Iron-containing layered silicate mineral nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of mineral materials and nano-enzyme catalysis, in particular to an iron-containing layered silicate mineral nano-enzyme as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out ball milling on the iron-containing layered silicate mineral, carrying out centrifugal washing, and drying to obtain the iron-containing layered silicate mineral nano-enzyme. According to the method, silicate minerals with a layered structure and rich iron content are adopted as raw materials, the ball-milled layered silicate mineral nano-enzyme is successfully prepared through a ball milling method, the crystallinity of the minerals is reduced, the surface acidity of the minerals is improved, and low-spin iron is generated, so that the affinity of the ball-milled minerals to H2O2 is enhanced; h2O2 is catalyzed to generate singlet oxygen and superoxide free radicals, so that the peroxidase-like activity of the ball-milled minerals is improved. The enzyme-like activity is improved by 10 times, high catalytic activity can still be kept under the neutral condition, and the method can be used for colorimetric sensing and antibacterial application of small biological molecules such as glucose, hydrogen peroxide and glutathione.

Description

Technical Field

[0001] The present invention relates to the technical fields of mineral materials and nanozyme catalysis, and particularly relates to an iron-containing layered silicate mineral nanozyme, a preparation method thereof, and an application thereof. Background Art

[0002] Nanozymes are a class of functional nanomaterials with enzyme-like catalytic activities. Among them, iron-based nanozymes, including iron oxide, iron sulfide, iron alloy, single-atom iron, and iron phosphate nanoparticles, have great application potential in the fields of green synthesis, environmental protection, and biomedicine due to their unique physical and chemical properties. The crystal structure of natural iron-containing silicate minerals is mainly composed of silicon-oxygen tetrahedrons and aluminum-oxygen octahedrons. The iron component enters the crystal structure through isomorphous substitution, showing certain catalytic ability. However, its iron component is masked by the external inert silicon-oxygen layer, resulting in low catalytic activity. Researchers usually use traditional liquid-phase exfoliation and cation-exchange methods to enhance their enzyme-like catalytic activity. The essence is to expose the iron component of the mineral itself or form new iron-containing substances based on the mineral. For example, Tang et al. prepared ultrathin vermiculite nanosheets with a thickness of about 1 nm by treating vermiculite through liquid-phase exfoliation, thereby activating its peroxidase-like activity. Wang et al. prepared Fe 3+ -montmorillonite by a cation-exchange strategy, which changed the surface acidity of montmorillonite and enhanced its peroxidase-like activity. Although the above methods all use minerals as raw materials, other raw materials need to be introduced at the same time, and the preparation methods are complex. Although these methods improve their catalytic ability to a certain extent, realizing large-scale, economical, and efficient production is still a challenge. Summary of the Invention

[0003] The purpose of the present invention is to propose an iron-containing layered silicate mineral nanozyme, a preparation method thereof, and an application thereof in view of the above deficiencies of the prior art.

[0004] The first object of the present invention is to provide a preparation method of an iron-containing layered silicate mineral nanozyme. The iron-containing layered silicate mineral is ball-milled, centrifuged, washed, and dried to obtain the iron-containing layered silicate mineral nanozyme; the rotation speed of the ball milling is 100 rpm - 800 rpm; the time of the ball milling is 1 - 72 h.

[0005] Further, the iron-containing layered silicate mineral includes one of biotite, vermiculite, nontronite, kaolinite, talc, and illite.

[0006] Further, the mass ratio of the media balls to the iron-containing layered silicate mineral is 20:1 - 100:1.

[0007] Further, the rotation speed of ball milling is 400 rpm - 600 rpm, the time is 1 - 48 h, during the ball milling process, ball milling is carried out for 5 - 30 min and paused for 1 - 10 min.

[0008] Further, wet ball milling or dry ball milling is adopted. For the wet ball milling, the solvent is one or more of ethanol, water, isopropanol, ethylene glycol and glycerol; the medium of the balls is ceramic medium balls, metal medium balls or non-metal medium balls.

[0009] The second object of the present invention is to provide an iron-containing layered silicate mineral nanozyme prepared by the above preparation method.

[0010] Further, the surface acidity is increased and low-spin iron is generated.

[0011] The third object of the present invention is to provide an application of an iron-containing layered silicate mineral nanozyme as described above in colorimetric sensing. In the presence of H2O2, a color reaction occurs between the ball-milled mineral with peroxidase-like activity and the chromogenic substrate, and the change in the absorbance value of the solution is correlated with the concentration of the target analyte, thereby establishing a colorimetric sensing platform for detecting the concentration of the target analyte to be detected.

[0012] Further, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt or o-phenylenediamine.

[0013] Further, the target analytes are biological small molecules such as glucose, hydrogen peroxide, glutathione, etc.

[0014] Further, the iron-containing layered silicate mineral nanozyme catalyzes the decomposition of H2O2 to generate hydroxyl radicals, singlet oxygen and superoxide radicals with strong oxidation ability, which can be used to efficiently kill bacteria.

[0015] The present invention uses a silicate mineral with a layered structure and rich iron content as a raw material, adopts a mechanical activation strategy, and successfully prepares a ball-milled layered silicate mineral nanozyme by ball milling method, reduces the crystallinity of the mineral, improves the surface acidity of the mineral and generates low-spin iron, thereby enhancing the affinity of the ball-milled mineral for H2O2, catalyzing H2O2 to generate singlet oxygen and superoxide radicals, and improving the peroxidase-like activity of the ball-milled mineral. Its enzyme-like activity is increased by 10 times and it can still maintain high catalytic activity under neutral conditions, overcoming the problem of low activity of nanozymes under neutral conditions. It can be used for colorimetric sensing and antibacterial applications of biological small molecules such as glucose, hydrogen peroxide, glutathione, etc., and it can also be used under neutral conditions, improving the utilization value of iron-containing layered silicate minerals.

[0016] The present invention provides a new method for the preparation of iron-containing layered silicate mineral nanozymes and the improvement of enzyme activity, provides a new way for the high-value utilization of iron-containing layered silicate minerals, and this method has a simple process, uses raw materials with low price, environmental protection and non-toxicity, and is easy to mass-produce. Description of the Drawings

[0017] Figure 1 Graph showing the test results of the peroxidase-like activity of the biotite nanozymes prepared in Example 1 and Example 2;

[0018] Figure 2 XRD patterns of unball-milled biotite, and the biotite nanozymes prepared in Example 1 and Example 2;

[0019] Figure 3 Electron microscope images of unball-milled biotite and the biotite nanozymes prepared in Example 1 and Example 2;

[0020] Figure 4 For the ball-milled biotite and biotite prepared in Example 1 57 Fe-Mössbauer spectra;

[0021] Figure 5 Graph showing the effect of pH on the peroxidase-like activity of the ball-milled biotite prepared in Example 1;

[0022] Figure 6 UV-visible absorption spectra of vermiculite and nontronite before and after ball milling in Example 3;

[0023] Figure 7 UV-visible absorption spectra of kaolinite, talc, and illite before and after ball milling in Example 3;

[0024] Figure 8 Graph showing the test results of the peroxidase-like activity of the biotite nanozymes prepared by dry and wet ball milling in Example 4 and unball-milled biotite;

[0025] Figure 9 XRD patterns of the biotite nanozymes prepared by dry and wet ball milling in Example 4 and unball-milled biotite;

[0026] Figure 10 Electron microscope image of the biotite nanozyme prepared by dry ball milling in Example 4;

[0027] Figure 11 Graph showing the test results of the peroxidase-like activity of the biotite nanozymes with different ball milling times prepared in Example 5;

[0028] Figure 12 Graph showing the test results of superoxide radicals tested by EPR in Example 6;

[0029] Figure 13Test result graph of hydroxyl radicals tested by EPR for Example 6;

[0030] Figure 14 Test result graph of singlet oxygen tested by EPR for Example 6. Detailed implementation manners

[0031] The following are specific embodiments of the present invention and, in combination with the accompanying drawings, further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0032] Example 1

[0033] Prepare ball-milled biotite nanozyme:

[0034] Use a ball-to-material ratio of 60:1 (3 g of biotite powder and 180 g of zirconia balls). Add both to a polytetrafluoroethylene ball-milling tank, and add 50 mL of ethanol. Then perform ball milling on a planetary ball mill, set the rotation speed to 400 rpm, and the ball-milling time to 24 h (during the ball-milling process, ball mill for 30 min and pause for 5 min) to prevent excessive wear and heat accumulation. After ball milling, centrifuge and wash the product 3 times with ultrapure water (centrifugation conditions: 600 rpm, 6 min) to ensure complete removal of residual ethanol and impurities. Finally, dry in a vacuum drying oven at 60 °C for 16 h to obtain the final product.

[0035] Example 2

[0036] Prepare ball-milled biotite nanozyme:

[0037] Change the ball-milling rotation speeds to 200 rpm and 600 rpm respectively, and keep other conditions the same as in Example 1 to obtain the final product.

[0038] Peroxidase-like detection: Use colorless 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogenic substrate. Its oxidized state has absorption at 652 nm. Use a 0.1 M acetic acid-sodium acetate buffer (ultrapure water, NaCl solution) with pH = 4.0, material concentration 66.66 μg / mL, H2O2 concentration 10 mM, and TMB concentration 1 mM. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of the solution at 652 nm every 30 s for 6 cycles.

[0039] Perform peroxidase-like activity tests on the products prepared in Example 1 and Example 2. The results are shown in the appendix Figure 1 The biotite ball-milled at 400 rpm has the highest peroxidase-like activity; see the appendix Figure 2At a rotational speed of 200 rpm, the diffraction peaks of biotite are still clearly visible and relatively sharp, indicating that the crystal structure has not changed significantly. At rotational speeds of 400 rpm and 600 rpm, the intensity of the diffraction peaks of biotite decreases significantly, and the crystallinity is poor, indicating that a higher rotational speed damages the crystal structure of biotite.

[0040] See the appendix Figure 3 , Biotite (a) and the biotite milled at rotational speeds of 200 rpm (b), 400 rpm (c), and 600 rpm (d) all exhibit a flaky structure, and the size changes from micrometers to nanometers.

[0041] See the appendix Figure 4 , During ball milling, Fe 2+ inside biotite is oxidized to form low-spin Fe 3+ .

[0042] See the appendix Figure 5 , Milled biotite has strong peroxidase-like activity at pH = 4 and still maintains relatively high peroxidase-like activity even under neutral conditions.

[0043] Example 3

[0044] Preparation of nanozymes from other types of layered silicate minerals:

[0045] Using a ball-to-material ratio of 60:1 (3 g of vermiculite / nonstronite / kaolinite / talc / illite powder and 180 g of zirconia balls), both are added to a polytetrafluoroethylene ball milling jar, and 50 mL of ethanol is added. Then, ball milling is carried out on a planetary ball mill with a set rotational speed of 400 rpm and a ball milling time of 24 h (ball milling for 30 min and pausing for 5 min during ball milling) to prevent excessive wear and heat accumulation. After ball milling, the product is centrifugally washed 3 times with ultrapure water (centrifugation conditions: 600 rpm, 6 min) to ensure complete removal of residual ethanol and impurities. Finally, it is dried in a vacuum drying oven at 60 °C for 16 h to obtain the final product.

[0046] Perform peroxidase-like activity tests on the minerals before and after ball milling. See the appendix Figure 6 , After ball milling, the peroxidase-like activity of Fe-rich vermiculite and nonstronite increases. See the appendix Figure 7 , After ball milling, the peroxidase-like activity of Fe-poor kaolinite, talc, and illite increases slightly.

[0047] Example 4

[0048] In this example, solvent-free (ethanol-free) milled biotite nanozymes are prepared:

[0049] Without adding ethanol as a solvent, the other steps are the same as in Example 1 to obtain the final product. Perform peroxidase-like activity tests.

[0050] See the appendix Figure 8 , the peroxidase-like activity of the ball-milled biotite nanozyme is the highest when wet ball milling (adding ethanol) is carried out.

[0051] See the appendix Figure 9 , whether it is dry ball milling or wet ball milling, the diffraction intensity of biotite is significantly weakened at 400 rpm, reducing its crystallinity.

[0052] See the appendix Figure 10 , after dry ball milling, the biotite structure transforms into spherical amorphous nanoparticles.

[0053] Example 5

[0054] In this example, biotite nanozymes prepared under different ball milling times are as follows:

[0055] Using a ball-to-material ratio of 60:1 (3 g of biotite powder and 180 g of zirconia balls), add both to a polytetrafluoroethylene ball milling jar, and add 50 mL of ethanol. Then, carry out ball milling on a planetary ball mill, set the rotation speed to 400 rpm, and the ball milling time to 0, 12, 24, 36, 48, 72 h (during the ball milling process, ball mill for 30 min and pause for 5 min) to prevent excessive wear and heat accumulation. After ball milling, centrifuge and wash the product 3 times with ultrapure water (centrifugation conditions: 600 rpm, 6 min) to ensure complete removal of residual ethanol and impurities. Finally, dry in a vacuum drying oven at 60 °C for 16 h to obtain the final product.

[0056] Perform peroxidase-like activity tests, and the results are shown in the appendix Figure 11 , the peroxidase-like activity of the biotite nanozyme is the best when ball milled for 24 h.

[0057] Use electron paramagnetic resonance spectroscopy (EPR) to test the reactive oxygen species, hydroxyl radicals, and superoxide radical species during the reaction process. Use 2,2,6,6-tetramethyl-4-piperidinol (TEMP) as the singlet oxygen ( 1 O2) scavenger, and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the hydroxyl radical (·OH) and superoxide radical (O2 .- ) scavenger.

[0058] See the appendix Figures 12 - 14 , in the ball-milled biotite nanozyme + H2O2 system, if DMPO-O2 is detected .-The characteristic quartet signal (1:1:1:1) indicates the generation of superoxide radicals; if the characteristic quartet signal (1:2:2:1) of DMPO-·OH is detected, it indicates the generation of hydroxyl radicals; if the characteristic triplet signal (1:1:1) of TEMP- 1 O2 is detected, it indicates the generation of singlet oxygen.

[0059] From Figure 12 it can be seen that ball-milled biotite nanozyme catalyzes H2O2 to produce O2 .- , Figure 13 it can be seen that ball-milled biotite nanozyme catalyzes H2O2 to produce ·OH, Figure 14 it can be seen that ball-milled biotite nanozyme catalyzes H2O2 to produce 1 O2. These generated reactive oxygen species have applications in antibacterial.

[0060] Where the above is not involved, it shall apply to the prior art.

[0061] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration and not for limiting the scope of the present invention. Those skilled in the technical field to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar ways to substitute, but will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of an iron-containing layered silicate mineral nanozyme, characterized in that: The iron-containing layered silicate minerals are ball-milled, centrifugally washed, and dried to obtain iron-containing layered silicate mineral nanozymes; the rotation speed of the ball milling is 100 rpm - 800 rpm; the ball milling time is 1 - 72 h.

2. The preparation method according to claim 1, characterized in that: The iron-containing layered silicate minerals include one of biotite, vermiculite, nontronite, kaolinite, talc, and illite.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the media balls to the iron-containing layered silicate minerals is 20:1 - 100:

1.

4. The preparation method according to claim 1, characterized in that: The rotation speed of the ball milling is 400 rpm - 600 rpm, the time is 1 - 48 h, during the ball milling, the ball milling is carried out for 5 - 30 min and paused for 1 - io min.

5. The preparation method according to claim 1, characterized in that: Wet ball milling or dry ball milling is adopted. For the wet ball milling, the solvent is one or more of ethanol, water, isopropanol, ethylene glycol, and glycerol; the media for the balls are ceramic media balls, metal media balls, and non-metal media balls.

6. An iron-containing layered silicate mineral nanozyme prepared by the preparation method according to any one of claims 1 - 5.

7. An iron-containing layered silicate mineral nanozyme as described in claim 6, characterized in that: The surface acidity is increased, and low-spin iron is generated.

8. Application of an iron-containing layered silicate mineral nanozyme as described in claim 6 or 7 in colorimetric sensing, characterized in that: In the presence of H2O2, the iron-containing layered silicate mineral nanozyme reacts with the chromogenic substrate to produce a chromogenic reaction. The change in the absorbance value of the solution is correlated with the concentration of the target analyte, thereby establishing a colorimetric sensing platform for detecting the concentration of the target analyte to be detected.

9. Use of the iron-containing layered silicate mineral nanozyme in colorimetric sensing according to claim 8, characterized in that: The chromogenic substrate is oxidized 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, or o-phenylenediamine.

10. The antibacterial application of a ball-milled layered silicate mineral nanozyme according to claim 7 or 8, characterized in that: The iron-containing layered silicate mineral nanozyme catalyzes the decomposition of H2O2 to generate hydroxyl radicals, singlet oxygen, and superoxide radicals with strong oxidation ability, and can be used to efficiently kill bacteria.

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