is 6.5-7.5, which includes but is not limited to ultrapure water, etc.

[0028] In an embodiment of the present application, the kit further comprises gold nanomaterials (AuNRs), and the gold nanomaterials are gold nanorods.

[0029] The present application also provides a method for detecting glutathione, glucose and / or glucose-6-phosphate dehydrogenase, which comprises the following steps:

[0030] The ultra-small trimetallic nanoszyme and the chromogenic substrate as described above are added into the sample to be tested, and the incubation reaction is carried out under the condition that the pH is 6.5-7.5; after the reaction is completed, the real-time absorbance is detected, and the concentration of glutathione, glucose and / or glucose-6-phosphate dehydrogenase in the sample to be tested is determined based on the preset relationship between the real-time absorbance and the concentration of the substance to be tested.

[0031] For example, the preset relationship between the absorbance and the concentration of the substance to be tested is a linear relationship between the absorbance change value and the concentration.

[0032] In an embodiment of the present application, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

[0033] In an embodiment of the present application, when used for detecting glucose, the method comprises the following steps: adding a known concentration of glucose oxidase into the sample to be tested to carry out a first incubation reaction; after the first incubation reaction is completed, the ultra-small trimetallic nanoszyme and the chromogenic substrate are added again to carry out a second incubation reaction under the condition that the pH is 6.5-7.5; after the second incubation reaction is completed, the absorbance is detected, and the concentration of glucose in the sample to be tested is determined according to a standard working curve. The first incubation reaction is carried out at room temperature, and the first incubation reaction time is 20-50 min, for example, 20 min, 30 min, 40 min and 50 min, etc.; the second incubation reaction is carried out at room temperature, and the second incubation reaction time is 20-50 min, for example, 20 min, 30 min, 40 min and 50 min, etc.

[0034] In an embodiment of the present application, when used for detecting glutathione, the method comprises the following steps: the ultra-small trimetallic nanoszyme, the chromogenic substrate and hydrogen peroxide are first incubated under the condition that the pH is 6.5-7.5 to carry out a first incubation reaction; after the first incubation reaction is completed, the sample to be tested is added to carry out a second incubation reaction under the condition that the pH is 6.5-7.5; after the second incubation reaction is completed, the absorbance is detected, and the concentration of glutathione in the sample to be tested is determined according to a standard working curve. The first incubation reaction is carried out at room temperature, and the first incubation reaction time is 5-20 min, for example, 5 min, 10 min, 15 min and 20 min, etc.; the second incubation reaction is carried out at room temperature, and the second incubation reaction time is 5-20 min, for example, 5 min, 10 min, 15 min and 20 min, etc.

[0035] In an embodiment of the present application, for detecting glucose-6-phosphate dehydrogenase, the method comprises the following steps: adding NAD and G6P into the sample to be detected to perform a first incubation reaction; adding the ultra-small trimetal nanoscale enzyme, a chromogenic substrate and hydrogen peroxide under the condition of pH 6.5-7.5 to perform a second incubation reaction; adding the reaction solution obtained in the second incubation reaction into the reaction solution obtained in the first incubation reaction, immediately detecting the absorbance, and determining the concentration of glucose-6-phosphate dehydrogenase in the sample to be detected according to a standard working curve. The first incubation reaction temperature is 37°C, and the first incubation reaction time is 20-50 min, for example, 20 min, 30 min, 40 min and 50 min, etc. The second incubation reaction is performed at room temperature, and the second incubation reaction time is 5-20 min, for example, 5 min, 10 min, 15 min and 20 min, etc.

[0036] The present application also provides a visual detection method of glutathione, glucose and / or glucose-6-phosphate dehydrogenase, comprising the following steps:

[0037] The ultra-small trimetal nanoscale enzyme and the chromogenic substrate as described above are added into the sample to be detected, and an incubation reaction is performed under the condition of pH 6.5-7.5. After the incubation reaction is completed, hydrochloric acid is added into the reaction solution to perform a reaction, and after the solution changes color, gold nanomaterials are added to perform an etching reaction. After the etching reaction is completed, a photo is taken. The real-time colorimetric signal is converted into a real-time visual signal by processing the photo, and the concentration of glutathione, glucose and / or glucose-6-phosphate dehydrogenase in the sample to be detected is determined according to the preset relationship between the real-time visual signal and the visual signal and the concentration of the sample to be detected.

[0038] In an embodiment of the present application, when the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, the color change of the solution refers to the color change of the solution from blue to yellow.

[0039] In an embodiment of the present application, the real-time colorimetric signal is converted into a real-time visual signal by processing the photo, and the concentration of glutathione, glucose and / or glucose-6-phosphate dehydrogenase in the sample to be detected is determined according to the preset relationship between the real-time visual signal and the visual signal and the concentration. The way is: the real-time RGB value is obtained by processing the photo, and the concentration of glutathione, glucose and / or glucose-6-phosphate dehydrogenase in the sample to be detected is determined according to the preset relationship between the real-time RGB value and the RGB value and the concentration of the sample to be detected. The photo shooting includes but is not limited to smart phones, tablet computers, cameras, etc., and the software for processing the photo to obtain the RGB value includes but is not limited to Colorpicker APP, etc. For example, the preset relationship between the RGB value and the concentration of the sample to be detected is a linear relationship between the RGB value and the concentration of the sample to be detected.

[0040] As described above, the ultra-small three-metal nanosensor of the present application and its preparation and application in colorimetric detection have the following beneficial effects:

[0041] The present application provides a three-metal nanosensor material with POD-like and OXD-like dual enzyme activities under neutral pH conditions and a multi-index colorimetric sensing platform constructed thereby, to overcome the technical bottleneck that the catalytic activity of existing nanosensors is limited to acidic environments and is difficult to adapt to physiological conditions. The present application introduces multivalent copper and iron elements to synthesize a CuFeMn nanosensor (CF4M) with excellent catalytic performance, and establishes a colorimetric detection method without the need to change buffers, which can efficiently catalyze reactions under neutral conditions and simplify the detection process. The platform can realize sensitive, stable and visual detection of various clinically relevant biomarkers such as GSH, Glu and G6PD, and combined with smartphone image analysis technology, it realizes portable and quantitative detection of multiple markers, and is expected to promote the wide application of nanosensors in precise diagnosis, metabolic disease monitoring and multi-index joint detection.

[0042] (1) The three-metal nanosensor CF4M with peroxidase (POD-like) and oxidase (OXD-like) activities under neutral pH conditions is constructed for the first time, which significantly breaks through the limitation of traditional nanosensors that require reactions under acidic conditions, and improves their application adaptability in biological samples and clinical environments.

[0043] (2) The multivalent synergistic effect of copper and iron in CF4M nanosensor significantly enhances the catalytic activity, so that it can efficiently catalyze substrate reactions without the need to change buffers, thereby simplifying the detection process and reducing the complexity of operation, facilitating clinical promotion and standardized application.

[0044] (3) Based on the multi-enzyme activity of CF4M, the present application establishes a colorimetric analysis platform that does not require labeling, has low cost and can quickly detect multiple markers (GSH, Glu, G6PD), which has good sensitivity and repeatability and good practicability.

[0045] (4) The present application first combines the CF4M colorimetric platform with the smartphone image analysis technology assisted by gold nanorods (AuNR) to realize visual and quantitative detection of multiple biomarkers, and the results can be obtained without expensive instruments, which expands the application prospect of nanosensors in portable, home and on-site detection and other scenarios.

[0046] (5) The method of the present application has good universality and expandability, and can be flexibly adapted to the detection of other related redox biomarkers by adjusting the substrate or cascade reaction system, providing an efficient and economical new solution for the application of nanosensors in early screening of diseases, metabolic monitoring and precise diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 TEM images (a), particle size distribution (b), elemental mapping (c) and XPS spectra (d-i) of CF4M ultrasmall nanoscale enzyme in the embodiments of the present application.

[0048] Figure 2 Schematic diagram of the application of CF4M-based biosensing platform in biomarker detection and visual analysis in the embodiments of the present application.

[0049] Figure 3 POD and OXD-like dual enzyme activity scheme (a, d) of CF4M ultrasmall nanoscale enzyme, Michaelis-Menten curves (b, e) and corresponding Lineweaver-Burk double-reciprocal plots (c, f) of catalyzing H2O2 and TMB in the embodiments of the present application.

[0050] Figure 4 Scheme (a, c, e) of CF4M ultrasmall nanoscale enzyme in detecting GSH, Glu and G6PD, and linear relationship (b, d, f) between absorbance change value and concentration.

[0051] Figure 5 Specificity and repeatability of CF4M ultrasmall nanoscale enzyme in detecting GSH, Glu and G6PD in the embodiments of the present application.

[0052] Figure 6 Corresponding (R+G) / B value and fitting linear curve of CF4M ultrasmall nanoscale enzyme integrated with AuNR-assisted smartphone RGB extraction technology in detecting H2O2, GSH, Glu and G6PD concentration in the embodiments of the present application. DETAILED DESCRIPTION

[0053] The embodiments of the present application are described below through specific and concrete examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present specification. The present application can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in the present specification based on different viewpoints and applications without departing from the spirit of the present application.

[0054] In the present application, the term "a plurality of" means two or more, unless otherwise specified.

[0055] The character " / " represents an "or" relationship between the objects before and after it. For example, A / B means: A or B.

[0056] The term "and / or" is a description of the association relationship between objects, which means that there can be three relationships. For example, A and / or B means: A or B, or, A and B, the three relationships.

[0057] Example 1

[0058] Preparation of ultra-miniature copper-iron-manganese nanozymes and detection of peroxidase-like (POD-like) and oxidase-like (OXD-like) activities.

[0059] (1) Preparation of ultra-miniature copper-iron-manganese nanozymes (CF4M):

[0060] 1 g of polyvinyl pyrrolidone (PVP) was dissolved in 5 mL of ultrapure water. 1 mL each of CuCl₂·2H₂O (0.02 M), FeCl₂·4H₂O (0.02 M), and MnCl₂·4H₂O (0.08 M) were mixed and added to the PVP solution. The mixture was stirred at 1200 rpm for 5 minutes. Then, 8 mL of NaOH (0.007 M) and 160 μL of 30% H₂O₂ were added to the solution, and the mixture was stirred at 1200 rpm for 30 minutes. After the reaction was complete, the nanoparticles were purified by ultracentrifugation and washed three times with ultrapure water. Finally, the precipitate was collected and stored at room temperature for later use.

[0061] Figure 1 The images shown are transmission electron microscopy (a), particle size distribution (b), elemental surface scan (c), and XPS images (d-i) of the CF4M ultra-small nanozyme in this embodiment.

[0062] like Figure 1 As shown, the synthesized CF4M ultraminiature nanozyme has an average diameter of 6.5 nm, and copper (Cu), iron (Fe), and manganese (Mn) are uniformly distributed and coexist within the nanoparticles. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of six elements in the material, including Cu, Fe, Mn, carbon (C), nitrogen (N), and oxygen (O). The two characteristic peaks of Cu are located at Cu2p... 3 / 2 (933.15eV) and Cu 2p 1 / 2 (952.90 eV), after peak decomposition, Cu was found to be in the form of Cu. + and Cu 2+ Mixed valence states exist. Similarly, for Fe 2p... 3 / 2 and 2p 1 / 2 Peak splitting results show that Fe also occurs as Fe 2+ and Fe 3+The mixed oxidation states exist. Although the content of Mn element is low, it is clearly detected in XPS, which confirms its participation in the construction of nano-enzyme structure. Further quantitative analysis shows that the mass fractions of Cu, Fe, and Mn are 43.97%, 55.87%, and 0.16%, respectively. The obvious oxygen vacancy characteristic peak appears in the XPS spectrum of O element, indicating that the material is a defect-rich multi-metal oxide system. These results together show that the metal elements in CF4M do not exist in the form of traditional single oxides (such as CuO or Fe2O3), but more likely form amorphous or spinel-like composite oxide nanoparticles. The detected C and N elements in the material come from the surface-coated polyvinylpyrrolidone (PVP). PVP acts as a stabilizer by coordinating with metal ions through the carbonyl group (–C=O) and pyrrole nitrogen (–N) in its molecules to form a surface adsorption layer. This organic layer helps to control the particle size, inhibit agglomeration, and improve water dispersibility. C and N elements are usually distributed on the outer surface of multi-metal oxide nanoparticles in an adsorbed / complexed state, forming a flexible coordination layer or amorphous coating layer, rather than a well-defined, dense and continuous “core-shell structure”.

[0063] (2) as shown in Figure 2 , Figure 3 and Figure 4 , a colorimetric sensor was constructed based on the CF4M nano-enzyme obtained by the above preparation method to realize the quantitative detection of glucose (Glu), reduced glutathione (GSH), and glucose-6-phosphate dehydrogenase (G6PD), and the standard curve was plotted:

[0064] 1) Glu detection: 50 μl of glucose oxidase (Gox) and different concentrations of glucose solution were mixed at a fixed concentration (1 mg / ml) and incubated at room temperature for 30 min, and the final concentration of glucose ranged from 0.05 mM to 1 mM. Then 50 μl of TMB (8 mM) and 100 μl of CF4M (2.5 mg / ml) were added to the above reaction solution, and the oxTMB absorbance was detected after incubation at room temperature for another 30 min. According to the linear relationship between the absorbance change value and the concentration of Glu, the Glu working curve was plotted.

[0065] 2) GSH detection: 50 μl of TMB (4 mM), 50 μl of H2O2 (2 mM), and 100 μl of CF4M (1 mg / ml) were incubated at room temperature for 10 min, and then 50 μl of different concentrations of GSH (final concentration range from 1 to 70 μM) were quickly added. The oxTMB absorbance was detected after 5 min, and according to the linear relationship between the absorbance change value and the concentration of GSH, the GSH working curve was plotted.

[0066] 3) G6PD detection: Add 5 μl of G6PD (concentration range from 0.25 to 4 U / ml) to a mixture of 220 μl NAD (0.36 mg / ml) and 25 μl G6P (40 mM), and incubate at 37 °C for 30 min. Mix 50 μl TMB (4 mM), 50 μl H2O2 (2 mM), and 100 μl CF4M (1 mg / ml) and incubate at room temperature for 10 min. Add 50 μl of the above G6PD mixture and immediately measure the absorbance of oxTMB. Based on the linear relationship between the absorbance change and the G6PD concentration, plot the G6PD working curve for the final concentration range of 1-16 mU / ml.

[0067] like Figure 3 As shown, the catalytic performance of CF4M nanozyme under neutral pH conditions, exhibiting both peroxidase-like (POD-like) and oxidase-like (OXD-like) properties, was investigated through kinetic experiments. The Km values ​​of CF4M for H2O2 and TMB were 0.1152 mM and 0.9141 mM, respectively. max They are 8.6059×10 -7 Ms -1 and 1.9387×10 -7 Ms -1 .

[0068] like Figure 4 As shown, 1) when the CF4M nanozyme detected GSH (concentration range of 1-70 μM), the absorbance of oxTMB decreased with increasing GSH concentration, conforming to the linear regression equation: Y = -0.006075X + 0.8291(R). 2 =0.994); 2) When detecting Glu (0.05-1mM concentration range), as the Glu concentration increases, the absorbance of oxTMB increases, and the working curve shows a bilinear region: Y = 2.397X + 0.0851 (0.05-0.6mM, R 2 =0.996) and Y = 1.135X + 0.8069(0.6-1mM, R 2 =0.994); 3) When detecting G6PD (concentration range of 1-16 mU / mL), as the G6PD concentration increases, the absorbance of oxTMB decreases, conforming to the linear regression equation: Y = -0.03344X + 0.7291, R 2 =0.987. (3) Specificity and repeatability in detecting GSH, Glu and G6PD.

[0069] like Figure 5 As shown, in GSH detection, the metal ion (K) was investigated. + Na + Ca 2+, amino acid (cysteine, Cys) and biomolecule (glutathione, GSH; ascorbic acid, AA; uric acid, UA; glucose, Glu). The results showed that except for GSH, AA and Cys, other substances did not cause significant absorbance changes. Although there was some interference of AA and Cys, it could be effectively eliminated by sample pretreatment. In the Glu detection, common interferents such as maltose, lactose, fructose were evaluated, and the results showed that the presence of these substances did not cause obvious absorbance changes compared with Glu. In the G6PD detection, enzyme interferents such as tyrosinase (TYR), alkaline phosphatase (ALP), uric acid oxidase (UOx) and glucose oxidase (GOx) were investigated, and the results showed that these interferents did not cause significant signal changes compared with G6PD.

[0070] The stability of CF4M nanozyme was also strictly evaluated. Through 28-day long-term monitoring, it was found that CF4M had no significant decrease in catalytic efficiency for H2O2 at low, medium and high substrate concentrations.

[0071] Example 2

[0072] This example is based on Example 1, combined with gold nanorods (AuNRs) and smart phone RGB acquisition, to realize the visual detection of different biomarkers, and the specific implementation process is as follows:

[0073] (1) Preparation of AuNRs:

[0074] First, prepare the seed solution, add 600 μl of NaBH4(0.01M) ice solution to the mixture of 9.75 ml of CTAB(0.1M) and 0.25 ml of HAuCl4·3H2O(0.01M), stir at 1200 rpm / min for 2 minutes, and then stand at 30°C for 30 minutes for standby. Second, prepare the growth solution, first mix 10 ml of CTAB(0.1M) and 0.5 ml of HAuCl4·3H2O(0.01M) by inversion, add 100 μl of AgNO3(0.01M) and mix again by inversion. Add 80 μl of ascorbic acid(0.1M) and 200 μL of hydrochloric acid(1M) to the above mixture in turn. Finally, add 13 μl of seed solution, mix by inversion, and stand at 30°C for 48 h. The prepared AuNRs are centrifuged at 7200 rpm / min for 30 min, and after removing the supernatant, they are dispersed in a CTAB(0.2M) solution for standby.

[0075] (2) Visual detection:

[0076] After completing the detection of H2O2, Glu, GSH and G6PD by the CF4M nanozyme sensor according to the steps of Example 1, 100 μL of reaction solution was mixed with 100 μL of HCl, and oxTMB was converted to TMB2+ The color of the solution changed from blue to yellow. Then 100 μΐ, of AuNRs (2 nM) was added and the etching reaction was carried out at room temperature for 35 min. The photos were taken by an Android smartphone and the RGB values were obtained by using Colorpicker APP. The colorimetric signal was further converted into visual signal for the detection of H2O2, GSH, Glu and G6PD.

[0077] As shown in Figure 6 , for the detection of H2O2, the concentration showed good linear correlation with the (R+G) / B value in the two concentration ranges of 0.125-0.5 mM and 0.5-2 mM, with the correlation coefficients (R 2 ) of 0.999 and 0.984, respectively. Meanwhile, for the detection of GSH, the concentration showed good linear correlation with the (R+G) / B value in the range of 1.0-70 μΜ, with the R 2 value of 0.986. The detection results of Glu showed that the Glu concentration showed linear correlation with the (R+G) / B value in the concentration range of 0.1-0.4 mM, with the R 2 value of 0.991. The detection results of G6PD also showed that the G6PD concentration showed good linear response with the (R+G) / B value in the range of 2-16 mU / mL, with the R 2 value of 0.994.

[0078] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. An ultra-miniature trimetallic nanozyme, characterized in that, This includes multi-metal oxide nanoparticles, wherein the multi-metal oxide nanoparticles contain monovalent copper Cu. + Divalent copper Cu 2+ Fe, divalent iron 2+ ferric iron (Fe) 3+ and divalent manganese Mn 2+ ; The surface of the multi-metal oxide nanoparticles has a coating layer in a certain area, and the coating layer includes a nitrogen-containing polymer; The particle size range of the ultra-small trimetallic nanozyme is 5-10 nm; The ultra-small trimetallic nanozyme exhibits both peroxidase-like and oxidase-like dual enzyme activities under pH conditions of 6.5-7.

5.

2. The ultra-miniature trimetallic nanozyme according to claim 1, characterized in that: In the polymetallic oxide nanoparticles, the total amount of metal elements is 100%, with copper having a mass fraction of 0.76% to 46.28%, iron having a mass fraction of 53.65% to 98.32%, and manganese having a mass fraction of 0.07% to 1.04%. And / or, the nitrogen-containing polymer includes polyvinylpyrrolidone.

3. A method for preparing an ultra-miniature trimetallic nanozyme as described in any one of claims 1-2, characterized in that, Includes the following steps: Copper, iron, and manganese salts were added to a polyvinylpyrrolidone solution, mixed well, and then sodium hydroxide and hydrogen peroxide solutions were added and stirred to react. After the reaction was completed, the nanoparticles were purified by ultracentrifugation, washed with water, and the resulting precipitate was collected to obtain the ultra-small trimetallic nanozyme.

4. The method for preparing ultra-miniature trimetallic nanozymes according to claim 3, characterized in that: The polyvinylpyrrolidone solution is an aqueous solution of polyvinylpyrrolidone with a concentration of 0.1-0.5 g / mL; And / or, the molar ratio of the copper salt, iron salt, manganese salt and sodium hydroxide is 1-4:1-4:1-4:0.02-0.04; And / or, the hydrogen peroxide solution is an aqueous solution of hydrogen peroxide with a concentration of 10%-40%; And / or, the stirring rate of the stirring reaction is 800-1600 rpm; And / or, the reaction time of the stirring reaction is 20-40 minutes.

5. A colorimetric assay kit for detecting glutathione, glucose, and / or glucose-6-phosphate dehydrogenase, characterized in that: The kit includes the ultra-miniature trimetallic nanozymes as described in any one of claims 1-2 and / or the ultra-miniature trimetallic nanozymes prepared by the method according to any one of claims 3-4, and also includes a chromogenic agent.

6. The colorimetric detection kit according to claim 5, characterized in that: The colorimetric reagent is 3,3',5,5'-tetramethylbenzidine; And / or, the kit further includes at least one of the following components: glucose oxidase, hydrogen peroxide, nicotinamide adenine dinucleotide, glucose-6-phosphate, diluent, and gold nanomaterials, wherein the diluent has a pH of 6.5-7.

5.

7. A method for detecting glutathione, glucose, and / or glucose-6-phosphate dehydrogenase, said method not for disease diagnosis or treatment purposes, characterized in that, The method includes the following steps: The ultra-miniature trimetallic nanozyme as described in any one of claims 1-2 and / or the ultra-miniature trimetallic nanozyme prepared by the method according to any one of claims 3-4, and the chromogenic substrate are added to the sample to be tested and incubated at pH 6.5-7.

5. After the reaction, the real-time absorbance is detected, and the concentrations of glutathione, glucose, and / or glucose-6-phosphate dehydrogenase in the sample to be tested are determined based on the real-time absorbance and the preset relationship between absorbance and the concentration of the analyte.

8. The method according to claim 7, characterized in that: The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

9. The method according to claim 7 or 8, characterized in that: When used for glucose detection, the method includes the following steps: adding a known concentration of glucose oxidase to the sample to be tested and performing a first incubation reaction; after the first incubation reaction, adding the ultra-small trimetallic nanozyme and chromogenic substrate, and performing a second incubation reaction under pH conditions of 6.5-7.5; after the second incubation reaction, detecting the absorbance, and determining the concentration of glucose in the sample to be tested according to the standard working curve; When used for the detection of glutathione, the method includes the following steps: the ultra-miniature trimetallic nanozyme, the chromogenic substrate, and hydrogen peroxide are first incubated at pH 6.5-7.5; after the first incubation, the sample to be tested is added, and a second incubation is performed at pH 6.5-7.5; after the second incubation, the absorbance is measured, and the concentration of glutathione in the sample to be tested is determined according to a standard working curve. When used to detect glucose-6-phosphate dehydrogenase, the method includes the following steps: adding nicotinamide adenine dinucleotide and glucose-6-phosphate to the sample to be tested for a first incubation reaction; adding the ultra-miniature trimetallic nanozyme, chromogenic substrate, and hydrogen peroxide for a second incubation reaction at pH 6.5-7.5; adding the reaction solution obtained from the second incubation reaction to the reaction solution obtained from the first incubation reaction, immediately detecting the absorbance, and determining the concentration of glucose-6-phosphate dehydrogenase in the sample to be tested according to the standard working curve.

10. A visual detection method for glutathione, glucose, and / or glucose-6-phosphate dehydrogenase, characterized in that, Includes the following steps: The ultra-miniature trimetallic nanozyme as described in any one of claims 1-2 and / or the ultra-miniature trimetallic nanozyme prepared according to the method described in any one of claims 3-4, and the chromogenic substrate are added to the sample to be tested and incubated at pH 6.5-7.

5. After the incubation reaction is completed, hydrochloric acid is added to the reaction solution to carry out the reaction. After the solution changes color, gold nanomaterials are added to carry out the etching reaction. After the etching reaction is completed, a photograph is taken. By processing the photograph, the real-time colorimetric signal is converted into a real-time visual signal. The concentrations of glutathione, glucose, and / or glucose-6-phosphate dehydrogenase in the sample to be tested are determined according to the real-time visual signal and the preset relationship between the visual signal and the concentration of the analyte.

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