Nanoprotease, preparation method and application thereof

Abstract

This invention provides a nanozyme, its preparation method, and its application, belonging to the field of analytical chemistry detection technology. The nanozyme is vanadium oxide doped with a metal, in the form of nanoribbons. The metal is titanium, which is atomically loaded onto the vanadium oxide, with an atomic percentage of 1%-2%. The nanozyme of this invention possesses dual-enzyme activity similar to NADH oxidase and peroxidase. Utilizing its dual-enzyme activity in combination with natural enzymes, the amount of hydrogen peroxide is converted into a final measurement standard. Through the color change of a 3,3',5,5'-tetramethylbenzidine solution system, rapid qualitative and quantitative analysis of biomarkers for rare genetic metabolic diseases is achieved, providing a rapid, sensitive, and reliable colorimetric detection method.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry detection, and in particular to a nanozyme, its preparation method, and its application. Background Technology

[0002] Enzyme colorimetry, with its advantages of simplicity and rapid reaction, has become an important method for biochemical detection. Enzyme-based visual detection essentially involves the reaction of hydrogen peroxide (H₂O₂) with a chromogenic substrate (such as 3,3',5,5'-tetramethylbenzidine, TMB) under the catalysis of peroxidase-like nanozymes. The solution changes from colorless to blue, converting the concentration of the target biomarker into a color signal. Among these methods, peroxidase-like enzyme activity has been the most extensively studied, providing broad applications for the visual detection of biomarkers such as glucose, uric acid, and cholesterol by generating H₂O₂, and significantly advancing the development of enzyme-based visual detection. However, this colorimetric system requires the biomarker itself to directly and rapidly generate H₂O₂, but many disease biomarkers cannot directly generate H₂O₂, which limits the application of this method.

[0003] In living organisms, the original nicotinamide adenine dinucleotide (NADH) and its oxidized form NAD are present. + As a core coenzyme in energy metabolism, NADH plays a crucial role in various redox reactions, participating in key metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the mitochondrial respiratory chain, and regulating cellular energy production and redox homeostasis. Therefore, NADH / NAD... + The dynamic changes in NADH not only reflect the metabolic state of cells but are also closely related to the occurrence and development of various diseases. Furthermore, NADH can act as an electron donor, reacting with O2 to generate H2O2 under the oxidation of NADH oxidase. In the past, the detection of NADH-related disease biomarkers was often quantified by measuring the production or consumption of NADH using ultraviolet spectrophotometry. However, this method is susceptible to sample quality issues, and its cumbersome detection process and strong dependence on instruments have limited its application in visual and on-site detection. Summary of the Invention

[0004] In view of this, in order to at least partially solve the aforementioned technical problems, the present invention provides a nanozyme, its preparation method, and its application.

[0005] According to one aspect of the present invention, a nanozyme is provided, wherein the nanozyme is vanadium oxide doped with a metal and is in the form of a nanoribbon, wherein the metal is titanium, and the titanium is loaded on the vanadium oxide in atomic form, wherein the atomic percentage of titanium is 1%-2%, and the nanozyme has a (110) crystal plane.

[0006] According to another aspect of the present invention, a method for preparing the above-mentioned nanozyme is provided: vanadium pentoxide, oxalic acid and titanium sulfate are dissolved in ultrapure water and heated to react to obtain nanozyme.

[0007] According to another aspect of the present invention, an application of the above-mentioned nanozyme in colorimetric detection of rare inherited metabolic diseases is provided, comprising: the nanozyme being used to detect disease marker A, which can be oxidized by its own related oxidase to generate hydrogen peroxide, or to detect disease marker B, which can react with reduced nicotinamide adenine dinucleotide under the action of its related dehydrogenase.

[0008] The nanozyme of this invention, designated Ti / VO2, possesses dual enzyme activities resembling both NADH oxidase and peroxidase (POD), exhibiting advantages such as high substrate affinity and strong catalytic activity. Utilizing this dual enzyme activity, it is possible to detect disease biomarkers A (such as glucose and uric acid) that directly affect hydrogen peroxide production and disease biomarkers B (such as pyruvate and α-ketoglutarate) that affect NADH levels. Compared to nanozyme detection systems based solely on peroxidase-like activity, the Ti / VO2 nanozyme provides a wider detection range for target analytes, enabling the detection of more rare genetic metabolic diseases. For example, traditional iron-copper alloy nanozymes only possess peroxidase-like activity; therefore, they can only catalyze the oxidation of TMB with hydrogen peroxide to produce color, and cannot oxidize NADH to generate hydrogen peroxide. Thus, they can only be used for the detection of disease biomarker A, not disease biomarker B. In contrast, the Ti / VO2 nanozyme of this invention can detect both disease biomarkers A and B. Attached Figure Description

[0009] Figure 1 The image shows a scanning electron microscope image of the nanozyme synthesized in Example 1.

[0010] Figure 2 The X-ray diffraction pattern of the nanozyme synthesized in Example 1;

[0011] Figure 3 This is the UV-Vis absorption curve of the nanozyme in Example 4, used for testing its peroxidase-like activity.

[0012] Figure 4 This is the UV-Vis absorption curve of NADH in Example 5;

[0013] Figure 5 This is the UV-Vis absorption curve of the nanozyme in Example 5, which is used to test the NADH oxidase-like activity.

[0014] Figure 6 The Lineweaver-Burk curve is the reaction kinetics of the nanozyme to H2O2 in Example 6.

[0015] Figure 7 The Lines-Burke curve is the reaction kinetics of nanozyme to TMB in Example 6.

[0016] Figure 8 The Lineweaver-Burk curve is the reaction kinetics of horseradish peroxidase to H2O2 in Example 6.

[0017] Figure 9 The Lines-Burke curve is the reaction kinetics of horseradish peroxidase to TMB in Example 6.

[0018] Figure 10 This is the UV-Vis absorption curve of the nanozyme for detecting lactic acid in Example 7;

[0019] Figure 11 This is a standard curve for the detection of lactic acid by nanozyme in Example 7;

[0020] Figure 12 The UV-Vis absorption curve of pyruvate detected by nanozyme in Example 7;

[0021] Figure 13 This is the standard curve for the detection of pyruvate by nanozyme in Example 7;

[0022] Figure 14 This is a comparison chart of the results of the standard sample and the sample to be tested when detecting lactic acid with nanozymes in Example 7;

[0023] Figure 15 This is a comparison chart of the results of the standard sample and the sample to be tested when the nanozyme was used to detect pyruvate in Example 7. Detailed Implementation

[0024] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0026] When using expressions such as "at least one of A, B or C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B or C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C, etc.).

[0027] Given the wide range and complexity of NADH-related reactions, constructing a nanozyme with dual enzyme activities resembling both NADH oxidase and peroxidase for a colorimetric rapid detection method for rare inherited metabolic diseases holds promise for playing a greater role in broader biomolecular recognition and metabolic state monitoring, thereby improving the applicability and accuracy of the detection.

[0028] Based on this, the present invention provides a nanozyme, wherein the nanozyme is vanadium oxide doped with metal and is in the form of nanoribbons, wherein the metal is titanium; titanium is loaded on vanadium oxide in atomic form, and the atomic percentage of titanium is 1%-2%.

[0029] The nanozyme of this invention possesses dual enzyme activities, similar to both NADH oxidase and peroxidase. Ti doping enhances its peroxidase-like activity, thereby accelerating the reaction rate of the catalytic process. Furthermore, the amount of Ti doping also affects the catalytic activity of the nanozyme; Ti doping levels exceeding 1%-2% can easily lead to problems such as lattice distortion, nanozyme particle aggregation, and a decrease in specific surface area.

[0030] According to an embodiment of the present invention, the above-mentioned nanozyme has a (110) crystal facet. In nanomaterials, a crystal facet refers to a specific arrangement of atoms on the crystal surface. Different crystal faces have different physicochemical properties, which directly affect the enzyme-like activity of the nanozyme. The atomic arrangement density, surface defects, oxygen vacancies, and other structures on different crystal faces are different. These structural differences affect the number and distribution of surface active sites of the material, thereby affecting its catalytic performance. In addition, the surface charge states of different crystal faces are different, which may affect their interaction with the substrate. For example, stronger polarity or electronegativity can promote electron transfer or substrate adsorption, thereby improving catalytic efficiency. The (110) crystal facet of the present invention has both peroxidase-like and NADH-like activities. Furthermore, the atoms on the (110) crystal facet are more densely packed, and the surface energy is relatively low, so it is more stable under certain conditions (such as high temperature or surface treatment). The (002) crystal facet has a higher surface energy and is prone to reconstruction or instability under external conditions, especially at high temperatures.

[0031] According to another aspect of the present invention, a method for preparing the above-mentioned nanozyme is provided, comprising: dissolving vanadium pentoxide, oxalic acid and titanium sulfate in ultrapure water, and heating to react to obtain nanozyme.

[0032] According to embodiments of the present invention, Ti / VO2 nanozymes with dual enzyme activities similar to NADH oxidase and peroxidase can be synthesized by a simple hydrothermal method. The synthesis method is simple, rapid, and highly efficient.

[0033] According to embodiments of the present invention, the mass ratio of vanadium pentoxide, oxalic acid, and titanium sulfate is 100-200:120-180:5-30, preferably 150:50:30. The reaction temperature is 150-200°C, for example, 150°C, 160°C, 170°C, 180°C, 190°C, or 200°C, preferably 180°C; the reaction time is 15-20 hours, for example, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours, preferably 16 hours.

[0034] In some specific embodiments of the present invention, a reaction temperature below 200°C can prevent vanadium dioxide from changing its configuration from B to A configuration if the temperature exceeds 200°C during synthesis.

[0035] According to an embodiment of the present invention, the post-processing method for nanozymes includes washing and drying. The washing agent used is ultrapure water and anhydrous ethanol, and the drying method is oven drying at a temperature of 60°C.

[0036] According to another aspect of the present invention, an application of the above-mentioned nanozyme in colorimetric detection of rare inherited metabolic diseases is provided, comprising: using the nanozyme to detect disease marker A, which can be oxidized by its own related oxidase to generate hydrogen peroxide, or using it to detect disease marker B, which can react with reduced nicotinamide adenine dinucleotide under the action of its related dehydrogenase. Specifically, disease marker A includes lactic acid, glucose, ethanol, uric acid, or tyrosine; disease marker B includes pyruvate, α-ketoglutarate, phenylalanine, α-ketoglutamate, acetoacetic acid, or oxaloacetic acid.

[0037] According to embodiments of the present invention, for hydrogen peroxide-related diseases, lactate is used as a disease marker to assess hyperlactatemia, glucose as a disease marker to assess diabetes, uric acid as a disease marker to assess hyperuricemia, and tyrosine as a disease marker to assess phenylketonuria; for NADH-related inherited metabolic rare diseases, pyruvate is used as a disease marker to assess hyperlactatemia, α-ketoglutarate as a disease marker to assess non-alcoholic fatty liver disease, phenylalanine as a disease marker to assess phenylketonuria, oxaloacetate as a disease marker to assess malonic acidemia, acetoacetate as a disease marker to assess diabetic ketoacidosis, and α-ketoglutamate as a disease marker to assess non-alcoholic fatty liver disease.

[0038] According to an embodiment of the present invention, a method for detecting disease biomarker A using nanozymes includes:

[0039] (1) Draw the standard curve

[0040] Disease biomarker A at different concentration gradients was co-incubated with disease biomarker A oxidase, and then TMB or ABTS, acetate-sodium acetate buffer and nanozyme were added. The absorbance value of the system at 652 nm was recorded, and a standard curve was plotted with the concentration of disease biomarker A as the x-axis and the absorbance value as the y-axis.

[0041] (2) Detection

[0042] Unknown concentrations of disease biomarker A and its oxidase were co-incubated, followed by the addition of 3,3',5,5'-tetramethylbenzidine, acetate-sodium acetate buffer, and nanozyme. The absorbance of the system at 652 nm was recorded, and the concentration of disease biomarker A was obtained through a standard curve.

[0043] In the detection of disease biomarker A, the oxidase for disease biomarker A specifically oxidizes it to generate hydrogen peroxide. Upon addition of nanozymes, it exhibits peroxidase-like activity, rapidly catalyzing the conversion of hydrogen peroxide into reactive oxygen species (ROS). The ROS then oxidizes TMB to generate a blue, oxidized form of 3,3',5,5'-tetramethylbenzidine (oxTMB) with strong absorption at 652 nm. The absorbance at 652 nm is then recorded using a UV-Vis spectrophotometer. A standard curve is plotted with the concentration of disease biomarker A on the x-axis and the absorbance at 652 nm on the y-axis. Using the same method, the concentration of disease biomarker A at unknown concentrations can be obtained by substituting its absorbance at 652 nm into the standard curve.

[0044] Furthermore, the dosage of each reagent can be:

[0045] (1) Draw the standard curve

[0046] 80-150 μL of 0.05-3 mM disease marker A and 30-100 μL of 50-100 μM disease marker A oxidase were co-incubated at 25-37°C for 5-15 min. Then, 50-100 μL of 10 mM TMB, 100-200 μL of pH 4-6 acetate-sodium acetate (HAc-NaAc) buffer, and 50-100 μL of 0.5 mg / mL nanozyme were added, and the mixture was incubated at 20-25°C for 15-30 min. The absorbance at 652 nm was recorded, and a standard curve was plotted with the concentration of disease marker A on the x-axis and the absorbance on the y-axis.

[0047] (2) Detection

[0048] 80-150 μL of disease biomarker A at an unknown concentration and 30-100 μL of 50-100 μM disease biomarker A oxidase were co-incubated. Then, 50-100 μL of 10 mM TMB, 100-200 μL of HAc-NaAc buffer (pH 4-6), and 50-100 μL of 0.5 mg / mL nanozyme were added and incubated together. The absorbance at 652 nm was then recorded using a UV-Vis spectrophotometer, and the concentration of disease biomarker A at the unknown concentration was obtained by substituting the values ​​into a standard curve.

[0049] According to an embodiment of the present invention, a method for detecting disease biomarker B using nanozymes includes:

[0050] (1) Draw the standard curve

[0051] Different concentration gradients of disease marker B, phosphate buffer, reduced nicotinamide adenine dinucleotide and disease marker B-related dehydrogenase were co-incubated, and then TMB or ABTS, acetate-sodium acetate buffer and the nanozyme were added. The absorbance value of the system at 652 nm was recorded, and a standard curve was plotted with the concentration of disease marker B as the x-axis and the absorbance value as the y-axis.

[0052] (2) Detection

[0053] An unknown concentration of disease biomarker B, phosphate buffer, reduced nicotinamide adenine dinucleotide, and disease biomarker B-related dehydrogenase were co-incubated. Subsequently, 3,3',5,5'-tetramethylbenzidine, acetate-sodium acetate buffer, and the nanozyme were added. The absorbance value of the system at 652 nm was recorded, and the concentration of the disease biomarker B was obtained through a standard curve.

[0054] In the detection of disease biomarker B, disease biomarker B-associated dehydrogenases catalyze the oxidation of disease biomarker B, consuming NADH and producing oxidized nicotinamide adenine dinucleotide (NAD). + After adding nanozymes, their NADH oxidase activity is first used to oxidize the remaining NADH from the previous steps to generate H₂O₂. Then, their peroxidase activity activates the generated hydrogen peroxide to form ROS. ROS oxidizes TMB to generate oxTMB, a blue substance with strong absorption at 652 nm. The absorbance of the system at 652 nm is recorded using a UV-Vis spectrophotometer. A standard curve is plotted with the concentration of disease biomarker B on the x-axis and the absorbance at 652 nm on the y-axis. The same method is used to detect unknown concentrations of disease biomarker B, and the absorbance at 652 nm is substituted into the standard curve to obtain the concentration of disease biomarker B.

[0055] Furthermore, the dosage of each reagent can be:

[0056] (1) Draw the standard curve

[0057] 80-150 μL of disease biomarker B (2-500 μM), 700-1000 μL of phosphate (Na₂HPO₄-KH₂PO₄) buffer (pH 6-9), 200-500 μL of 10 mM NADH, and 30-100 μL of 50-100 μM disease biomarker B-related dehydrogenase were co-incubated at 25-37°C for 5-10 min. Then, 50-100 μL of 10 mM TMB, 100-200 μL of HAc-NaAc buffer (pH 4-6), and 50-100 μL of 0.5 mg / mL nanozyme were added, and the mixture was incubated at 20-25°C for 15-30 min. The absorbance at 652 nm was recorded, and a standard curve was plotted with the concentration of disease biomarker B on the x-axis and the absorbance on the y-axis.

[0058] (2) Detection

[0059] 80-150 μL of disease biomarker B at an unknown concentration, 700-1000 μL of phosphate buffer (pH 6-9), 200-500 μL of 10 mM NADH, and 30-100 μL of 50-100 μM disease biomarker B-related dehydrogenase were co-incubated. Then, 50-100 μL of 10 mM TMB, 100-200 μL of HAc-NaAc buffer (pH 4-6), and 30-100 μL of 0.5 mg / mL nanozyme were added and co-incubated. The absorbance at 652 nm was recorded using a UV-Vis spectrophotometer, and the concentration of disease biomarker B at the unknown concentration was obtained by substituting the values ​​into a standard curve.

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Example 1: Synthesis of Nanozymes

[0062] 0.15 g V₂O₅, 0.12 g H₂C₂O₄, and 30 mg Ti(SO₄)₂ were dissolved in 50 mL of ultrapure water and stirred at room temperature for 1 hour. The solution was then transferred to a reaction vessel and reacted in an oven at 180 °C for 16 hours. The mixture was then washed three times with ultrapure water and ethanol, centrifuged, and dried in a vacuum oven at 60 °C to obtain the Ti / VO₂ nanozyme.

[0063] The elemental composition of the nanozyme was determined by inductively coupled plasma spectroscopy, revealing an atomic percentage of titanium of 1.491%. This 1.491% metal doping concentration ensures uniform distribution of the doped metal element within the crystal lattice, contributing to enhanced catalytic activity and ensuring structural stability. Figure 1 The scanning electron microscope images show that metal doping did not affect the morphology of the material.

[0064] The nanozymes were characterized by X-ray diffraction (XRD), and the results are as follows: Figure 2 As shown, the present invention successfully synthesized Ti-doped VO2, and the main crystal plane is (110) crystal plane.

[0065] Example 2: A method for synthesizing nanozymes

[0066] 0.1 g V₂O₅, 0.12 g H₂C₂O₄, and 5 mg Ti(SO₄)₂ were dissolved in 50 mL of ultrapure water and stirred at room temperature for 1 hour. The solution was then transferred to a reaction vessel and reacted in an oven at 200 °C for 18 hours. The mixture was then washed three times with ultrapure water and ethanol, collected by centrifugation, and dried in a vacuum oven at 60 °C to obtain the Ti / VO₂ nanozyme.

[0067] Example 3: A method for synthesizing nanozymes

[0068] 0.2 g V₂O₅, 0.18 g H₂C₂O₄, and 30 mg Ti(SO₄)₂ were dissolved in 50 mL of ultrapure water and stirred at room temperature for 1 hour. The solution was then transferred to a reaction vessel and reacted in an oven at 150 °C for 20 hours. The mixture was then washed three times with ultrapure water and ethanol, collected by centrifugation, and dried in a vacuum oven at 60 °C to obtain the Ti / VO₂ nanozyme.

[0069] Example 4: Testing the peroxidase-like activity of nanozymes

[0070] Three groups (a, b, and c) were set up. The following solutions were added to a pH 4 HAc-NaAc buffer: a) 100 μL 10 mM TMB + 100 μL 10 mM H2O2; b) 50 μL 10 mM Ti / VO2 + 100 μL 10 mM TMB; c) 50 μL 10 mM Ti / VO2 + 100 μL 10 mM TMB + 100 μL 10 mM H2O2. The reactions were allowed to proceed for 10 min, and the absorbance at 652 nm was recorded for each system. The results are shown below. Figure 3 See Table 1.

[0071] Table 1

[0072]

[0073] like Figure 3 As shown in Table 1, the solution in group a did not turn blue under the TMB+H2O2 condition; the absorbance of the system in group b under the TMB+Ti / VO2 condition approached 0; the solution in group c under the Ti / VO2+TMB+H2O2 condition turned blue and showed a significant absorption peak at 652 nm. This confirms that Ti / VO2 has peroxidase-like activity and can catalyze the decomposition of H2O2 to generate reactive oxygen species, driving the oxidation of TMB to turn blue.

[0074] Example 5: Testing the NADH oxidase-like activity of nanozymes

[0075] (1) Experimental group: 350 μL of 10 mM NADH and 50 μL of 10 mM Ti / VO2 were added to 700 μL of HAc-NaAc buffer solution with pH 4 and reacted for 15 min. The UV-Vis absorption spectrum was recorded.

[0076] (2) Control group: 400 μL of 10 mM NADH was added to 700 μL of HAc-NaAc buffer solution with pH 4, and its UV-Vis absorption spectrum was recorded.

[0077] The results are as follows Figure 4 As shown in Table 2.

[0078] Table 2

[0079]

[0080] according to Figure 4 As shown in Table 2, the characteristic absorbance of the experimental group decreased at 340 nm, indicating that NADH was consumed by the reaction, and Ti / VO2 successfully catalyzed the oxidation of NADH to NAD. + .

[0081] Further, three groups were set up: a, b, and c. a) 350 μL 10 mM NADH + 50 μL 10 mM HRP + 50 μL 10 mM TMB; b) 50 μL 10 mM Ti / VO2 + 50 μL 10 mM HRP + 100 μL 10 mM TMB; c) 350 μL 10 mM NADH + 50 μL 10 mM Ti / VO2 + 50 μL 10 mM HRP + 100 μL 10 mM TMB. All three groups were added to 700 μL of HAc-NaAc buffer at pH 4, and their UV absorbance values ​​were measured. The results are as follows: Figure 5 As shown in Table 3.

[0082] Table 3

[0083]

[0084] according to Figure 5As shown in Table 3, Ti / VO2 can only oxidize NADH to hydrogen peroxide when NADH and Ti / VO2 coexist. Then, the oxidizing property of HRP is used to activate the generated hydrogen peroxide to form ROS. ROS oxidizes TMB to generate oxTMB with strong absorption at a wavelength of 652 nm.

[0085] This example demonstrates the NADH-like oxidase activity of Ti / VO2, which can effectively catalyze the oxidation of NADH by O2 to generate NAD. + And hydrogen peroxide.

[0086] Example 6: Testing the steady-state kinetics of nanozymes

[0087] (1) In this embodiment, the reaction kinetics of nanozyme with H2O2 were first investigated. The concentration of substrate TMB was kept at 1 mM, while the concentration of H2O2 was changed. The specific steps are as follows:

[0088] 100 μL of 0.5 mg / mL nanozyme, 100 μL of 10 mM TMB, and 100 μL of H2O2 at concentrations of 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM were added to 700 μL of HAc-NaAc buffer at pH 4. The mixture was incubated at 35 °C for 15 min, and the absorbance at 652 nm was recorded for each system. A standard curve was plotted. The results are shown below. Figure 6 As shown.

[0089] (2) This embodiment also investigated the reaction kinetics of nanozymes with the substrate TMB, keeping the concentration of H2O2 at 1 mM and changing the concentration of TMB. The specific steps are as follows:

[0090] 100 μL of 0.5 mg / mL nanozyme, 100 μL of TMB at gradient concentrations of 0.5 mM, 1 mM, 2.5 mM, 5 mM, 7.5 mM, and 10 mM, and 100 μL of 10 mM H2O2 were added to 700 μL of HAc-NaAc buffer at pH 4. The mixture was incubated at 35 °C for 15 min, and the absorbance values ​​at 652 nm were recorded. A standard curve was plotted, and the results are shown below. Figure 7 As shown.

[0091] (3) This embodiment also investigated the reaction kinetics of the natural enzyme horseradish peroxidase (HRP) with H2O2, keeping the concentration of substrate TMB at 1 mM and changing the concentration of H2O2. The specific steps are as follows:

[0092] Add 100 μL of 0.5 mg / mL HRP, 100 μL of 10 mM TMB, and 100 μL of H2O2 with gradient concentrations of 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM to 700 μL of HAc-NaAc buffer at pH 4. Incubate at 35 °C for 15 min and record the absorbance values ​​of the corresponding systems at 652 nm. Plot a standard curve. The results are shown below. Figure 8 As shown.

[0093] (4) This embodiment also investigated the reaction kinetics of HRP on the substrate TMB, keeping the concentration of H2O2 at 1 mM and changing the concentration of TMB. The specific steps are as follows:

[0094] Add 100 μL of 0.5 mg / mL HRP, 100 μL of TMB at gradient concentrations of 0.5 mM, 1 mM, 2.5 mM, 5 mM, 7.5 mM, and 10 mM, and 100 μL of 10 mM H2O2 to 700 μL of HAc-NaAc buffer at pH 4. Incubate at 35 °C for 15 min and record the absorbance values ​​of the corresponding systems at 652 nm. Plot a standard curve. The results are shown below. Figure 9 As shown.

[0095] To quantitatively assess the differences in the binding ability of nanozymes to substrates and their catalytic activity, steady-state kinetic parameters were determined based on the Michaelis equation: V=Vmax[S]Km+[S].

[0096] Where V is the initial reaction rate, Vmax is the maximum reaction rate, [S] is the substrate concentration, and Km is the Michaelis constant.

[0097] After recording the absorbance of the system at 652 nm using a UV-Vis spectrophotometer, curves were plotted by changing the substrate concentration, and Km and Vmax were calculated by fitting the data. The smaller the Km value, the better the affinity of the material for the substrate; the higher the Vmax, the faster the catalytic reaction rate.

[0098] like Figure 6 and Figure 7 As shown, the Km values ​​of the Ti / VO2 nanozymes with dual enzyme activity for H2O2 and TMB are 0.96 mM and 0.12 mM, respectively, both lower than those of the natural enzyme horseradish peroxidase (HRP) (3.70 mM and 0.43 mM). The lower Km values ​​indicate that the Ti / VO2 nanozymes have a high affinity for their respective substrates.

[0099] The maximum reaction rate Vmax was calculated to be 6.17 × 10⁻⁶ for H₂O₂ and TMB, respectively. -7 M·s -1 and 4.75×10 -7M·s -1 All were higher than the 0.87 × 10⁻⁶ of the natural enzyme HRP. -7 M·s -1 and 1.00×10 -7 M·s -1 This clearly demonstrates that Ti doping promotes enhanced POD-like activity, further accelerating the reaction rate of the catalytic process.

[0100] Example 7: Detection of lactate and pyruvate, markers of hyperlactatemia, by nanozymes

[0101] (1) This embodiment first explored the feasibility analysis of nanozymes for lactic acid detection, as follows:

[0102] A lactate detection system was established using lactate oxidase and a Ti / VO2 cascade reaction.

[0103] Add 150 μL of lactate at different concentrations (0.1, 0.3, 0.5, 0.7, 1.0, 1.2 mM) and 100 μL of 50 μM lactate oxidase, and incubate at 37°C for 10 minutes. In this step, lactate oxidase specifically oxidizes lactate to produce hydrogen peroxide.

[0104] 700 μL of HAc-NaAc buffer (pH 4), 100 μL of 0.5 mg / mL nanozyme, and 100 μL of 10 mM TMB were added sequentially, and the reaction was carried out at room temperature for 15 min. The nanozyme exhibited peroxidase-like activity, rapidly catalyzing the conversion of hydrogen peroxide to ROS. Subsequently, the ROS oxidized TMB to generate oxTMB, which has strong absorption at 652 nm. The absorbance of the system at 652 nm was recorded using a UV-Vis spectrophotometer, and the results are as follows: Figure 8 As shown in Table 4.

[0105] Table 4

[0106]

[0107] A standard curve was plotted with lactic acid concentration on the x-axis and absorbance at 652 nm on the y-axis. The results are as follows: Figure 9 As shown.

[0108] according to Figure 8 , Figure 9 As shown in Table 4, TMB is oxidized to form the blue product oxTMB. As the lactic acid concentration increases, the blue color of the solution gradually deepens, and the absorbance at 652 nm gradually increases.

[0109] To detect lactate of unknown concentration: Add 150 μL of lactate of unknown concentration and 100 μL of 50 μM lactate oxidase, and incubate at 37 °C for 10 min. After the reaction, add 700 μL of HAc-NaAc buffer (pH 4), 100 μL of 0.5 mg / mL nanozyme, and 100 μL of 10 mM TMB sequentially, and react at room temperature for 15 min. Record the absorbance at 652 nm and substitute it into the standard curve to obtain the lactate concentration.

[0110] (2) This embodiment also explored the feasibility analysis of nanozymes for the detection of pyruvate, as follows:

[0111] A colorimetric detection system for pyruvate was established using a cascade reaction of L-lactic acid dehydrogenase (LDH) and nanozyme.

[0112] Add 150 μL of pyruvate at different concentrations (0, 20, 60, 100, 150, 200 μM), 700 μL of phosphate buffer (pH 8), 100 μL of NADH (10 mM), and 50 μL of lactate dehydrogenase (100 μM), and incubate at 37°C for 5 min. In the first step, LDH catalyzes the oxidation of pyruvate, consuming NADH and producing NAD. + .

[0113] 700 μL of HAc-NaAc buffer (pH 4), 100 μL of 0.5 mg / mL nanozyme, and 100 μL of 10 mM TMB were added sequentially, and the mixture was reacted at room temperature for 15 minutes. The nanozyme, exhibiting NADH oxidase-like activity, oxidized the remaining NADH to generate H₂O₂. Then, its peroxidase-like activity rapidly catalyzed the conversion of hydrogen peroxide to ROS. Subsequently, the ROS oxidized TMB to generate oxTMB, which exhibits strong absorption at 652 nm. The absorbance at 652 nm was recorded using a UV-Vis spectrophotometer. The results are shown below. Figure 10 As shown in Table 5.

[0114] Table 5

[0115]

[0116] With a fixed initial NADH concentration, increasing pyruvate concentration promotes the conversion of NADH to LDH, thus reducing the NADH available to the nanozyme. This competition leads to a decrease in H₂O₂ production, resulting in a drop in absorbance at 652 nm. Pyruvate concentration is inversely proportional to the intensity of the colorimetric signal, making the quantification of pyruvate possible. The absorbance at 652 nm was recorded, and a standard curve was plotted with pyruvate concentration on the x-axis and absorbance at 652 nm on the y-axis. The results are shown below. Figure 11 As shown.

[0117] according to Figure 10 , Figure 11 As shown in Table 5, the absorbance at 652 nm gradually decreases with the increase of pyruvate concentration.

[0118] To detect pyruvate of unknown concentration: Add 150 μL of pyruvate of unknown concentration, 700 μL of phosphate buffer (pH 8), 100 μL of 10 mM NADH, and 50 μL of 100 μM lactate dehydrogenase, and incubate at 37°C for 5 min. After the reaction, add 700 μL of HAc-NaAc buffer (pH 4), 100 μL of 0.5 mg / mL nanozyme, and 100 μL of 10 mM TMB sequentially, and react at room temperature for 15 min. Record the absorbance at 652 nm and substitute it into the standard curve to obtain the concentration of pyruvate.

[0119] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanozyme, characterized in that: The nanozyme is vanadium oxide doped with metal and exists in the form of nanoribbons; the metal is titanium. The titanium is atomically loaded onto the vanadium oxide, and the atomic percentage of titanium is 1%-2%. The nanozyme has a (110) crystal plane.

2. A method for preparing nanozymes as described in claim 1, characterized in that, The nanozyme was obtained by dissolving vanadium pentoxide, oxalic acid, and titanium sulfate in ultrapure water and heating the mixture.

3. The preparation method according to claim 2, characterized in that: The mass ratio of vanadium pentoxide, oxalic acid and titanium sulfate is 100-200:120-180:5-30.

4. The preparation method according to claim 2, characterized in that: The temperature of the heating reaction is 150-200℃.

5. The preparation method according to claim 2, characterized in that: The reaction time is 15-20 hours.

6. The application of a nanozyme as described in claim 1 or 2 in the colorimetric detection of rare inherited metabolic diseases, characterized in that: The nanozyme is used to detect disease marker A, which can be oxidized by its own related oxidase to generate hydrogen peroxide, or to detect disease marker B, which can react with reduced nicotinamide adenine dinucleotide under the action of its related dehydrogenase.

7. The application according to claim 6, characterized in that: The disease marker A includes lactic acid, glucose, ethanol, uric acid, or tyrosine; The disease marker B includes pyruvate, α-ketoglutarate, phenylalanine, α-ketoglutamate, acetoacetic acid, or oxaloacetic acid.

8. The application according to claim 6, characterized in that: The method for detecting disease biomarker A using nanozymes includes: (1) Draw the standard curve Disease biomarker A at different concentration gradients was co-incubated with disease biomarker A oxidase, followed by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) or 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), acetate-sodium acetate buffer, and the nanozyme. The absorbance value of the system at 652 nm was recorded, and a standard curve was plotted with the concentration of disease biomarker A on the x-axis and the absorbance value on the y-axis. (2) Detection Unknown concentrations of disease biomarker A and its oxidase were co-incubated, followed by the addition of 3,3',5,5'-tetramethylbenzidine, acetate-sodium acetate buffer, and the nanozyme. The absorbance of the system at 652 nm was recorded, and the concentration of disease biomarker A was obtained through a standard curve. The method for detecting disease biomarker B using nanozymes includes: (1) Draw the standard curve Different concentration gradients of disease marker B, phosphate buffer, reduced nicotinamide adenine dinucleotide and disease marker B-related dehydrogenase were co-incubated, and then TMB or ABTS, acetate-sodium acetate buffer and the nanozyme were added. The absorbance value of the system at 652 nm was recorded, and a standard curve was plotted with the concentration of disease marker B as the x-axis and the absorbance value as the y-axis. (2) Detection An unknown concentration of disease biomarker B, phosphate buffer, reduced nicotinamide adenine dinucleotide, and disease biomarker B-related dehydrogenase were co-incubated. Subsequently, 3,3',5,5'-tetramethylbenzidine, acetate-sodium acetate buffer, and the nanozyme were added. The absorbance value of the system at 652 nm was recorded, and the concentration of the disease biomarker B was obtained through a standard curve.

9. The application according to claim 8, characterized in that: In the method for detecting disease biomarker A using nanozymes, the incubation temperature is 25-37°C, the incubation time is 5-15 min, and the pH of the acetate-sodium acetate buffer solution is 4-6.

10. The application according to claim 8, characterized in that: In the method for detecting disease biomarker B using nanozymes, the pH of the phosphate buffer is 6-9, the incubation temperature is 25-37℃, the incubation time is 5-10 min, and the pH of the acetate-sodium acetate buffer is 4-6.

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