Tannic acid functionalized micro-motor with functions of enhancing bacterium capture and bacteriostasis, and preparation method and application of tannic acid functionalized micro-motor

Abstract

The invention discloses a tannic acid functionalized micromotor with functions of enhancing bacterium capture and bacteriostasis, and a preparation method and application thereof. The micromotor takes Cu7S4 / vSiO2 particles with a virus-like structure as a carrier, and tannic acid is loaded on the surface of the carrier through a self-assembly method. On one hand, TA has an antibacterial effect, and rich peptidoglycan on cell walls of gram-positive bacteria has a high affinity effect; on the other hand, a virus-like structure with a spike structure has relatively high adhesion to bacteria, and the micromotor Cu7S4 / vSiO2 / TA has an enhanced capture effect on gram-positive bacteria; in addition, the photothermal effect and self-driving generated by Cu7S4 under the irradiation of infrared light can cooperate with TA to realize efficient sterilization.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, and a preparation method and application thereof. Background Art

[0002] Gram-positive bacteria impose a huge burden on human health, healthcare and social economy. Although various antimicrobial agents such as metal or metal oxide nanoparticles currently developed can be used to kill pathogenic microorganisms in water bodies, they have the disadvantages of low targeting efficiency and the need for macroscopic stirring to effectively contact pathogenic microorganisms. For example, antimicrobial materials based on advanced oxidation technology (AOP) have insufficient ability to capture pathogenic microorganisms in water bodies, making it difficult to actively capture, enrich and kill pathogenic microorganisms in water bodies in situ, and cannot achieve high sterilization efficiency.

[0003] Antibacterial micro / nanomotors have attracted extensive attention from researchers due to their high antibacterial efficiency, multifunctional integration (such as photothermal performance, photodynamic performance, etc.), precise delivery and enrichment. In addition, researchers currently mainly functionalize nanomaterials through electrostatic effects and substances that can be bound to the bacterial surface (such as antibodies, aptamers, antibiotics, etc.) to achieve the purpose of rapid capture of pathogenic microorganisms in water bodies, but they have unsatisfactory problems such as high cost. The natural product tannic acid (TA) has antioxidant, antibacterial and anti-inflammatory effects, and has high affinity with the abundant peptidoglycan on the cell wall of Gram-positive bacteria, and can selectively capture Gram-positive bacteria. In addition, inspired by the local "capture and kill" effect caused by the spike structure on the surface of natural bacteriophages, a tannic acid-functionalized virus-like micromotor with photothermal activity was constructed. On the one hand, it can efficiently capture pathogenic microorganisms in situ, and on the other hand, it can achieve efficient sterilization through photothermal action, realizing the "active capture-in situ inactivation" of Gram-positive bacteria.

[0004] So far, there has been no relevant research and reports on tannic acid-functionalized photothermal antibacterial micromotors with virus-like structures. Summary of the invention

[0005] In view of this, the present invention provides a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, which is used to solve the technical problem of low performance of traditional bactericides in killing pathogenic microorganisms in the prior art.

[0006] The first aspect of the present invention provides a method for preparing a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects.

[0007] To this end, the first technical solution provided by the present invention is as follows:

[0008] A tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects comprises Cu7S4 / vSiO2 particles with a virus-like structure and an antibacterial natural product, wherein the natural product is loaded on the surface of the Cu7S4 / vSiO2 particles with a virus-like structure.

[0009] Furthermore, in the above-mentioned tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, SiO2 coats Cu7S4 in the virus-like structured Cu7S4 / vSiO2 particles.

[0010] Furthermore, in the above-mentioned tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, the natural product is tannic acid.

[0011] Furthermore, in the above-mentioned tannic acid functionalized micromotor with enhanced bacterial capture and antibacterial effect, the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = (20-80) mg: (10-60) mg: (3-6) mmol. More preferably, the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 50 mg: 34 mg: 4.5 mmol.

[0012] Furthermore, in the above-mentioned tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, the particle size of the virus-like structured Cu7S4 / vSiO2 particles is 450nm.

[0013] The second technical solution provided by the present invention is a method for preparing the above-mentioned tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect, comprising the steps of:

[0014] Step S1: dissolving copper salt in deionized water, adding hydrazine hydrate for reduction, and then adding sodium sulfide to obtain Cu7S4 particles;

[0015] Step S2: Disperse Cu7S4 particles, hexadecyltrimethylammonium bromide and sodium hydroxide in water, add tetraethoxysilane prepared with cyclohexane, and perform hydrothermal reaction at 30-90° C. for 24-72 hours to obtain Cu7S4 / vSiO2 particles with virus-like structure;

[0016] Step S3: Mix the virus-like structure Cu7S4 / vSiO2 particle dispersion and tannic acid solution, and load tannic acid on the surface of the virus-like structure Cu7S4 / vSiO2 particles through electrostatic self-assembly to obtain an antibacterial micromotor with bacteria capture ability.

[0017] In step S3, the virus-like structure Cu7S4 / vSiO2 particle dispersion is obtained by mixing Cu7S4 / vSiO2 particles with deionized water at a mass volume ratio of (50-200) mg: (10-30) mL, and ultrasonically dispersing for 10-30 min;

[0018] The tannic acid solution is obtained by mixing tannic acid TA and deionized water in a mass volume ratio of (20-100) mg: (10-30) mL through a vortex mixer;

[0019] The electrostatic self-assembly is to place the mixed solution in a constant temperature shaker, the reaction temperature is 20-60° C., and the reaction time is 1-4 hours.

[0020] Furthermore, in the above-mentioned method for preparing a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect, in step S1, the copper salt is copper nitrate hexahydrate;

[0021] The last technical solution provided by the present invention is the application of the above-mentioned tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect in capturing and killing pathogenic microorganisms.

[0022] The pathogenic microorganisms are Escherichia coli and methicillin-resistant Staphylococcus aureus.

[0023] The third aspect of the present invention provides an application of a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects in the field of killing pathogenic microorganisms. Preferably, the pathogenic microorganisms include Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

[0024] The novel tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect prepared by the present invention has the following advantages and beneficial effects:

[0025] (1) Tannic acid TA has a high affinity to the abundant peptidoglycan on the cell wall of Gram-positive bacteria; on the other hand, the virus-like structure with spike structure has a higher adhesion to bacteria. Therefore, the micromotor Cu7S4 / vSiO2 / TA has an enhanced capture effect on Gram-positive bacteria (methicillin-resistant Staphylococcus aureus).

[0026] (2) The photothermal effect and self-driving of Cu7S4 under infrared light irradiation can cooperate with TA to achieve efficient sterilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 TEM image of the micromotor Cu7S4 / vSiO2 / TA;

[0028] Figure 2FT-IR and Zeta potential diagrams of the samples;

[0029] Figure 3 The temperature rise cycle image (a), motion trajectory (b), motion rate at different powers (c) and motion time-lapse image (d) of the micromotor Cu7S4 / vSiO2 / TA under near-infrared light 1064nm;

[0030] Figure 4 The growth status diagram of MRSA: (a) blank control group; (b) blank control group + 1064nm infrared light; (c) micromotor Cu7S4 / vSiO2 / TA + 1064nm infrared light;

[0031] Figure 5 Comparison of the capture rates of MRSA by different samples (a) and TEM image of MRSA captured by the micromotor Cu7S4 / vSiO2 / TA (b). DETAILED DESCRIPTION

[0032] The technical solution of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0033] Example 1

[0034] This embodiment 1 provides a tannic acid functionalized micromotor with enhanced bacterial capture and antibacterial effects, wherein Cu7S4 particles are prepared by a reduction method, Cu7S4 / vSiO2 particles are prepared by a hydrothermal reaction method, and a micromotor Cu7S4 / vSiO2 / TA is prepared by an electrostatic self-assembly method, wherein the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 50 mg: 34 mg: 4.5 mmol.

[0035] It is prepared by the following method:

[0036] 1) The steps of preparing Cu7S4 particles by reduction method are as follows: dissolve copper salt copper nitrate hexahydrate (0.28 g) in deionized water, add dispersant 2.5 mL PVP (50 mg / mL), stir magnetically until fully dissolved, add 35 mL hydrazine hydrate for reduction, stir magnetically for 25 min, add 1.5 mL sodium sulfide solution (7.8 mg / mL), stir magnetically for 10 min; after the reaction is completed, collect the product, wash it with ethanol and deionized water respectively, and dry it to obtain Cu7S4 particles;

[0037] 2) The steps of preparing Cu7S4 / vSiO2 particles by hydrothermal reaction are as follows: Disperse Cu7S4 particles (50 mg), hexadecyltrimethylammonium bromide (250 mg) and 0.15 mL of sodium hydroxide (0.1 mol / mL) in a round-bottom flask filled with deionized water (10 mL), place in an oil bath pot at 60°C and use magnetic stirring to react for 2 hours. Then add a mixture of 1 mL of tetraethoxysilane and 9 mL of cyclohexane, react in an oil bath pot at 60°C for 48 hours, cool to room temperature after the reaction, collect the product, wash with anhydrous ethanol and deionized water, and dry to obtain Cu7S4 / vSiO2 particles.

[0038] 3) The steps for preparing Cu7S4 / vSiO2 / TA micromotors by electrostatic self-assembly method are as follows: ultrasonically disperse 100 mg of virus-like structured Cu7S4 / vSiO2 particles in 20 mL of deionized water, add 20 mL of TA (2.5 mg / mL) and mix, place it in a constant temperature shaker, shake it at 200 r / min and 40°C for 2 h, collect the product, wash it with anhydrous ethanol and deionized water, and dry it to obtain the virus-like structured micromotors Cu7S4 / vSiO2 / TA.

[0039] Among them, the TEM images of Cu7S4 particles, Cu7S4 / vSiO2 particles and micromotor Cu7S4 / vSiO2 / TA are as follows: Figure 1 As shown in Figures a to c, it can be seen from the figures that the Cu7S4 prepared in Example 1 of the present application is irregular in shape and has a particle size of about 100 nm; Cu7S4 / vSiO2 is a virus-like structure, with a spike structure on the surface and is relatively rough; the morphology of the micromotor Cu7S4 / vSiO2 / TA is similar to that of Cu7S4 / vSiO2, showing a virus-like structure with a spike structure, indicating that the loading of TA has not changed its morphology.

[0040] The FT-IR and Zeta potential diagrams of the micromotor Cu7S4 / vSiO2 / TA are shown in Figure 2 As shown, from Figure 2 It can be concluded from a that the FT-IR image of Cu7S4 / vSiO2 / TA has typical characteristic peaks of Cu7S4 / vSiO2 and TA, proving that TA has been successfully loaded on the surface of Cu7S4 / vSiO2. Figure 2 b It can be concluded that Cu7S4 / vSiO2 and TA have opposite potentials. The micromotor Cu7S4 / vSiO2 / TA can be prepared by electrostatic self-assembly, and the surface of the final product has a negative charge.

[0041] Example 2

[0042] This embodiment 2 provides a tannic acid functionalized micromotor with enhanced bacterial capture and antibacterial effects, wherein Cu7S4 particles are prepared by a reduction method, Cu7S4 / vSiO2 particles are prepared by a hydrothermal reaction method, and a micromotor Cu7S4 / vSiO2 / TA is prepared by an electrostatic self-assembly method, wherein the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 20 mg: 10 mg: 3 mmol.

[0043] It is prepared by the following method:

[0044] 1) The steps of preparing Cu7S4 particles by reduction method are as follows: dissolve copper salt copper nitrate hexahydrate (0.28 g) in deionized water, add dispersant 2.5 mL PVP (50 mg / mL), stir magnetically until fully dissolved, add 35 mL hydrazine hydrate for reduction, stir magnetically for 25 min, add 1.5 mL sodium sulfide solution (7.8 mg / mL), stir magnetically for 10 min; after the reaction is completed, collect the product, wash it with ethanol and deionized water respectively, and dry it to obtain Cu7S4 particles;

[0045] 2) The steps of preparing Cu7S4 / vSiO2 particles by hydrothermal reaction are as follows: Disperse Cu7S4 particles (20 mg), hexadecyltrimethylammonium bromide (166.7 mg) and 0.15 mL of sodium hydroxide (0.07 mol / mL) in a round-bottom flask filled with deionized water (10 mL), place in an oil bath pot at 60°C and use magnetic stirring to react for 2 hours. Then add a mixture of 0.7 mL of tetraethoxysilane and 6 mL of cyclohexane, react in an oil bath pot at 60°C for 48 hours, cool to room temperature after the reaction, collect the product, wash it with anhydrous ethanol and deionized water, and dry it to obtain Cu7S4 / vSiO2 particles.

[0046] 3) The steps for preparing Cu7S4 / vSiO2 / TA micromotors by electrostatic self-assembly method are as follows: ultrasonically disperse 100 mg of virus-like structured Cu7S4 / vSiO2 particles in 20 mL of deionized water, add 20 mL of TA (0.75 mg / mL) and mix, place it in a constant temperature shaker, shake it at 200 r / min and 40°C for 2 h, collect the product, wash it with anhydrous ethanol and deionized water, and dry it to obtain the virus-like structured micromotors Cu7S4 / vSiO2 / TA.

[0047] Example 3

[0048] This Example 3 provides a tannic acid functionalized micromotor with enhanced bacterial capture and antibacterial effects, wherein Cu7S4 particles are prepared by a reduction method, Cu7S4 / vSiO2 particles are prepared by a hydrothermal reaction method, and a micromotor Cu7S4 / vSiO2 / TA is prepared by an electrostatic self-assembly method, wherein the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 80 mg: 60 mg: 6 mmol.

[0049] It is prepared by the following method:

[0050] 1) The steps of preparing Cu7S4 particles by reduction method are as follows: dissolve copper salt copper nitrate hexahydrate (0.28 g) in deionized water, add dispersant 2.5 mL PVP (50 mg / mL), stir magnetically until fully dissolved, add 35 mL hydrazine hydrate for reduction, stir magnetically for 25 min, add 1.5 mL sodium sulfide solution (7.8 mg / mL), stir magnetically for 10 min; after the reaction is completed, collect the product, wash it with ethanol and deionized water respectively, and dry it to obtain Cu7S4 particles;

[0051] 2) The steps of preparing Cu7S4 / vSiO2 particles by hydrothermal reaction are as follows: Disperse Cu7S4 particles (80 mg), hexadecyltrimethylammonium bromide (333.3 mg) and 0.15 mL of sodium hydroxide (0.13 mol / mL) in a round-bottom flask filled with deionized water (10 mL), place in an oil bath pot at 60°C and use magnetic stirring to react for 2 hours. Then add a mixture of 1.3 mL of tetraethoxysilane and 12 mL of cyclohexane, react in an oil bath pot at 60°C for 48 hours, cool to room temperature after the reaction, collect the product, wash it with anhydrous ethanol and deionized water, and dry it to obtain Cu7S4 / vSiO2 particles.

[0052] 3) The steps for preparing Cu7S4 / vSiO2 / TA micromotors by electrostatic self-assembly are as follows: ultrasonically disperse 100 mg of virus-like structured Cu7S4 / vSiO2 particles in 20 mL of deionized water, add 20 mL of TA (4.4 mg / mL) and mix, place it in a constant temperature shaker, shake it at 200 r / min and 40°C for 2 h, collect the product, wash it with anhydrous ethanol and deionized water, and dry it to obtain the virus-like structured micromotors Cu7S4 / vSiO2 / TA.

[0053] Example 4

[0054] In this embodiment, the micromotor Cu7S4 / vSiO2 / TA prepared in Example 1 was subjected to motion testing.

[0055] The 0.1 mg Cu7S4 / vSiO2 / TA micromotor provided in Example 1 was placed in a 96-well plate containing 100 μL of water, and its motion characteristics in water were observed with or without the application of 1064 nm infrared light.

[0056] The results are as follows Figure 3 As shown, from Figure 3 a It can be seen that the micromotor Cu7S4 / vSiO2 / TA heats up from 25°C to 75°C within 8 minutes and can be recycled 3 times, indicating that Cu7S4 / vSiO2 / TA has good photothermal effect and recyclability. Figure 3 b is its motion trajectory under near-infrared light irradiation. The motion rate of the micromotor Cu7S4 / vSiO2 / TA under near-infrared light is positively correlated with the light power, such as Figure 3 As shown in c, when the optical power increases from 1 to 2 mW / cm 2 When the speed of the movement increases from 2 to 7um / s. Figure 3 d is the time-lapse image of the micromotor Cu7S4 / vSiO2 / TA moving in water.

[0057] Example 5

[0058] In this example, the antibacterial property of the micromotor Cu7S4 / vSiO2 / TA prepared in Example 1 against MRSA was tested.

[0059] The process of testing the bactericidal performance of the micromotor Cu7S4 / vSiO2 / TA is as follows: 0.1 mg of the micromotor Cu7S4 / vSiO2 / TA provided in Example 1 was added to the MRSA bacterial solution of the experimental group, wherein the concentration of the micromotor in the bacterial solution after addition was 0.5 mg / mL, and a 1064 nm infrared laser was used as the photothermal light source at an optical power of 1.5 mW / cm 2 After irradiation for 8 minutes, 100 μL of bacterial solution was taken out, diluted 10×10 times, and 100 μL was evenly spread on LB solid culture medium. Each sterilization plate was repeated 3 times, and then cultured in a 37°C constant temperature incubator for 24 hours. No antibacterial micromotors were added to the MRSA bacterial solution of the control group. The test process was the same. The antibacterial activity of Cu7S4 / vSiO2 / TA with micromotors was evaluated by observing the number of MRSA colonies in the experimental group and the control group.

[0060] The antibacterial results of the micromotor Cu7S4 / vSiO2 / TA are as follows Figure 4 As shown in Figure 2, Figure c shows excellent antibacterial effect compared to Figure a and Figure b. At the same time, Figure b also proves that at 1.5 mW / cm 2 Irradiation with 1064nm infrared laser for 8min did not affect the normal growth of MRSA.

[0061] Example 6

[0062] In this example, the MRSA capture performance of the sample prepared in Example 1 was tested.

[0063] The test process of the micromotor's MRSA capture performance is as follows: 1 mg of the micromotor Cu7S4 / vSiO2 / TA provided in Example 1 was added to the MRSA bacterial solution of the experimental group, wherein the concentration of the antibacterial micromotor in the bacterial solution after addition was 0.5 mg / mL. A 1064 nm infrared laser was used as the photothermal light source at an optical power of 1.5 mW / cm 2 After the reaction, the supernatant was taken from the bacterial solution and the OD600 absorbance was tested for comparison with the initial bacterial solution. At the same time, 1 mL of the bacterial solution after the reaction was taken and fixed with 4% paraformaldehyde for 6 hours, then centrifuged, and then graded and dehydrated with 30%, 50%, 70% and 90% methanol, and finally stored with 100% methanol.

[0064] The test results of the sample's MRSA capture performance are as follows: Figure 5 As shown, from Figure 5 a It can be seen that compared with Cu7S4 and Cu7S4 / vSiO2, the capture rate of MRSA by the micromotor Cu7S4 / vSiO2 / TA is significantly improved. Figure 5 b is the TEM image of the micromotor Cu7S4 / vSiO2 / TA capturing MRSA. It can be seen that Cu7S4 / vSiO2 / TA is tightly aggregated on MRSA, intuitively demonstrating its capture ability.

[0065] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, characterized in that: The invention comprises Cu7S4 / vSiO2 particles with a virus-like structure and antibacterial natural products, wherein the natural products are loaded on the surface of the Cu7S4 / vSiO2 particles with a virus-like structure.

2. The tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 1, characterized in that: In the virus-like structured Cu7S4 / vSiO2 particles, SiO2 coats Cu7S4.

3. The tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 2, characterized in that: The natural product is tannic acid.

4. The tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 3, characterized in that: The mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = (20-80) mg: (10-60) mg: (3-6) mmol.

5. The tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 2, characterized in that: The particle size of the virus-like structure Cu7S4 / vSiO2 particles is 450nm.

6. The method for preparing a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to any one of claims 1 to 5, characterized in that: Includes steps: Step S1: dissolving copper salt in deionized water, adding hydrazine hydrate for reduction, and then adding sodium sulfide to obtain Cu7S4 particles; Step S2: Disperse Cu7S4 particles, hexadecyltrimethylammonium bromide and sodium hydroxide in water, add tetraethoxysilane prepared with cyclohexane, and perform hydrothermal reaction at 30-90° C. for 24-72 hours to obtain Cu7S4 / vSiO2 particles with virus-like structure; Step S3: Mix the dispersion of the virus-like structured Cu7S4 / vSiO2 particles with the tannic acid solution to obtain a mixed solution, and load the tannic acid on the surface of the virus-like structured Cu7S4 / vSiO2 particles through electrostatic self-assembly to obtain an antibacterial micromotor with the ability to capture bacteria.

7. The method for preparing a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 6, characterized in that: In step S1, the copper salt is copper nitrate hexahydrate.

8. The method for preparing a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 6, characterized in that: In step S3, the virus-like structure Cu7S4 / vSiO2 particle dispersion is obtained by mixing Cu7S4 / vSiO2 particles with deionized water at a mass volume ratio of (50-200) mg: (10-30) mL, and ultrasonically dispersing for 10-30 min; The tannic acid solution is obtained by mixing tannic acid TA and deionized water in a mass volume ratio of (20-100) mg: (10-30) mL through a vortex mixer; The electrostatic self-assembly is to place the mixed solution in a constant temperature shaker, the reaction temperature is 20-60° C., and the reaction time is 1-4 hours.

9. Use of the tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effect as described in any one of claims 1 to 5 in capturing and killing pathogenic microorganisms.

10. The use according to claim 9, characterized in that: The pathogenic microorganisms are Escherichia coli and methicillin-resistant Staphylococcus aureus.

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