Glucose oxidase-driven swimming nanorobot and preparation method thereof

Abstract

The application discloses a glucose oxidase driven swimming nanorobot and a preparation method thereof, and belongs to the technical field of swimming nanorobots. The application constructs a small-scale swimming nanorobot with biocompatibility. The application uses a sol-gel method to prepare mesoporous silica balls with small scales, and then constructs a yin-yang type mesoporous silica-gold nanoparticle after unilateral modification of metal gold. After modification of glucose oxidase and polyethylene glycol on the gold side, the swimming nanorobot has certain biocompatibility, and can perform self-propelled motion in a glucose solution. The application effectively avoids biological adhesion, can meet the application scene requirement when applied in a biological environment, and has a good application prospect in the fields of active drug delivery, biosensing and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of swimming nanorobots, and particularly relates to a glucose oxidase driven swimming nanorobot and a preparation method thereof. BACKGROUND

[0002] Swimming nanorobots refer to nanosystems capable of converting chemical energy or light, electricity, magnetism and other energy in the surrounding environment into their own mechanical movement in a fluid. The size of swimming nanorobots is usually in the micro-nano scale, and is in the low Reynolds number region. Due to the existence of viscous effect, swimming nanorobots cannot maintain the inertial force of macroscopic object movement, and need to overcome viscous resistance and Brownian motion, so that driving and motion control of swimming nanorobots are challenging. Through sustainable conversion (input) of energy, effective displacement can occur in a low Reynolds number environment. Unlike other colloidal particles of micro-nano scale that only make Brownian motion, swimming nanorobots can break symmetry and time reversal through continuous conversion of energy in the surrounding environment, and produce effective displacement in a low Reynolds number. At present, swimming nanorobots can be used for drug targeted transport, tumor treatment and other biomedical fields after chemical modification. The application requirements of swimming nanorobots in a biocompatible environment are becoming higher and higher. The construction of small-scale swimming nanorobots with biocompatibility can be better applied to medical treatment. SUMMARY

[0003] So far, swimming nanorobots have been developed for decades. Among various fuels reported for driving swimming nanorobots, the most commonly used fuel is hydrogen peroxide. Water, acid, base, urea and glucose can also be used as chemical fuels, and these fuels have the advantages of good biocompatibility and low toxicity. Enzyme-driven swimming nanorobots have good application prospects in the fields of drug targeted transport and biosensing due to their small size, flexible movement and clear driving mechanism.

[0004] The application develops a glucose oxidase driven swimming nanorobot. In the method of the application, mesoporous silica spheres are asymmetrically modified, metal gold is deposited on one side of the surface of the mesoporous silica by physical vapor deposition technology of metal vacuum sputtering, thereby forming a yin-yang type mesoporous silica-gold nanoparticle, and after the glucose oxidase and polyethylene glycol are grafted on the gold side of the mesoporous silica sphere, the swimming nanorobot can perform self-driven movement in a glucose solution. The motor of the application has the advantages that mesoporous silica spheres with small size are prepared by a sol-gel method, and after one side is modified with metal gold, a yin-yang type mesoporous silica-gold nanoparticle is constructed. After the glucose oxidase and polyethylene glycol are modified on the gold side, the swimming nanorobot has certain biocompatibility, and can perform self-propelled movement in a glucose solution.

[0005] To solve the above technical problems, the present application adopts the following technical solutions:

[0006] The present application aims to provide a preparation method of glucose oxidase driven swimming nanorobot, which is carried out according to the following steps:

[0007] Step one, disperse the mesoporous silica spheres in deionized water, ultrasonic dispersion to uniform, then drop on the hydrophilic substrate, after natural spreading into a single layer of particles, dry;

[0008] Step two, then use vacuum sputtering physical vapor deposition technology, deposit metal on one side of the mesoporous silica sphere surface, then use deionized water to wash the particles on the hydrophilic substrate, centrifugal, get MSN@Au yin and yang mesoporous silica-metal nanoparticles;

[0009] Step three, disperse the MSN@Au yin and yang mesoporous silica-gold nanoparticles prepared in step two in a volume concentration of 90% to 100% ethanol solution, add mercapto polyethylene glycol and mercapto propionic acid, oscillate for at least 30 min, centrifugal water washing for at least 3 times, get MSN@Au-COOH-PEG particles;

[0010] Step four, disperse the MSN@Au-COOH-PEG particles in PBS buffer, add 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide, react for at least 30 min, centrifugal for at least 15 min, then disperse in PBS buffer, add glucose oxidase and react for at least 30 min, centrifugal for at least 15 min, get glucose oxidase driven swimming nanorobot.

[0011] Further limited, in step one, the mesoporous silica spheres are prepared by the following steps:

[0012] Step 1, mix 200mg-250mg cetyltrimethylammonium bromide, 50mg-80mg sodium hydroxide and 100mL-120mL deionized water, heat to 80℃, then add 1mL-2mL tetraethyl orthosilicate dropwise, after dropwise addition, heat for 2h, then centrifugal water washing for 3 times under the condition of 7000r / min-8000r / min, dry at 80℃ to get white solid powder;

[0013] Step 2, place the powder obtained in step 1 in a quartz boat and in a tube furnace, under air atmosphere, the temperature rising rate is 1℃ / min-2℃ / min, the temperature is raised to 500℃-550℃, then calcine for 4h-5h under the condition of calcination temperature of 500℃-550℃, finally wash, centrifugal and dry, get mesoporous silica spheres.

[0014] Further limit, in step two, the metal is gold, and the deposition thickness is 10nm-20nm.

[0015] Further limit, in step two, the vacuum sputtering parameters are: current 20mA-30mA, and sputtering time 1min-2min.

[0016] Further limit, in step three, the concentration of mercapto polyethylene glycol in the reaction system is 0.2mg / mL-0.5mg / mL, and the concentration of mercaptopropionic acid in the reaction system is 0.2mg / mL-0.5mg / mL.

[0017] Further limit, in step three, under the condition that the rotation speed is 7000r / min-8000r / min, centrifugation is carried out for 15min-20min.

[0018] Further limit, in step three, the volume concentration of the ethanol solution is 90%-100%, and the concentration of the mesoporous silica sphere dispersion solution is 0.2mg / mL-1mg / mL.

[0019] Further limit, in step four, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide in the reaction system is 0.01M-0.02M, and the concentration of N-hydroxysuccinimide in the reaction system is 0.05M-0.01M.

[0020] Further limit, in step four, the concentration of glucose oxidase in the reaction system is 1mg / mL-2mg / mL.

[0021] The glucose oxidase driven swimming nanorobot prepared by the above method.

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

[0023] The preparation method of the present application is simple to operate, and the prepared swimming nanorobot has small size and good chemical stability and dispersity, and by loading glucose oxidase to convert glucose in the surrounding environment into driving force, the swimming nanorobot is partially modified with polyethylene glycol, which can effectively avoid biological adhesion, and can meet the application scene requirements when applied in the biological environment, and has good application prospect in the fields of active drug delivery and biological sensing.

[0024] The present application prepares swimming nanorobots by a physical vapor deposition method of vacuum sputtering, deposits metal on one side of the colloidal particle surface, introduces asymmetry, and prepares swimming nanorobots with certain functions. Then, different chemical modifications are made on the swimming nanorobots according to the requirements of different application scenes, so that the swimming nanorobots have different motion characteristics.

[0025] The glucose oxidase is modified by gold, polyethylene glycol and glucose, the glucose in the organism can be used for chemical driving, and the glucose oxidase can be better applied in the biomedical field.

[0026] The application prepares a swimming nanorobot with good stability and biocompatibility and capable of moving in a glucose solution, and has important significance for promoting the practical application of the swimming nanorobot in a biomedical environment.

[0027] In order to further understand the features and technical contents of the application, please refer to the following detailed description of the application and the accompanying drawings, however, the accompanying drawings are provided for reference and illustration only, and are not used to limit the application. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A preparation method flowchart of the glucose oxidase driven swimming nanorobot of the application;

[0029] Figure 2 A transmission electron microscope image of the MSN@Au-GOx-PEG swimming nanorobot prepared in Example 1;

[0030] Figure 3 An energy dispersive X-ray spectrum of the MSN@Au-GOx-PEG swimming nanorobot prepared in Example 1, a is the Si element, b is the Au element, and c is the S element;

[0031] Figure 4 A glucose oxidase activity test image of the MSN@Au-GOx-PEG swimming nanorobot prepared in Example 1;

[0032] Figure 5 A graph of the relationship between the translational diffusion coefficient and the glucose solution concentration of the MSN@Au-GOx-PEG swimming nanorobot prepared in Example 1;

[0033] Figure 6 A principle diagram of the enzyme catalytic driving of the MSN@Au-GOx-PEG swimming nanorobot prepared in Example 1. DETAILED DESCRIPTION

[0034] The application will be further described below in combination with specific examples, and it should be understood that the examples are only used to illustrate the application and not used to limit the scope of the application.

[0035] Example 1: In this embodiment, a preparation method of a glucose oxidase driven swimming nanorobot is realized according to the following steps:

[0036] Step one: preparing mesoporous silica spheres

[0037] Step 1, 250 mg of cetyltrimethylammonium bromide, 60 mg of sodium hydroxide solid and 120 mL of deionized water were heated to 80℃, then 1.25 mL of tetraethyl orthosilicate was added dropwise, and heated for 2 h. Centrifugal water washing 3 times, 80℃ drying to obtain white solid powder;

[0038] Step 2, the powder obtained in step 1 was placed in a quartz boat and placed in a tube furnace, the temperature was raised to 550℃ at a rate of 1℃ / min in an air atmosphere, then calcined at a calcination temperature of 550℃ for 5h, and finally washed, centrifuged and dried to obtain mesoporous silica spheres.

[0039] Step 2, then use vacuum sputtering physical vapor deposition technology to deposit gold on one side of the mesoporous silica sphere surface under the condition of current 20 mA, sputtering time 1 min, then use deionized water to wash the particles on the hydrophilic substrate, centrifuge at a speed of 8000 r / min for 15 min, to obtain MSN@Au ambipolar mesoporous silica-gold nanoparticles;

[0040] Step 3, the MSN@Au ambipolar mesoporous silica-gold nanoparticles prepared in step 2 were dispersed in a 90% volume concentration ethanol solution, the mesoporous silica sphere dispersion liquid concentration was 0.2 mg / mL, mercapto polyethylene glycol and mercapto propionic acid were added, the concentration of mercapto polyethylene glycol added to the reaction system was 0.3 mg / mL, and the concentration of mercapto propionic acid added to the reaction system was 0.3 mg / mL, and the mixture was oscillated for 30 min. Centrifugal water washing at least 3 times, each time at a speed of 7000 r / min for 15 min, to obtain MSN@Au-COOH-PEG particles;

[0041] Step 4, 0.5 mg of MSN@Au-COOH-PEG particles were dispersed in 5 mL to 10 mL of PBS buffer, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide were added, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide added to the reaction system was 0.01 mg / mL, and the concentration of N-hydroxysuccinimide added to the reaction system was 0.01 mg / mL, the reaction was carried out for 30 min, and then the mixture was centrifuged for 15 min and dispersed in PBS buffer. Then 1 mg / mL of glucose oxidase was added and the mixture was reacted for 30 min. The mixture was centrifuged at a speed of 7000 r / min for 15 min to obtain glucose oxidase-driven swimming nanorobot.

[0042] To verify the self-driving nature of the glucose oxidase-driven swimming nanorobot, the motor was placed in glucose solutions of different concentrations and moved. The motion was detected using a Malvern dynamic light scattering instrument. After motion analysis, motion parameters such as the translational diffusion coefficient proved that the swimming nanorobot could move self-drivingly in the glucose solution.

[0043] The transmission electron microscope image of the MSN@Au-GOx-PEG swimming nanorobots prepared in this embodiment is shown below. Figure 2 As shown; by Figure 2 The morphological characteristics of glucose oxidase-driven swimming nanorobots can be seen. The mesoporous silica spheres have a uniform pore structure, and one side of the mesoporous silica spheres is covered with metallic gold with a thickness of 10 nm, which confirms the yin-yang structure of glucose oxidase-driven swimming nanorobots.

[0044] The energy dispersive X-ray spectrum of the MSN@Au-GOx-PEG swimming nanorobot prepared in this embodiment is shown below. Figure 3 As shown, by Figure 3 Elemental analysis of the glucose oxidase-driven swimming nanorobots revealed that silicon was present in the mesoporous silica spheres, and the distribution of gold indicated that the mesoporous silica spheres were covered with metallic gold on one side. The distribution of sulfur indicated the binding sites of thiol polyethylene glycol and glucose oxidase on the gold side of the mesoporous silica spheres. In other words, thiol polyethylene glycol and glucose oxidase were distributed on one side of the surface of the swimming nanorobots, meaning that glucose oxidase and thiol polyethylene glycol were simultaneously covered on the metallic gold side.

[0045] The glucose oxidase activity test diagram of the MSN@Au-GOx-PEG swimming nanorobot prepared in this embodiment is shown in the figure below. Figure 4 As shown, by Figure 4 The relationship between glucose oxidase activity and time was investigated using UV-Vis spectroscopy. Glucose oxidase decomposes glucose solution to produce gluconic acid and hydrogen peroxide solution. Then, peroxidase decomposes hydrogen peroxide to produce free radicals. TMB reacts with the generated free radicals to form a colorimetric reaction, producing an absorption peak. A 2 mL mixture containing 250 μM 3,3',5,5'-tetramethylbenzidine (TMB), 175 mM glucose aqueous solution, and 4 μg peroxidase (HRP) was prepared. Glucose oxidase was added to the mixture to drive the mobile nanorobots. After mixing and dispersion, UV-Vis and near-infrared spectroscopy was performed at wavelengths of 400-800 nm. The concentration of hydrogen peroxide in the solution changed over time. Within 40 min, the concentration of hydrogen peroxide gradually increased and stabilized, indicating that glucose oxidase was modified on the surface of the mobile nanorobots and was active within 40 min.

[0046] The relationship between the translational diffusion coefficient of the MSN@Au-GOx-PEG motile nanorobot prepared in the embodiment and the concentration of the glucose solution is shown in the graph of Figure 5 As shown in Figure 5 It can be seen that when the glucose solution is increased from 0 mM to 75 mM, the translational diffusion coefficient of the MSN@Au-GOx-PEG motile nanorobot is increased from 0.57 μm 2 / s to 1.34 μm 2 / s. The translational diffusion coefficient of the MSN@Au-PEG microparticle in the glucose solution of 0 mM to 75 mM is increased from 0.64 μm 2 / s to 0.52 μm 2 / s. The translational diffusion coefficient of the MSN@Au-GOx-PEG motile nanorobot is increased with the increase of the concentration of the glucose solution, while the translational diffusion coefficient of the MSN@Au-PEG microparticle is basically unchanged;

[0047] The principle diagram of the enzyme catalytic driving of the MSN@Au-GOx-PEG motile nanorobot prepared in the embodiment is shown in Figure 6 The glucose oxidase on the surface of the MSN@Au-GOx-PEG motile nanorobot catalytically decomposes the glucose solution in the surrounding environment, and the products are gluconic acid and hydrogen peroxide, and the glucose oxidase provides the driving force.

Claims

1. A method for preparing a glucose oxidase-driven swimming nanorobot, characterized by The preparation method is carried out according to the following steps: Step one, disperse the mesoporous silica spheres in deionized water, ultrasonic dispersion to uniform, then drop on the hydrophilic substrate, after natural spread into a single layer of particles, drying; Step two, then use vacuum sputtering physical vapor deposition technology, deposit gold on one side of the mesoporous silica sphere surface, then use deionized water to wash the particles on the hydrophilic substrate, centrifugal, get MSN@Au yin and yang mesoporous silica-gold nanoparticles; Step three, disperse the MSN@Au yin and yang mesoporous silica-gold nanoparticles prepared in step two in a volume concentration of 90%~100% ethanol solution, add mercapto polyethylene glycol and mercapto propionic acid, oscillate at least 30 min, centrifugal cleaning at least 3 times, get MSN@Au-COOH-PEG particles; Step four, disperse the MSN@Au-COOH-PEG particles in PBS buffer, add 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide, react at least 30 min, centrifugal at least 15 min, then disperse in PBS buffer, add glucose oxidase, react at least 30 min, centrifugal at least 15 min, get glucose oxidase driven swimming nanorobot.

2. The production method according to claim 1, characterized by, In step one, the mesoporous silica spheres are prepared by the following steps: Step 1, mix 200mg~250mg cetyltrimethylammonium bromide, 50mg~80mg sodium hydroxide and 100mL~120mL deionized water, heat to 80℃, then add 1mL~2mL tetraethyl orthosilicate dropwise, after dropwise addition, heat for 2h, then centrifugal water washing 3 times at a rotation speed of 7000r / min~8000r / min, dry at 80℃ to obtain white solid powder; Step 2, place the powder obtained in step 1 in a quartz boat and in a tube furnace, under air atmosphere, the temperature is raised at a rate of 1℃ / min~2℃ / min, then the temperature is raised to 500℃~550℃, then calcine at a calcination temperature of 500℃~550℃ for 4h~5h, finally wash, centrifugal and dry to obtain mesoporous silica spheres.

3. The preparation method according to claim 1, characterized in that, In step two, the deposition thickness is 10nm~20nm.

4. The production method according to claim 1, characterized by, In step two, the vacuum sputtering parameters: current 20mA~30mA, sputtering time 1min~2min. In step three, the concentration of mercapto polyethylene glycol in the reaction system is 0.2mg / mL~0.5mg / mL and the concentration of mercapto propionic acid in the reaction system is 0.2mg / mL~0.5mg / mL.

5. The preparation method according to claim 1, characterized in that, In step three, centrifugal at a rotation speed of 7000r / min~8000r / min for 15min~20min.

6. The method of claim 1, wherein, In step three, the volume concentration of the ethanol solution is 90%~100%, and the concentration of the MSN@Au yin and yang mesoporous silica-gold nanoparticles dispersion is 0.2mg / mL~1mg / mL. ​ 7. The preparation method according to claim 1, characterized in that, ​ ​ 8. The method of claim 1, wherein, In step four, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the reaction system is 0.01M-0.02M, and the concentration of N-hydroxysuccinimide in the reaction system is 0.05M-0.01M.

9. The method of claim 1, wherein, In step four, the concentration of glucose oxidase in the reaction system is 1mg / mL-2mg / mL.

10. A glucose oxidase driven swimming nanorobot prepared by the method of any one of claims 1-9.

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