Preparation method of manganese dioxide nanozyme and its application in disease detection kit

Abstract

The present invention discloses a method for preparing a manganese dioxide nanozyme and an application of the manganese dioxide nanozyme in a disease detection kit. The method for preparing the manganese dioxide nanozyme comprises the following steps: uniformly mixing a fish peptone solution containing fish peptone powder with a potassium permanganate aqueous solution to obtain a mixed solution, stirring the mixed solution until it turns brown, and sequentially centrifuging, washing, and drying to obtain the manganese dioxide nanozyme. The manganese dioxide nanozyme has an ultrasmall particle size of 2-5 nm, overcoming the problem of small particle size and easy agglomeration of traditional nanozymes, and has excellent dispersibility and stability. In addition, the manganese dioxide nanozyme prepared using fish peptone powder has high peroxidase-like activity and stability, and is 2.36 times higher than the HRP enzyme activity in an ELISA kit for brucellosis detection.

Description

Technical Field

[0001] The present invention belongs to the fields of material technology and biomedical detection, and specifically relates to a method for preparing a manganese dioxide nanozyme and its application in a disease detection kit. Background Art

[0002] Enzyme-linked immunosorbent assay (ELISA) kits are highly sensitive detection technologies based on antigen-antibody specific reactions and are widely used in biology, medicine, and food testing. Horseradish peroxidase (HRP) is commonly used as a colorimetric marker, co-catalyzed with hydrogen peroxide to react with a substrate (such as 3,3',5,5'-tetramethylbenzidine, TMB) to detect antigen or antibody levels. However, natural horseradish peroxidase is expensive, has weak catalytic activity, and is unstable. This high price, in particular, has severely limited the promotion and use of ELISA kits for early screening of diseases such as brucellosis.

[0003] In recent years, manganese dioxide (MnO2) nanozymes have shown promising application prospects in nanomedicine. MnO2 nanozymes exhibit unique peroxidase-like activity. They interact with substrates in the presence of hydrogen peroxide, generating a colorimetric reaction through the generation of free radicals (ROS) and electron transfer. While similar to horseradish peroxidase, their catalytic mechanism and performance are more dependent on surface properties. A previous study (Wenping Yang, Xi Yang, Longjiao Zhu, Huashuo Chu, Xiangyang Li, Wentao Xu. Nanozyme: Activity origin, catalytic mechanism, and biological application. Coordination Chemistry Reviews. 448(2021)214170.) has shown that nanozyme activity depends on its size, morphology, and external stimuli (such as light, sound, and heat). Smaller nanozyme particles significantly increase their specific surface area. A high specific surface area alters the number of surface atoms and surface energy, significantly affecting the catalytic activity of the nanozyme particles. Therefore, the particle size of manganese dioxide nanozymes is closely related to their catalytic activity. However, existing studies of manganese dioxide nanozymes generally have the problem of large particle size (usually >50 nm). For example, the manganese dioxide nanozyme disclosed in Chinese patent publication number CN113461063A has a diameter of approximately 200 nm. For another example, Chinese patent publication number CN116173249A discloses the use of protamine sulfate as a stabilizer to prepare manganese dioxide nanoparticles. The particle size of the manganese dioxide nanoparticles is approximately 90 to 350 nm. The above high particle size limits its specific surface area and the exposure of surface active sites. The internal active sites also have difficulty contacting the substrate, which causes the color reaction kinetics to lag, thereby affecting the catalytic efficiency. However, when pursuing ultra-small particle sizes (e.g., <10 nm), the high surface energy of small-particle manganese dioxide nanozymes results in interparticle attractions (e.g., van der Waals forces) far outweighing repulsions, leading to spontaneous aggregation and formation of aggregates. Particle agglomeration leads to uneven dispersion, reducing the effective specific surface area and accessibility of active sites, resulting in a significant decrease in catalytic efficiency. Furthermore, while biomineralization overcomes the biosafety concerns associated with the use of chemical reagents, its raw materials, such as bovine serum albumin, protamine sulfate, sericin, and human serum albumin, are also expensive. Therefore, finding inexpensive, biosafe biological materials as raw materials and developing simple preparation methods to obtain manganese dioxide nanozymes with small particle size, good dispersibility, and stability are of great significance for their application in disease detection kits. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for preparing manganese dioxide nanozyme.

[0005] Another object of the present invention is to provide an application of manganese dioxide nanozyme in a disease detection kit.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] A method for preparing manganese dioxide nanozyme comprises the following steps:

[0008] A fish peptone solution containing fish peptone powder is evenly mixed with an aqueous potassium permanganate solution to obtain a mixed solution. The mixed solution is stirred until it turns brown, and is successively centrifuged, washed, and dried to obtain manganese dioxide nanozyme, wherein the ratio of potassium permanganate to fish peptone powder in the mixed solution is 0.158:(0.02~2) by mass.

[0009] In the above technical solution, the ratio of potassium permanganate to fish peptone powder in the mixed solution is preferably 0.158:(1~2) by mass.

[0010] In the above technical solution, the concentration of potassium permanganate in the potassium permanganate aqueous solution is 0.02~1 mol / L.

[0011] In the above technical solution, the operation of obtaining the fish peptone solution includes: dissolving the fish peptone powder in water, stirring for 5 to 30 minutes until uniform, and obtaining the fish peptone solution.

[0012] In the above technical solution, the mixed solution is stirred for 5 to 60 minutes until it turns brown.

[0013] In the above technical solution, the ratio of the mass fraction of the fish peptone powder to the volume fraction of water in the fish peptone solution is (0.02~2):(19~190), the unit of the mass fraction is g, and the unit of the volume fraction is mL.

[0014] In the above technical solution, the operation of obtaining the potassium permanganate aqueous solution includes: dissolving potassium permanganate in water and stirring for 5 to 30 minutes to obtain the potassium permanganate aqueous solution.

[0015] In the above technical solution, the stirring speed is 100~900 r / min.

[0016] In the above technical solution, the centrifugal speed is 1000~9000 r / min, and the centrifugal time is 7~10 minutes.

[0017] In the above technical solution, the cleaning operation includes: centrifuging with pure water at least 3 times.

[0018] In the above technical solution, the drying temperature is 30-60° C., and the drying time is at least 24 hours.

[0019] The manganese dioxide nanozyme obtained by the above preparation method has a particle size of 2~10nm.

[0020] Application of the above-mentioned manganese dioxide nanozyme in disease detection kits.

[0021] Use of fish peptone in reducing the particle size of manganese dioxide.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The manganese dioxide nanozyme of the present invention has an ultra-small particle size of 2-5 nm, which overcomes the problem of small particle size and easy agglomeration of traditional nanozymes and has excellent dispersibility and stability. In addition, the manganese dioxide nanozyme prepared by the present invention using fish peptone powder has a high peroxidase-like activity, which is 2.36 times higher than the HRP enzyme activity in the brucellosis detection ELISA kit. It is expected to become an HRP replacement enzyme in the brucellosis detection ELISA kit for early screening of brucellosis, thereby further reducing the price of the brucellosis detection ELISA kit.

[0024] (2) The preparation method of the present invention is simple and easy to operate. It does not require complex post-processing operations and has no requirements for reaction temperature. Only simple stirring is required to prepare manganese dioxide nanozymes on a large scale. In addition, the raw material (fish peptone) used is inexpensive and non-toxic, making its preparation cost low and convenient for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are photos of the mixed solutions after stirring in Examples 1-2 and Comparative Examples 1-3;

[0026] Figure 2 The UV-visible absorption spectra of the mixed solution before and after stirring in Example 1;

[0027] Figure 3 This is a high-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 1;

[0028] Figure 4 This is a high-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 2;

[0029] Figure 5 The XPS graph of the manganese dioxide nanozyme prepared in Example 1, wherein (a) is the full spectrum, (b) is the C1s graph, (c) is the O1s graph, and (d) is the Mn2p graph;

[0030] Figure 6The UV-visible absorption curves of the test solution containing the manganese dioxide nanozyme prepared in Example 1 and the test solution not containing the manganese dioxide nanozyme prepared in Example 1;

[0031] Figure 7 The UV-visible absorption curves of the test solution containing different concentrations of the manganese dioxide nanozyme prepared in Example 1;

[0032] Figure 8 The UV-visible absorption curves of the test solution containing the manganese dioxide nanozyme prepared in Example 1 after being placed for different times;

[0033] Figure 9 These are the UV-visible absorption curves of the test solution containing the manganese dioxide nanozyme and HRP enzyme prepared in Example 1 and the test solution without adding nanozyme. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0035] In the following examples, the operation of obtaining the potassium permanganate aqueous solution includes: dissolving potassium permanganate in pure water and stirring for 10 minutes to obtain the potassium permanganate aqueous solution.

[0036] Examples 1-2 and Comparative Examples 1-3

[0037] A method for preparing manganese dioxide nanozyme comprises the following steps:

[0038] The fish peptone powder was dissolved in pure water and stirred at a speed of V1r / min for H1 min until uniform to obtain a transparent fish peptone solution; a purple potassium permanganate aqueous solution was added to the fish peptone solution to obtain a mixed solution, and the mixture was stirred at a speed of V1r / min for H2 min until the mixed solution turned brown (reduction reaction occurred), and the mixed solution was centrifuged at a speed of V2r / min for 10 min to obtain a crude product, and the crude product was centrifuged with pure water at a speed of V2r / min for 3 times, and dried at 60°C for 24 h to obtain manganese dioxide nanozyme, wherein the ratio of potassium permanganate to fish peptone powder in the mixed solution is W by mass; the ratio of the mass fraction of fish peptone powder to the volume fraction of pure water in the fish peptone solution is G, the unit of mass fraction is g, and the unit of volume fraction is mL; the concentration of potassium permanganate in the potassium permanganate aqueous solution is 0.02 mol / L.

[0039] The W, G, V1, H1, H2 and V2 of the manganese dioxide nanozymes prepared in Examples 1-2 and Comparative Examples 1-3 are shown in Table 1.

[0040] Table 1

[0041]

[0042] After the above reduction reaction is completed (after stirring H2min), the mixed solution is observed and analyzed, such as Figure 1 As shown, it was found that the manganese dioxide nanozymes prepared in Examples 1 to 2 could be evenly dispersed in the reaction system, and the mixed solution was brown, while the mixed solutions of Comparative Examples 1 to 3 showed obvious sedimentation, and the manganese dioxide nanozymes could not be evenly dispersed in the reaction system, indicating that the manganese dioxide nanozymes prepared in Comparative Examples 1 to 3 were unstable and difficult to meet the application requirements of the disease detection kit. It can be seen that the feed ratio of fish peptone powder and potassium permanganate in the potassium permanganate aqueous solution plays a vital role in the dispersion performance of manganese dioxide nanozymes and is one of the key factors affecting its stability.

[0043] Figure 2 is the UV-visible absorption spectrum of the mixed solution before and after stirring in Example 1, Figure 2 As can be seen, when the mixed solution is free of MnO2 nanozymes (before stirring), it appears purple, similar to potassium permanganate, with a distinct absorption peak between 450 and 600 nm on the UV-visible absorption spectrum. However, after MnO2 nanozymes are formed (after stirring), the mixed solution turns brown, and the absorption peak between 450 and 600 nm disappears, indicating the successful preparation of MnO2 nanozymes.

[0044] Figure 3 This is a high-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 1. Figure 3 It can be seen that the particle size of the manganese dioxide nanozyme prepared in Example 1 is an ultra-small nanoparticle of about 2 to 5 nm (such as Figure 3 circled in the middle). Figure 4 This is a high-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 2. Figure 4 It can be seen that the size of the manganese dioxide nanozyme prepared in Example 2 is an ultra-small nanoparticle of about 2 to 9 nm (such as Figure 4 Compared with Example 1, the difference in the conditions (G, V1, H1, H2, and V2) in the preparation method of Example 2 only slightly changes the size of the manganese dioxide nanozyme, which has little effect on the synthesis of the manganese dioxide nanozyme, and the manganese dioxide nanozyme can be successfully prepared.

[0045] The manganese dioxide nanozyme prepared in Example 1 was further subjected to XPS. The XPS graph is shown in FIG. Figure 5 As shown by Figure 5 It can be seen that the Mn and O elements appeared clearly in the XPS full spectrum, which indicates that the manganese dioxide nanozyme prepared in Example 1 was successfully synthesized, especially Figure 5 (d), Figure 5 The dotted line in (d) indicates the original peak. Figure 5The solid line in (d) represents the fitting peak, and the two characteristic peaks of Mn appear at 654.2eV and 642.3eV, indicating that Mn exists in the 4+ valence state. Figure 5 As shown in (b), the manganese dioxide nanozyme prepared in Example 1 also exhibits a characteristic C peak, indicating that fish peptone participates in the formation of the manganese dioxide nanozyme, resulting in the presence of a certain amount of carbon in the manganese dioxide nanozyme, indicating the presence of a large number of carboxyl and hydroxyl groups on the surface of the manganese dioxide nanozyme. In summary, fish peptone can be used for the stable reduction preparation of manganese dioxide nanozyme.

[0046] Example 3

[0047] In an ELISA kit, taking the detection of brucellosis as an example, the antigen of Brucella is coated on the microporous surface of an enzyme-labeled plate, and the sample to be tested is added. If the sample to be tested contains Brucella antibodies, they will bind to the antigen characteristics on the enzyme-labeled plate (unbound substances are removed by washing), forming an antigen-antibody complex. HRP enzyme is used as a marker, and its labeled secondary antibody (targeting the antigen in the sample to be tested) can specifically recognize and bind to the antigen-antibody complex. By adding a color-developing solution containing hydrogen peroxide and a substrate (such as 3,3',5,5'-tetramethylbenzidine (TMB)), the oxidase catalytic activity of the HRP enzyme and hydrogen peroxide jointly catalyze the substrate to produce a color reaction. The depth of the color reflects the content of the antigen or antibody, thereby achieving qualitative or quantitative detection of the target substance. Therefore, the present invention uses TMB to evaluate whether the manganese dioxide nanozyme has peroxidase-like catalytic activity and judge whether it can cause the substrate to produce a color reaction.

[0048] Peroxidase-like activity test: A certain amount of nanozyme was mixed with 80 μL of TMB-DMSO solution to obtain a stock solution (the concentration of the nanozyme in the stock solution was 2 mg / mL). The stock solution was omitted or 80 μL of the stock solution was taken and diluted to 8 mL with HAc-NaAc buffer solution (purchased from Aladdin, pH = 4.0). The solution was mixed thoroughly to obtain the test solution. After standing at room temperature for 5 minutes, the UV-visible absorption curve of the test solution was measured. Figure 6 As shown in the figure, "without MnO2 nanozyme" means no mother liquor was taken, and "with MnO2 nanozyme" means 80 μL of mother liquor was taken. The nanozyme is the manganese dioxide nanozyme prepared in Example 1, and the TMB-DMSO solution is a mixed solution of 3,3',5,5'-tetramethylbenzidine (TMB) and dimethyl sulfoxide (DMSO). The concentration of TMB in the TMB-DMSO solution is 10 mM.

[0049] Depend on Figure 6It can be seen that when no manganese dioxide nanozyme is added (i.e., no mother liquor is taken), the UV-visible absorption curve of the test solution has no absorption peak, indicating that TMB is not catalyzed to produce a blue substance. However, after the addition of manganese dioxide nanozyme (the mother liquor is added in an amount of 80 μL), TMB in the test solution is catalyzed to produce a blue substance. The UV-visible absorption graph of the test solution clearly shows an absorption peak at 652 nm. The appearance of the absorption peak proves that the manganese dioxide nanozyme can catalyze the color reaction of TMB, resulting in a clear absorption peak at 652 nm. This shows that the manganese dioxide nanozyme prepared by stable reduction of fish peptone has peroxidase-like activity.

[0050] In addition, in order to study the peroxidase-like activity under different manganese dioxide nanozyme concentrations, the above peroxidase-like activity test was referred to and the mother solution addition amounts were set to 0 μL, 12.5 μL, 25 μL, 50 μL and 100 μL respectively. Figure 7 As shown in the figure, the higher the concentration of manganese dioxide nanozyme, the higher the absorption peak at 652 nm. This indicates that the peroxidase-like activity of the synthesized manganese dioxide nanozyme gradually increases with increasing concentration, and the catalytic effect on TMB is also stronger.

[0051] Manganese dioxide nanozymes prepared using biological protein substances often contain a large number of carboxyl and hydroxyl groups on their surfaces. When encountering brucellosis antibodies containing amino groups on their surfaces, they can be connected to the antibodies through a simple amidation reaction (Zhang Zhiwei. Preparation of MnO2-based oxidase active materials and dual-mode detection research. Northeast Normal University, 2022. DOI:10.27011 / d.cnki.gdbsu.2022.000258.). The present invention uses the above Figure 5 Analysis shows that the surface of manganese dioxide nanozyme also has a large number of carboxyl groups and hydroxyl groups. Therefore, when using the ELISA kit, the manganese dioxide nanozyme can be combined with the secondary antibody containing amino groups through an amidation reaction, replacing the HRP enzyme for color development reaction for early disease screening.

[0052] Example 4

[0053] The stability of manganese dioxide nanozymes is of great significance for the storage and application of manganese dioxide nanozymes. The manganese dioxide nanozymes prepared in Example 1 and the newly synthesized manganese dioxide nanozymes prepared in Example 1 that were placed under sealed conditions at room temperature for 14 days were used as the "nanozymes" in Example 3 for peroxidase-like activity tests (the amount of mother liquor added was 110 μL). The test results after 0 days and 14 days are shown as follows: Figure 8 As shown by Figure 8It can be seen that the absorption peak of the manganese dioxide nanozyme prepared in Example 1 after 14 days of storage is basically consistent with that of the manganese dioxide nanozyme prepared in Example 1 just after synthesis (0 days of storage), without obvious decrease or shift, which indicates that the manganese dioxide nanozyme not only has good peroxidase-like activity but also has good stability.

[0054] Example 5

[0055] HRP enzyme (purchased from Aladdin) and manganese dioxide nanozyme prepared in Example 1 (mother solution added in an amount of 100 μL) were used as the "nanozyme" in Example 3 to perform peroxidase-like activity tests, and the peroxidase-like activity test results without adding nanozyme (without taking mother solution) were used as controls. Figure 9 As shown by Figure 9 It can be seen that at the same concentration, the absorption peak of manganese dioxide nanozyme is higher than that of HRP enzyme. The maximum absorption peak of manganese dioxide nanozyme at 652 nm is 0.618, and the maximum absorption value of HRP enzyme at 652 nm is 0.262. Therefore, the peroxidase-like activity of MnO2 nanozyme is 2.36 times that of HRP enzyme. This indicates that the synthesized manganese dioxide nanozyme has better peroxidase-like activity and can replace HRP enzyme for use in disease detection ELISA kits.

[0056] Example 6

[0057] As shown in Table 2, the price of the raw material fish peptone used in the present invention is very low compared to other proteins. The price of 1g of fish peptone is 1.429 RMB, which is even much lower than the price of HRP enzyme (the price of 1g of HRP enzyme is 5999 RMB). This shows that the cost price of using fish peptone to stably reduce and prepare manganese dioxide nanozymes will also be very low, and can be used for large-scale preparation of manganese dioxide nanozymes. This greatly reduces the price of ELISA kits for brucellosis detection.

[0058] Table 2

[0059]

[0060] The above is an exemplary description of the present invention. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by other skilled in the art without expending creative labor falls within the scope of protection of the present invention.

Claims

1. An application of a manganese dioxide nanozyme in a disease detection kit, characterized in that: The particle size of the manganese dioxide nanozyme is 2-10 nm. The preparation method of the manganese dioxide nanozyme comprises the following steps: A fish peptone solution containing fish peptone powder is evenly mixed with a potassium permanganate aqueous solution to obtain a mixed solution. The mixed solution is stirred until it turns brown, and is successively centrifuged, washed, and dried to obtain manganese dioxide nanozyme, wherein the ratio of potassium permanganate to fish peptone powder in the mixed solution is 0.158:(1~2) by mass, and the concentration of potassium permanganate in the potassium permanganate aqueous solution is 0.02~1 mol / L.

2. The use according to claim 1, characterized in that The process of obtaining the fish peptone solution comprises dissolving the fish peptone powder in water, stirring for 5 to 30 minutes until the mixture is uniform, and obtaining the fish peptone solution.

3. The use according to claim 1, characterized in that The mixed solution was stirred for 5-60 min until it turned brown.

4. The use according to claim 1, characterized in that The ratio of the mass fraction of the fish peptone powder to the volume fraction of water in the fish peptone solution is (0.02-2):(19-190), the unit of the mass fraction is g, and the unit of the volume fraction is mL.

5. The use according to claim 1, characterized in that The operation of obtaining the potassium permanganate aqueous solution includes: dissolving potassium permanganate in water and stirring for 5 to 30 minutes to obtain the potassium permanganate aqueous solution.

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