Nanozyme and its preparation method and application

Abstract

The present invention discloses a nanozyme, its preparation method, and application. The nanozyme comprises a metal alloy and cuprous oxide bound to the surface of the metal alloy. This nanozyme is a superoxide dismutase nanozyme, which exhibits excellent stability, catalytic efficacy, and high catalytic rate, making it suitable for scavenging reactive oxygen species. This nanozyme addresses the prior art issues of liquid metals being unused in enzyme-mimicking catalysis and the low catalytic activity of existing superoxide dismutase mimics.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme catalysis technology, and more specifically, to a nanozyme, a preparation method thereof, and an application thereof. Background Art

[0002] Most natural enzymes are protein structures with extremely high catalytic activity and selectivity. However, their protein structure makes them easily inactivated and unstable, and the cost of large-scale industrialization is too high.

[0003] These limitations of natural biological enzymes have greatly restricted their practical applications in biomedicine, food safety and other fields. It is precisely for these reasons that the emergence and development of various enzyme mimics (or artificial enzymes) have been promoted.

[0004] Nanozymes are a class of nanomaterials that can catalyze enzyme substrates under mild or extreme conditions and convert them into products following enzyme kinetics (such as the Michaelis-Menten equation). They have gradually become a new generation of enzyme mimics that can replace natural enzymes. Compared with natural biological enzymes, nanozymes can catalyze chemical reactions efficiently and stably under mild physiological conditions, and have the advantages of being easy to prepare and store, and can be prepared on a large scale. Therefore, high-performance nanomaterial enzyme mimics have the potential to make significant progress in the fields of biotechnology and catalytic science. The development of new enzyme mimics has broad biological scientific significance and biomedical practical application value, and will have a huge impact in areas such as cell detection, biosensors, immunoassays, cancer diagnosis and treatment.

[0005] As the first line of defense against damage mediated by reactive oxygen species (ROS), superoxide dismutase (SOD) is expected to play an important role in the treatment of oxidative stress-related diseases. However, its unstable structure and high cost severely limit its clinical application. Nanozymes, on the other hand, perfectly address these shortcomings, offering advantages such as greater stability, greater cost-effectiveness, easier modification, and adjustable activity. Due to their excellent properties, nanozymes have received widespread attention in recent years and are expected to become effective alternatives to natural enzymes in many applications. Importantly, several nanozymes with SOD-like activity have been developed and demonstrated to have promising therapeutic effects on diseases caused by oxidative stress. As of January 2021, there were over 3,000 studies related to nanozymes, while fewer than 200 studies related to SOD-like activity. Clearly, SOD nanozymes are severely underdeveloped compared to other nanozymes. Furthermore, the catalytic activity of SOD nanozymes reported to date is not high, and generally does not exceed the activity of natural SOD enzymes. Summary of the Invention

[0006] Based on the above facts, the present invention aims to provide a nanozyme, a preparation method, and applications thereof. The nanozyme is a superoxide dismutase nanozyme with good stability, good catalytic effect, and high catalytic rate, suitable for scavenging reactive oxygen species. This nanozyme solves the problem that liquid metals have not been applied to the field of enzyme mimics in the prior art, and the catalytic activity of existing superoxide dismutase mimics is low.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides a nanozyme, wherein the structure of the nanozyme comprises a metal alloy and cuprous oxide bound to the surface of the metal alloy.

[0009] Furthermore, the metal alloy is selected from one of gallium-tin alloy, gallium-indium alloy, and gallium-indium-tin alloy. The metal alloy is preferably gallium-indium alloy, in which case the SOD enzyme activity of the nanozyme is the highest.

[0010] There is no research on the use of liquid metal and cuprous oxide in enzyme catalysis. After in-depth research, the inventors found that the combination of liquid metal and cuprous oxide can act as a superoxide dismutase mimic to achieve ultra-high catalytic effects, while also creating a new field for superoxide dismutase simulation. In addition, after in-depth research, it was found that the original cuprous oxide and liquid metal often lack catalytic active sites and do not have superoxide dismutase activity, which greatly limits the application of liquid metal and cuprous oxide in enzyme catalysis. Therefore, in order to fully develop and utilize the excellent performance of liquid metal and cuprous oxide in the field of nanozymes, and to enrich the field of superoxide dismutase simulation and improve the current superoxide dismutase mimic activity, the above situation is made possible by regulating the structure and performance of the two. In addition, in the technical solution of the present invention, the nanozyme shows excellent superoxide dismutase activity, has a low cost, and can achieve the ability to efficiently decompose reactive oxygen species that is difficult to achieve with other materials, which is conducive to expanding the synthesis method of ultra-high-efficiency superoxide dismutase nanozymes and their further application and promotion.

[0011] Furthermore, the mass ratio of cuprous oxide to metal alloy is 130:1-270:1. Exemplarily, the mass ratio of cuprous oxide to metal alloy includes but is not limited to 130:1, 190:1-270:1, 190:1-250:1, 270:1, etc. By controlling the mass ratio of the two, the nanozyme exhibits excellent superoxide dismutase activity.

[0012] Furthermore, in the metal alloy, the mass percentage of gallium is 70%-80%.

[0013] Furthermore, in the metal alloy, the mass ratio of gallium to indium is 75.5:24.5.

[0014] Furthermore, the size of the nanozyme is 200-600 nm. It is understood that the size of the nanozyme generally refers to its diameter.

[0015] In another aspect, the present invention provides a method for preparing the nanozyme as described above, comprising the following steps:

[0016] The liquid metal alloy is mixed with an aqueous solution of copper sulfate, and then subjected to ultrasonic crushing, centrifugation, and freeze-drying to obtain the nanozyme.

[0017] The liquid metal alloy is the aforementioned metal alloy in liquid form and can be purchased commercially or prepared using a publicly available method.

[0018] Furthermore, when the liquid metal alloy is a gallium-indium alloy, the preparation method thereof comprises the following steps:

[0019] Mixing 99.9% pure metal gallium and indium;

[0020] The resulting mixture is heated under vacuum until melted;

[0021] The heated and molten metal mixture is stirred until no precipitate appears to obtain a liquid gallium-indium alloy.

[0022] Furthermore, the vacuum heating temperature is 100° C. and the heating time is 3 hours.

[0023] Furthermore, the aqueous solution of copper sulfate is an acidified aqueous solution of copper sulfate, with a pH of 2-4, and a concentration of 0.2-0.4 mol / L. The use of an aqueous solution of copper sulfate of this concentration can make the obtained nanozyme have higher activity. Exemplarily, the concentration of the aqueous solution of copper sulfate includes but is not limited to 0.2 mol / L, 0.3-0.4 mol / L, 0.4 mol / L, etc.

[0024] Acidifying the aqueous solution of copper sulfate can prevent oxidation of the liquid metal alloy. Preferably, the acidifying acid is a strong acid or a weak acid. To avoid the introduction of other ionic impurities, the acid is preferably sulfuric acid.

[0025] Furthermore, the preparation of the aqueous solution of acidified copper sulfate comprises the following steps:

[0026] S1, adding dilute sulfuric acid dropwise into a copper sulfate aqueous solution to obtain a preliminarily acidified copper sulfate aqueous solution;

[0027] S2. Ultrasonicate the preliminary copper sulfate aqueous solution in S1 to obtain the acidified copper sulfate aqueous solution.

[0028] Furthermore, it should be noted that, in the above S1, the concentration of dilute sulfuric acid is 0.2 mol / L.

[0029] Furthermore, the volume ratio of the copper sulfate aqueous solution to the liquid metal alloy is 50:(0.05-0.5), preferably 50:(0.1-0.5).

[0030] Furthermore, the ultrasonic disruption method is: using 70% power continuous ultrasonication for 24 minutes.

[0031] Furthermore, the temperature of the ultrasonic crushing is lower than 150°C.

[0032] Furthermore, the centrifugation specifically comprises the following steps:

[0033] The mixture obtained after ultrasonic fragmentation was centrifuged at 900 rpm for 3 minutes, and the supernatant was taken and continued to be centrifuged at 3000 rpm; the supernatant was removed, and deionized water was added to obtain a second mixed solution, the second mixed solution was ultrasonicated, and the second mixed solution after the ultrasonication was completed was centrifuged at 4000 rpm to obtain a cuprous oxide-gallium indium nanozyme mixed solution; the supernatant was removed to obtain wet cuprous oxide-gallium indium nanozyme.

[0034] Furthermore, the freeze-drying conditions are to freeze-dry the wet nanozyme in a freeze drying chamber for 4 hours.

[0035] In the preparation method of the present invention, ultrasonic crushing is directly carried out in a copper sulfate aqueous solution. Even in the ultrasonic crushing process in a high-temperature oxygen environment, Cu 2+ Can also be reduced to Cu + , and finally made into composite nanoparticles.

[0036] Furthermore, the ultrasonic crushing time is set to 24 minutes, because a shorter time will lead to incomplete reaction of the gallium-indium alloy, and a longer time will lead to oxidation of some cuprous metals. The ultrasonic crusher includes but is not limited to selecting the CONTANT mode, i.e., continuous ultrasonication.

[0037] Furthermore, during the ultrasonic fragmentation process, the reaction will be affected due to the heat generated by ultrasound. To eliminate the influence, the present invention chooses to perform ultrasonic fragmentation in an ice bath environment, and the ice bath is replaced every 12 minutes.

[0038] In another aspect, the present invention provides use of the nanozyme as described above in scavenging reactive oxygen species.

[0039] Furthermore, the application includes using the nanozyme to catalyze active oxygen to generate hydrogen peroxide.

[0040] Furthermore, nanozymes are used to mimic superoxide dismutase.

[0041] Furthermore, when the nanozyme is used as a superoxide dismutase simulation, the nanozyme is dispersed in water, ultrasonicated for 10 minutes, and shaken to allow the nanozyme to be better dispersed in the solution and fully exert its catalytic effect.

[0042] Furthermore, the step of determining the rate at which the active enzyme scavenges reactive oxygen species comprises:

[0043] dissolving the nanozyme in deionized water to obtain an aqueous solution of the nanozyme;

[0044] The aqueous solution of the nanozyme was added to a total SOD activity detection kit (WST-8 method) to carry out the reaction;

[0045] The absorbance of the total SOD activity detection kit (WST-8 method) added with cuprous oxide-gallium indium nanozyme was measured at 450 nm using a microplate reader;

[0046] The specific activity value can be obtained by calculating the test results.

[0047] Furthermore, in the application, the reaction temperature is 37°C.

[0048] Furthermore, in the application, the reaction time is 10-50 min. Exemplarily, the reaction time includes but is not limited to 20-40 min, 30-40 min, 40 min, etc., in which case the catalytic activity of the nanozyme is higher.

[0049] Furthermore, in the application, the concentration of the nanozyme in the aqueous solution is 0.5-3 mg / ml. Exemplarily, the concentration of the nanozyme includes but is not limited to 0.5-2 mg / ml, 0.5-1 mg / ml, 1-3 mg / ml, 1-2 mg / ml, etc. In this case, the nanozyme has the best effect in catalyzing the removal of reactive oxygen species.

[0050] The beneficial effects of the present invention are as follows:

[0051] The nanozyme structure provided by the present invention incorporates cuprous oxide coating on a metal alloy, overcoming the limitations of existing superoxide dismutase nanozymes, which suffer from low catalytic activity, achieving efficient and rapid scavenging of reactive oxygen species. Furthermore, the nanozyme, with its nanoscale size and large specific surface area, provides abundant binding and catalytic sites for the catalytic reaction. Its activity far exceeds that of natural superoxide dismutase, overcoming the limitations of natural enzymes and demonstrating enormous potential for application in industry, medicine, biology, and other fields.

[0052] The preparation method of the nanozyme provided in the present invention is simple to prepare, avoids the complicated preparation process of existing nanozymes, and is low-cost and easy to scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0054] Figure 1 The ICP-AES test results of the nanozyme described in Example 1 are shown.

[0055] Figure 2 The XRD pattern of the nanozyme described in Example 1 is shown.

[0056] Figure 3 (a) and (b) are the SEM test results of the nanozyme prepared in Example 1; (c) is the TEM test result of the nanozyme prepared in Example 9.

[0057] Figure 4 A diagram showing the gallium-based liquid metal cuprous oxide-indium nanozyme of the present invention and its catalytic decomposition process of reactive oxygen species. DETAILED DESCRIPTION

[0058] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the following detailed description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0059] Example 1

[0060] A nanozyme, the preparation method of which is as follows:

[0061] Prepare 50mL of a 0.4mol / L copper sulfate aqueous solution, add 1mL of a 0.2mol / L sulfuric acid solution for acidification, add 100μl of a gallium-indium liquid metal alloy (the mass ratio of gallium to indium is 75.5:24.5) to the above solution, and use an ultrasonic crusher to continuously sonicate for 24 minutes at 70% amplitude in an ice bath environment, and replace the ice bath every 12 minutes; the sonicated liquid metal particle suspension is centrifuged at low speed (900rpm for 3 minutes; the supernatant is taken and continued to be centrifuged at 3000rpm; the supernatant is removed and 40ml of deionized water is added to obtain a mixed solution, which is placed in an ultrasonic cleaning machine and sonicated at maximum power for 3 minutes) to remove large particle molecules, and then centrifuged at 4000rpm, the supernatant is removed, and freeze-dried to obtain the nanozyme.

[0062] The ICP-AES test results of the nanozyme are shown in FIG. Figure 1 As shown in the figure, it can be seen that in the nanozyme, the proportions of copper, gallium and indium are 99.33%, 0.56% and 0.11% respectively.

[0063] Figure 2The XRD pattern of the nanozyme described in Example 1 is shown. As can be seen from the figure, the nanozyme components include Cu2O in addition to the metal alloy.

[0064] Figure 3 Figures a and b show the SEM images of the nanozyme described in Example 1, respectively.

[0065] Figure 4 A diagram showing the gallium-based liquid metal cuprous oxide-indium nanozyme of the present invention and its catalytic decomposition process of reactive oxygen species.

[0066] A method for using liquid gallium-indium alloy, cuprous oxide, and the nanozyme as simulated peroxidase to test its catalytic activity:

[0067] (1) Dispersing the liquid gallium-indium alloy, cuprous oxide, and the nanozyme into water (the mass-to-volume ratio of the original liquid metal, cuprous oxide, the nanozyme, and water was 1 mg / mL), and ultrasonicating for a period of time to obtain the original liquid metal dispersion (hereinafter referred to as 1# dispersion), the cuprous oxide dispersion (hereinafter referred to as 2# dispersion), and the nanozyme dispersion (hereinafter referred to as 3# dispersion); wherein the original liquid metal, cuprous oxide, and the nanozyme were dispersed into water and ultrasonicated for 20 minutes;

[0068] (2) 20 μL of each of the 1# dispersion, 2# dispersion, and 3# dispersion was added to the total SOD activity detection kit (WST-8 method) and incubated at 37°C for 30 min;

[0069] (3) The incubated total SOD activity detection kit (WST-8 method) was used to measure its absorbance at 450 nm using a microplate reader. The test results were calculated using the formula provided with the kit to obtain the SOD enzyme activity. The results were as follows: the catalytic activity of the original liquid gallium-indium alloy was -802 U / mg, which was a negative value, i.e., no catalytic activity; the catalytic activity of cuprous oxide was 7.1 U / mg, and the activity of cuprous oxide-gallium-indium nanozyme was 1995.4 U / mg, indicating that the cuprous oxide-gallium-indium nanozyme prepared using gallium-indium liquid metal had specific superoxide dismutase activity and was extremely active.

[0070] The formula for the above kit is as follows:

[0071] Inhibition percentage = [(A blank control 1-A blank control 2)-(A sample-A blank control 3)] / (A blank control 1-A blank control 2)×100%

[0072] SOD enzyme activity units in the test sample = SOD enzyme activity units in the detection system = inhibition percentage / (1-inhibition percentage) units

[0073] A is the light intensity.

[0074] In addition, the activity of superoxide dismutase in the liver, the most active part of the human body, tested using the above method was 324 U / mg, which was also far lower than the effect in Example 1.

[0075] Example 2-Example 5

[0076] The same as Example 1, except that when the prepared nanozyme was used as a simulated peroxidase to test its catalytic activity, the incubation time at 37°C in step (2) was changed to 10 min, 20 min, 40 min, and 50 min, respectively, while the other conditions remained unchanged. The measured activities of the nanozyme were 1143.8 U / mg, 1483.8 U / mg, 2965 U / mg, and 945.4 U / mg, respectively.

[0077] Example 6-Example 8

[0078] The same as Example 1, except that when the prepared nanozyme was used as a simulated peroxidase to test its catalytic activity, the mass volume ratio of nanozyme to water in the nanozyme dispersion prepared in step (1) was 0.25 mg / mL, 0.5 mg / mL and 2 mg / mL, respectively, and the other conditions remained unchanged. The measured activities of the nanozyme were -99.4 U / mg (negative value means no activity), 178.6 U / mg and 27.1 U / mg, respectively.

[0079] Example 9-Example 10

[0080] The same as Example 1, except that the concentrations of the copper sulfate aqueous solution prepared during the nanozyme preparation were 0.2 mol / L and 0.3 mol / L, respectively, and all other conditions remained unchanged. The measured nanozyme activities were 423.3 U / mg and 765.4 U / mg, respectively. Figure 3 Middle c shows the TEM image of the nanozyme described in Example 9.

[0081] Example 11

[0082] A nanozyme, the preparation method of which is as follows:

[0083] A 50 mL 0.2 mol / L copper sulfate aqueous solution was prepared, and 1 mL 0.2 mol / L sulfuric acid solution was added for acidification. 100 μL of a gallium-tin liquid metal alloy (gallium:tin mass ratio of 75.5:24.5) was added to the solution. The solution was sonicated continuously in an ice bath at 70% amplitude using an ultrasonic disruptor for 24 minutes, with the ice bath replaced every 12 minutes. The sonicated liquid metal particle suspension was subjected to low-speed centrifugation (900 rpm for 3 minutes; the supernatant was removed and further high-speed centrifugation at 3000 rpm; the supernatant was removed and 40 mL of deionized water was added to obtain a mixed solution. The mixed solution was then placed in an ultrasonic cleaner and sonicated at maximum power for 3 minutes) to remove large particles. The solution was then subjected to high-speed centrifugation at 4000 rpm, the supernatant removed, and freeze-dried to obtain the nanozyme. The activity of the nanozyme was measured to be 15.17781 U / mg.

[0084] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing a nanozyme, characterized in that: The structure of the nanozyme comprises a metal alloy and cuprous oxide bound to the surface of the metal alloy; the metal alloy is selected from one of a gallium-tin alloy, a gallium-indium alloy, and a gallium-indium-tin alloy; The preparation method comprises the following steps: The liquid metal alloy is mixed with an aqueous solution of copper sulfate, and then subjected to ultrasonic crushing, centrifugation, and freeze-drying to obtain the nanozyme; The aqueous solution of copper sulfate is an acidified copper sulfate aqueous solution with a pH of 2-4; The ultrasonic disruption was carried out by continuous ultrasonication for 24 minutes at 70% amplitude in an ice bath environment using an ultrasonic disruptor.

2. The preparation method according to claim 1, characterized in that The mass ratio of the cuprous oxide to the metal alloy is 130:1-270:

1.

3. The preparation method according to claim 2, characterized in that In the metal alloy, the mass percentage of gallium is 70%-80%.

4. The preparation method according to any one of claims 1 to 3, characterized in that The size of the nanozyme is 200-600nm.

5. The preparation method according to claim 1, characterized in that The concentration of the copper sulfate aqueous solution is 0.2-0.4 mol / L; and / or The volume ratio of the copper sulfate aqueous solution to the liquid metal alloy is 50:(0.1-0.5).

6. The preparation method according to claim 1, characterized in that The centrifugation specifically comprises the following steps: The mixture obtained after ultrasonic disruption was centrifuged at 900 rpm for 3 minutes, and the supernatant was taken and further centrifuged at 3000 rpm; the supernatant was removed, and deionized water was added to obtain a second mixed solution, the second mixed solution was ultrasonicated, and the second mixed solution after ultrasonication was centrifuged at 4000 rpm to obtain a cuprous oxide-gallium indium nanozyme mixed solution; The supernatant was removed to obtain wet copper oxide-gallium indium nanozyme.

7. Use of the nanozyme prepared by the preparation method according to any one of claims 1 to 6 in the scavenging of reactive oxygen species.

8. The use according to claim 7, characterized in that The application is to use the nanozyme to catalyze active oxygen to generate hydrogen peroxide.

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