Preparation method of manganese dioxide nano-enzyme and application of manganese dioxide nano-enzyme in disease detection kit

Abstract

The invention discloses a preparation method of manganese dioxide nano-enzyme and application of the manganese dioxide nano-enzyme in a disease detection kit.The preparation method of the manganese dioxide nano-enzyme comprises the following steps that a fish peptone solution containing fish peptone powder and a potassium permanganate aqueous solution are mixed to be uniform, a mixed solution is obtained, the mixed solution is stirred to be brown, and a manganese dioxide nano-enzyme solution is obtained; the manganese dioxide nano-enzyme has an ultra-small particle size of 2-5nm, overcomes the problem that the traditional nano-enzyme is small in particle size and easy to agglomerate, and has excellent dispersity and stability; in addition, the manganese dioxide nano-enzyme prepared from the fish peptone powder has high peroxidase-like activity and stability, and the enzyme activity of the manganese dioxide nano-enzyme is 2.36 times higher than that of HRP in a brucellosis detection ELISA kit.

Description

Technical Field

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

[0002] Enzyme-linked immunosorbent assay (ELISA) kits are a highly sensitive detection technology based on antigen-antibody specific reactions, and are widely used in fields such as biology, medicine, and food detection. Horseradish peroxidase (HRP) is commonly used as a chromogenic marker to catalyze the substrate (such as 3,3',5,5'-tetramethylbenzidine, TMB) to develop color in the presence of hydrogen peroxide to detect the content of antigens or antibodies. However, natural horseradish peroxidase has problems such as high price, weak catalytic ability, and poor stability. In particular, the high price seriously restricts the popularization and use of ELISA kits in the early screening of diseases (such as brucellosis).

[0003] In recent years, manganese dioxide (MnO2) nanozymes have gradually shown good application prospects in the field of nanomedicine treatment. Manganese dioxide nanozymes have unique peroxidase-like activity and can interact with substrates in the presence of hydrogen peroxide to achieve color reactions through free radical (ROS) generation and electron transfer mechanisms, which is similar to horseradish peroxidase. However, the catalytic mechanism and performance of manganese dioxide nanozymes are more dependent on surface properties. The literature (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 also shown that the activity of nanozymes depends on their size, morphology, and external stimuli (such as light, sound, and heat). The smaller the size of nanozyme particles, the significantly increased specific surface area. The high specific surface area will change the number of surface atoms and surface energy, greatly affecting the catalytic activity of nanozyme particles. Therefore, the particle size of manganese dioxide nanozymes is closely related to their catalytic activity. However, in existing studies, manganese dioxide nanozymes generally have the problem of relatively large particle sizes (usually >50 nm). For example, the manganese dioxide nanozymes disclosed in the Chinese patent with publication number CN113461063A have a diameter of about 200 nm. Another example is that the Chinese patent with publication number CN116173249A discloses the preparation of manganese dioxide nanoparticles using protamine sulfate as a stabilizer, and the particle size of the manganese dioxide nanoparticles is about 90 - 350 nm. The above large particle sizes limit their specific surface area and the exposure of surface active sites, and the internal active sites are also difficult to contact the substrate, resulting in a lag in the color reaction kinetics and thus affecting the catalytic efficiency. However, when pursuing ultra-small particle sizes of manganese dioxide nanozymes (such as <10 nm), the high surface energy of small-sized nanozymes leads to the attractive force (such as van der Waals force) between particles being much greater than the repulsive force, easily causing spontaneous aggregation to form aggregates, resulting in uneven dispersion due to particle aggregation, thereby reducing the effective specific surface area and the accessibility of active sites, leading to a significant decrease in catalytic efficiency. In addition, although the preparation of manganese dioxide nanozymes by biomineralization overcomes the biosafety problems brought by the use of chemical reagents, its raw materials such as bovine serum albumin, protamine sulfate, sericin, and human serum albumin also have the problem of relatively high prices. Therefore, finding low-cost and highly biosafe biological substances as raw materials and developing simple preparation methods to obtain manganese dioxide nanozymes with small particle sizes, good dispersibility, and stability are of great significance for their application in disease detection kits. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method of manganese dioxide nanozyme.

[0005] Another purpose 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 realized through the following technical solutions.

[0007] A preparation method of manganese dioxide nanozyme includes the following steps: Mix a peptone solution containing peptone powder with an aqueous potassium permanganate solution evenly to obtain a mixed solution, stir the mixed solution until it turns brown, and then perform centrifugation, washing, and drying in sequence to obtain manganese dioxide nanozyme. Among them, by mass fraction, the ratio of potassium permanganate to peptone powder in the mixed solution is 0.158:(0.02 - 2).

[0008] In the above technical solution, preferably, by mass fraction, the ratio of potassium permanganate to peptone powder in the mixed solution is 0.158:(1 - 2).

[0009] In the above technical solution, the concentration of potassium permanganate in the aqueous potassium permanganate solution is 0.02 - 1 mol / L.

[0010] In the above technical solution, the operation of obtaining the peptone solution includes: dissolving peptone powder in water and stirring for 5 - 30 min until it is uniform to obtain a peptone solution.

[0011] In the above technical solution, stir the mixed solution for 5 - 60 min until it turns brown.

[0012] In the above technical solution, the ratio of the mass fraction of the peptone powder to the volume fraction of water in the 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.

[0013] In the above technical solution, the operation of obtaining the aqueous potassium permanganate solution includes: dissolving potassium permanganate in water and stirring for 5 - 30 min to obtain an aqueous potassium permanganate solution.

[0014] In the above technical solution, the rotation speed of the stirring is 100 - 900 r / min.

[0015] In the above technical solution, the rotation speed of the centrifugation is 1000 - 9000 r / min, and the centrifugation time is 7 - 10 min.

[0016] In the above technical solution, the operation of the washing includes: centrifuging with pure water at least 3 times.

[0017] In the above technical solution, the drying temperature is 30~60°C, and the drying time is at least 24 h.

[0018] The manganese dioxide nanozyme obtained by the above preparation method has a particle size of 2~10 nm.

[0019] The application of the above manganese dioxide nanozyme in a disease detection kit.

[0020] The use of peptone in reducing the particle size of manganese dioxide.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The manganese dioxide nanozyme of the present invention has an ultra-small particle size of 2~5 nm, overcomes the problem that traditional nanozymes are prone to aggregation due to their small particle size, and has excellent dispersibility and stability. In addition, the manganese dioxide nanozyme prepared by using peptone powder in the present invention 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 alternative enzyme in the Brucellosis detection ELISA kit for early screening of Brucellosis, thereby further reducing the price of the Brucellosis detection ELISA kit.

[0022] (2) The preparation method of the present invention is simple and easy to operate, does not require complex post-treatment operations, has no requirement for the reaction temperature, and only needs simple stirring to prepare manganese dioxide nanozymes on a large scale. Moreover, the raw material used (peptone) is inexpensive and non-toxic, making its preparation cost relatively low and facilitating large-scale production and application. Description of the Drawings

[0023] Figure 1 Photographs of the stirred mixed solutions in Examples 1~2 and Comparative Examples 1~3; Figure 2 Ultraviolet-visible absorption spectra of the mixed solution before and after stirring in Example 1; Figure 3 High-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 1; Figure 4 High-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 2; Figure 5 XPS spectra of the manganese dioxide nanozyme prepared in Example 1, where (a) is the full-spectrum, (b) is the C1s spectrum, (c) is the O1s spectrum, and (d) is the Mn2p spectrum; Figure 6 Ultraviolet-visible absorption curves of the test solution containing the manganese dioxide nanozyme prepared in Example 1 and the test solution without the manganese dioxide nanozyme prepared in Example 1; Figure 7The UV-Vis absorption curve of the test solution containing manganese dioxide nanozyme prepared in Example 1 with different concentrations; Figure 8 The UV-Vis absorption curve of the test solution containing manganese dioxide nanozyme prepared in Example 1 with different storage times; Figure 9 The UV-Vis absorption curves of the test solutions containing manganese dioxide nanozyme and HRP enzyme prepared in Example 1 and the test solution without adding nanozyme. Specific Embodiments

[0024] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0025] In the following embodiments, the operation of obtaining the potassium permanganate aqueous solution includes: dissolving potassium permanganate in pure water and stirring for 10 min to obtain the potassium permanganate aqueous solution.

[0026] Examples 1-2 and Comparative Examples 1-3 A preparation method of manganese dioxide nanozyme includes the following steps: Dissolve the peptone powder in pure water, stir at a speed of V1 r / min for H1 min until uniform to obtain a transparent peptone solution; add the purple potassium permanganate aqueous solution to the peptone solution to obtain a mixed solution, stir at a speed of V1 r / min for H2 min until the mixed solution turns brown (reduction reaction occurs), centrifuge the mixed solution at a speed of V2 r / min for 10 min to obtain a crude product, and then centrifuge the crude product with pure water 3 times at a speed of V2 r / min, and dry at 60 °C for 24 h to obtain manganese dioxide nanozyme, wherein, by mass fraction, the ratio of potassium permanganate to peptone powder in the mixed solution is W; the ratio of the mass fraction of peptone powder to the volume fraction of pure water in the 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.

[0027] The values of 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.

[0028] Table 1

[0029] After the above reduction reaction ends (after stirring for H2 min), observe and analyze the mixed solution, such as Figure 1As shown, it was found that the manganese dioxide nanozymes prepared in Examples 1-2 could be uniformly dispersed in the reaction system, and the mixed solution was brown. However, obvious sedimentation phenomena occurred in the mixed solutions of Comparative Examples 1-3, and the manganese dioxide nanozymes could not be uniformly dispersed in the reaction system, indicating that the manganese dioxide nanozymes prepared in Comparative Examples 1-3 were unstable and difficult to meet the application requirements of disease detection kits. Thus, it can be seen that the feeding ratio of potassium permanganate in peptone powder and potassium permanganate aqueous solution plays a crucial role in the dispersion performance of manganese dioxide nanozymes and is one of the key factors affecting their stability.

[0030] Figure 2 is the ultraviolet-visible absorption spectrum of the mixed solution before and after stirring in Example 1. From Figure 2 it can be seen that when there was no manganese dioxide nanozyme generated in the mixed solution (before stirring), the mixed solution was purple due to potassium permanganate and had obvious absorption peaks at 450-600 nm in the ultraviolet-visible absorption spectrum. When manganese dioxide nanozymes were generated (after stirring), the mixed solution turned brown and the absorption peaks at 450-600 nm on the absorption spectrum disappeared significantly, indicating that manganese dioxide nanozymes were successfully prepared.

[0031] Figure 3 is the high-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 1. From Figure 3 it can be seen that the particle size of the manganese dioxide nanozyme prepared in Example 1 was ultra-small nanoparticles about 2-5 nm in size (as shown by the circles in Figure 3 ). Figure 4 is the high-resolution transmission electron microscopy image of the manganese dioxide nanozyme prepared in Example 2. From Figure 4 it can be seen that the size of the manganese dioxide nanozyme prepared in Example 2 was ultra-small nanoparticles about 2-9 nm in size (as shown by the circles in Figure 4 ). Compared with Example 1, the differences in conditions (G, V1, H1, H2, and V2) in the preparation method of Example 2 only caused slight changes in the size of the manganese dioxide nanozyme and had little impact on the synthesis of the manganese dioxide nanozyme, and manganese dioxide nanozymes could be successfully prepared.

[0032] Furthermore, XPS was performed on the manganese dioxide nanozyme prepared in Example 1. The XPS graph is as shown in Figure 5 . From Figure 5 it can be seen that obvious Mn and O elements appeared in the XPS full spectrum, indicating that the manganese dioxide nanozyme prepared in Example 1 was successfully synthesized. Especially in Figure 5 (d), Figure 5 (d), the dotted line represents the original peak, Figure 5 (d), the solid line represents the fitted peak. The appearance of two characteristic peaks of Mn at 654.2 eV and 642.3 eV indicates that Mn exists in the 4+ valence state. From Figure 5As can be seen from (b) of , the C characteristic peak also appears in the manganese dioxide nanozyme prepared in Example 1, indicating that peptone participated in the formation of the manganese dioxide nanozyme, resulting in the presence of a certain amount of C element in the manganese dioxide nanozyme, which shows that there are a large number of carboxyl and hydroxyl groups on the surface of the manganese dioxide nanozyme. In summary, peptone can stably reduce and prepare manganese dioxide nanozyme.

[0033] Example 3 In the ELISA kit, taking the detection of brucellosis as an example, the antigen of Brucella is coated on the micropore surface of the enzyme-labeled plate, and the test sample is added. If the test sample contains Brucella antibody, it will specifically bind to the antigen on the enzyme-labeled plate (the unbound substances are removed by washing), forming an antigen-antibody complex. The HRP enzyme-labeled secondary antibody (against the antigen in the test sample) can specifically recognize and bind to the antigen-antibody complex. By adding a chromogenic solution containing hydrogen peroxide and a substrate (such as 3,3',5,5'-tetramethylbenzidine (TMB)), the peroxidase catalytic activity of the HRP enzyme and hydrogen peroxide are used together to catalyze the substrate to undergo a chromogenic reaction, and the content of the antigen or antibody is reflected by the depth of the color, so as to realize the qualitative or quantitative detection of the target substance. Therefore, in the present invention, TMB is used to evaluate whether the manganese dioxide nanozyme has peroxidase-like catalytic activity and to judge whether it can cause the substrate to undergo a chromogenic reaction.

[0034] Peroxidase-like activity test: A certain amount of nanozyme and 80 μL of TMB-DMSO solution are mixed to obtain a mother liquor (the concentration of nanozyme in the mother liquor is 2 mg / mL). Without taking the mother liquor or taking 80 μL of the mother liquor, it is made up to 8 mL with HAc-NaAc buffer solution (purchased from Aladdin, pH = 4.0), and mixed well to obtain the test solution. After standing at room temperature for 5 min, the ultraviolet-visible absorption curve of the test solution is measured, as Figure 6 shown, where "without MnO2 nanozyme" represents not taking the mother liquor, and "adding MnO2 nanozyme" represents taking 80 μL of the mother liquor. Among them, the nanozyme is the manganese dioxide nanozyme prepared in Example 1, the TMB-DMSO solution is a mixed solution of 3,3',5,5'-tetramethylbenzidine (TMB) and dimethyl sulfoxide (DMSO), and the concentration of TMB in the TMB-DMSO solution is 10 mM.

[0035] From Figure 6It can be seen that when manganese dioxide nanozyme is not added (i.e., the mother liquor is not taken), there is no absorption peak in the ultraviolet-visible absorption curve of the test solution, indicating that TMB is not catalyzed to generate blue substances. However, when manganese dioxide nanozyme is added (the addition amount of the mother liquor is 80 μL), TMB in the test solution is catalyzed to generate blue substances, and it can be clearly seen that an absorption peak appears at 652 nm in the ultraviolet-visible absorption diagram of the test solution. The appearance of the absorption peak proves that manganese dioxide nanozyme can catalyze the chromogenic reaction of TMB, resulting in an obvious absorption peak at 652 nm. This shows that the manganese dioxide nanozyme prepared by stabilizing and reducing with peptone has peroxidase-like activity.

[0036] In addition, in order to study the peroxidase-like activity at different concentrations of manganese dioxide nanozyme, referring to the above peroxidase-like activity test, the addition amounts of the mother liquor were set to 0 μL, 12.5 μL, 25 μL, 50 μL, and 100 μL respectively. The test results are as Figure 7 shown. 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 the increase of concentration, and the catalytic effect on TMB is also stronger.

[0037] When manganese dioxide nanozyme is prepared using biological protein substances, its surface often contains a large number of carboxyl and hydroxyl groups. When encountering Brucella antibodies with amino groups on the surface, the antibody can be linked through a simple amidation reaction (Zhang Zhiwei. Preparation of MnO2-based peroxidase-like activity materials and dual-mode detection research. Northeast Normal University, 2022. DOI: 10.27011 / d.cnki.gdbsu.2022.000258.). Through the above Figure 5 analysis, it can be known that the surface of manganese dioxide nanozyme also has a large number of carboxyl and hydroxyl groups. Therefore, when applying an ELISA kit, manganese dioxide nanozyme can be combined with the secondary antibody containing amino groups through an amidation reaction to replace HRP enzyme for chromogenic reaction for early disease screening.

[0038] Example 4 The stability of manganese dioxide nanozyme is of great significance for the storage and application of manganese dioxide nanozyme. The manganese dioxide nanozyme prepared in Example 1 placed at room temperature under sealed conditions for 14 days and the manganese dioxide nanozyme prepared in Example 1 just synthesized (placed for 0 days) were respectively used as the "nanozyme" in Example 3 for peroxidase-like activity test (the addition amount of the mother liquor is 110 μL). The test results of placing for 0 days and 14 days are as Figure 8 shown. From Figure 8It can be seen that the absorption peak of the manganese dioxide nanozyme prepared in Example 1 after being placed for 14 days is basically the same as that of the manganese dioxide nanozyme prepared in Example 1 just after synthesis (placed for 0 days), without obvious decrease and shift. This indicates that the manganese dioxide nanozyme not only has good peroxidase-like activity but also has good stability.

[0039] Example 5 The HRP enzyme (purchased from Aladdin) and the manganese dioxide nanozyme prepared in Example 1 (the added amount of the mother liquor was 100 μL) were respectively used as the "nanozyme" in Example 3 for peroxidase-like activity testing, and the peroxidase-like activity test result without adding nanozyme (without taking the mother liquor) was used as the control. As Figure 9 shown, from Figure 9 it can be seen that at the same concentration, the absorption peak of the manganese dioxide nanozyme is higher than that of the HRP enzyme. The maximum absorption peak of the manganese dioxide nanozyme at 652 nm is 0.618, and the maximum absorption value of the HRP enzyme at 652 nm is 0.262. Therefore, the peroxidase-like activity of the MnO2 nanozyme is 2.36 times that of the HRP enzyme, which indicates that the synthesized manganese dioxide nanozyme has good peroxidase-like activity and can replace the HRP enzyme and be applied to the disease detection ELISA kit.

[0040] Example 6 As shown in Table 2, the price of the raw material peptone used in the present invention is very low compared with the prices of other proteins. The price of 1 g of peptone is 1.429 RMB, even much lower than that of the HRP enzyme (the price of 1 g of HRP enzyme is 5999 RMB). This indicates that the cost price of preparing the manganese dioxide nanozyme by stabilizing and reducing with peptone will also be very low, and it can be used for the large-scale preparation of the manganese dioxide nanozyme. Thus, the price of the brucellosis detection ELISA kit can be greatly reduced.

[0041] Table 2

[0042] 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 equivalent replacement that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. A preparation method of manganese dioxide nanozyme, characterized in that It includes the following steps: Mix the peptone solution containing peptone powder with potassium permanganate aqueous solution evenly to obtain a mixed solution. Stir the mixed solution until it turns brown, and then perform centrifugation, washing, and drying in sequence to obtain manganese dioxide nanozyme. Among them, by mass fraction, the ratio of potassium permanganate to peptone powder in the mixed solution is 0.158:(0.02 - 2).

2. The preparation method according to claim 1, characterized in that, By mass fraction, the ratio of potassium permanganate to peptone powder in the mixed solution is 0.158:(1 - 2).

3. The preparation method according to claim 1, characterized in that, The concentration of potassium permanganate in the potassium permanganate aqueous solution is 0.02 - 1 mol / L.

4. The preparation method according to claim 1, characterized in that, The operation of obtaining the peptone solution includes: dissolving peptone powder in water and stirring for 5 - 30 min until it is uniform to obtain the peptone solution.

5. The preparation method according to claim 1, characterized in that, Stir the mixed solution for 5 - 60 min until it turns brown.

6. The preparation method according to claim 1, characterized in that, The ratio of the mass fraction of the peptone powder to the volume fraction of water in the 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.

7. The preparation method 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 - 30 min to obtain the potassium permanganate aqueous solution.

8. The manganese dioxide nanozyme obtained by the preparation method according to claim 1, characterized in that, The particle size of the manganese dioxide nanozyme is 2 - 10 nm.

9. Use of the manganese dioxide nanozyme according to claim 8 in a disease detection kit.

10. Use of peptone in reducing the particle size of manganese dioxide.

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