Photo-acid-driven single-hole colloid motor and preparation method thereof

Abstract

The invention discloses a photo-acid-driven single-hole colloid motor and a preparation method thereof, and belongs to the field of colloid motors. The colloid motor takes a polyelectrolyte single-hole microcapsule loaded with photoacid as a core, and a capsule-like vesicle film is assembled on the surface of the polyelectrolyte single-hole microcapsule. The method comprises the following steps: extracting the thylakoid vesicles embedded with the biomolecular motor FlFo-ATP synthase by virtue of methods of homogenizing and homogenizing, gradient centrifugation and extrusion; 2, preparing a single-hole microcapsule by a heterogeneous nucleation, self-expansion and selective etching method; 3, carrying out layer-by-layer self-assembly by taking the single-hole microcapsule as a template to prepare a polyelectrolyte single-hole microcapsule; and 4, assembling the capsule-like vesicles on the surface of the polyelectrolyte single-hole microcapsule. By utilizing a unique single-hole channel structure, precise regulation and control on light-operated proton release and transmembrane transportation are realized, and the proton driving efficiency is remarkably improved, so that rotation of FlFo-ATP synthase is efficiently activated to synthesize ATP, a light response motion behavior is realized, and directional motion and energy metabolism regulation and control in a complex biological medium can be realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of colloidal motors, and particularly relates to a light-acid-driven single-hole colloidal motor and a preparation method thereof. BACKGROUND

[0002] Chemically-driven colloidal motors can convert chemical energy into directional motion at the microscale, showing great potential in the construction of non-physical equilibrium models and biomedical nanorobots. However, in complex biological media such as pathological sites, the motion performance of such colloidal motors is often severely limited. For example, enzyme-driven colloidal motors are mostly dependent on self-diffusiophoresis or self-electrophoresis mechanisms for propulsion, and the ions in the biological environment can interfere with the diffusion of products and reactants, significantly reducing the chemical potential difference and propulsion force. In contrast, protein motors (such as rotary F l F o -ATP synthase) in living organisms can work efficiently in physiological environments. In recent years, researchers have attempted to introduce F l F o -ATP synthase into synthetic systems to construct colloidal motors that respond to light or chemical gradients, but there is still a lack of effective means to regulate the behavior of proton transmembrane transport, which restricts the controllability of their motion and the integration of biological functions. Therefore, developing a colloidal motor system that can precisely regulate proton flow and has both motion control and ATP synthesis capabilities has become a key problem that needs to be broken through in this field. SUMMARY

[0003] To solve the above technical challenges, the application provides a light-acid-driven single-hole colloidal motor and a preparation method thereof. The method of the application is simple and has strong universality. The single-hole colloidal motor prepared by the method has a unique structure and can realize the integration of light-controlled motion and ATP synthesis, and has good motion control ability and biocompatibility in complex biological environments.

[0004] To achieve the above object, the technical scheme adopted by the application is as follows:

[0005] A light-acid-driven single-hole colloidal motor, wherein the colloidal motor takes a polyelectrolyte single-hole microcapsule loaded with a light acid as a core, and a vesicle membrane is assembled on the surface of the colloidal motor, wherein F l F o -ATP synthase is distributed on the membrane.

[0006] Further, the light acid is a photoacid generator 2-hydroxybenzaldehyde (2-NBA) loaded in the single-hole microcapsule, and the light acid has the property of decomposing and releasing protons in response to ultraviolet light; the single-hole microcapsule has a single-hole structure, a particle size of 2-3 um, preferably 2.2 um, and a pore size of 300-350 nm, and the single-hole channel is used to realize the directional release and transmembrane transport of the light-controlled protons.

[0007] Further, the polyelectrolyte is assembled by alternating cationic polyelectrolyte polyallylamine hydrochloride and anionic polyelectrolyte sodium polystyrene sulfonate, and the thickness is 50-150 nm.

[0008] Further, the polyelectrolyte microcapsule is assembled by alternating 30 layers of cationic polyelectrolyte and 30 layers of anionic polyelectrolyte on the single-hole microcapsule.

[0009] Further, the thylakoid vesicle is extracted from the thylakoid membrane of chloroplasts of spinach, kale, celery or romaine lettuce, the size of the thylakoid vesicle is 100±10 nm, and the thickness of the assembled membrane is 3-7 nm.

[0010] A preparation method of the above-mentioned photoacid-driven single-hole colloidal motor, the method comprising the following preparation steps:

[0011] Step one: extracting thylakoid vesicles;

[0012] Step two: obtaining single-hole microcapsules by using a heterogeneous nucleation, self-swelling and selective etching method; the heterogeneous nucleation uses polystyrene with a size of 300-400 nm (preferably 350 nm) as a heterogeneous seed, the self-swelling occurs in 110 mM sodium hydroxide solution, and the etching solvent is tetrahydrofuran.

[0013] Step three: alternately assembling cationic polyelectrolyte and anionic polyelectrolyte on the single-hole microcapsule particles by using a template-assisted layer-by-layer self-assembly method to obtain polyelectrolyte single-hole microcapsules with a single-hole channel inside; and loading photoacid into the polyelectrolyte single-hole microcapsules by using a solvent exchange method;

[0014] Step four: mixing the thylakoid vesicles obtained in step one with the polyelectrolyte single-hole microcapsules loaded with photoacid obtained in step three by using a vesicle fusion method to obtain a photoacid-driven single-hole colloidal motor.

[0015] Further, the step one is specifically:

[0016] (1) prepare three kinds of buffer solution: chloroplast buffer solution, chloroplast lysis solution and thylakoid protection solution; wherein, the chloroplast buffer solution is composed of 50-80 g sucrose, 0.2-0.5 g potassium chloride (KCl), 0.5-1.2 g sodium hydrogen phosphate (Na2HPO4), 1.5-2.8 g potassium dihydrogen phosphate (KH2PO4), 0.15-0.34 g magnesium chloride (MgCl2) dissolved in water and made up to 500 mL; the chloroplast lysis solution is composed of 8-12 mL of 10 mM Hepes-NaOH (pH=7.5), 0.3-0.6 g magnesium chloride (MgCl2) dissolved in water and made up to 500 mL; the thylakoid protection solution is composed of 1.5-3.6 mL of 10 mM Hepes-NaOH (pH=7.5), 0.16-0.36 g magnesium chloride (MgCl2), 1.2-2.4 g potassium chloride (KCl) dissolved in water and made up to 500 mL, and stored in a 4°C refrigerator for standby;

[0017] (2) after the 500 g fresh spinach leaves are washed and the stems are removed, they are stored in a 4°C refrigerator overnight, 300-600 mL of chloroplast buffer solution is added, homogenized and crushed, the mesophyll is destroyed, and the chloroplasts are separated out;

[0018] (3) the homogenate solution obtained in step (2) is filtered with 8 layers of gauze to collect the filtrate, and the precipitate is collected by centrifugation at 3500-4500 rpm for 3-6 min and resuspended in 10-30 mL of chloroplast buffer solution;

[0019] (4) 8-12 mL of 60%(w / v) sucrose, 8-12 mL of 40%(w / v) sucrose and 8-12 mL of 20%(w / v) sucrose are sequentially added to the centrifuge tube from bottom to top, then the mixed solution obtained in step (3) is added above the 20%(w / v) sucrose, and centrifugation is performed at 3500-4800 rpm for 4-6 min at 4°C, and the dark green solution between the 20%(w / v) and 40%(w / v) sucrose solutions is taken;

[0020] (5) the solution obtained in step (4) is washed with chloroplast buffer solution at 3500-4800 rpm for 4-6 min at 4°C, the supernatant is removed, the precipitate is collected, and the washing is repeated three times to obtain purified chloroplasts;

[0021] (6) the purified chloroplasts obtained in step (5) are centrifuged at 4500-5800 rpm for 4-6 min at 4°C, the supernatant is removed, the precipitate is left, 25-45 mL of chloroplast lysis solution is added, and the mixture is uniformly mixed and then placed in a 4°C refrigerator for 1-2 h;

[0022] (7) the mixture obtained in step (6) is centrifuged at 4500-5800 rpm for 4-6 min at 4°C, the supernatant is removed, and the precipitate is obtained to be dispersed in 6-12 mL of thylakoid protection solution.

[0023] (8) Pour the suspension obtained in step (7) into a mortar, add ultra-fine quartz sand, the quartz sand should not exceed the surface of the suspension, uniformly mix and grind for 30-60 min, centrifuge at 1000-1500 rpm for 2-3 min at 4°C, remove the quartz sand precipitate at the bottom, centrifuge the supernatant at 7000-8500 rpm for 4-6 min, remove the precipitate, recover the supernatant to obtain a thylakoid membrane fragment liquid;

[0024] (9) The thylakoid membrane fragment liquid obtained in step (8) is sequentially extruded through a cell membrane extruder with polycarbonate membranes with pore diameters of 1 μm, 800 nm, 400 nm, 200 nm and 100 nm from large to small, and each pore diameter needs to be extruded for 21-42 cycles to obtain a thylakoid vesicle suspension.

[0025] Further, the step two is specifically:

[0026] (1) The styrene (PS) is repeatedly washed with 8-15 wt% sodium hydroxide (NaOH) in a separatory funnel, then the styrene after alkali washing is mixed with deionized water for washing to remove excess sodium hydroxide, and the solution is washed to be neutral, and finally mixed with anhydrous sodium sulfate (Na2SO4) or anhydrous magnesium sulfate (MgSO4) and stand for 30-60 min to remove excess deionized water, and the upper clear light yellow purified styrene is removed for standby;

[0027] (2) 10-15 mL of the purified styrene obtained in step (1) is added to 100-150 mL of deionized water, and the nitrogen gas is continuously purged for 20-40 min, and then gradually heated to 70°C, and then 0.15-0.4 g of potassium persulfate (KPS) is added, and the mixture is stirred for 24 h to obtain a 300-350 nm polystyrene seed, which is centrifuged and washed with deionized water for 3 times, and then the polystyrene seed is dispersed in deionized water and freeze-dried for 24 h, and then resuspended in deionized water to form a 6-10 wt% polystyrene seed liquid, which is stored at room temperature for standby;

[0028] (3) 4-8 mL of 3-(trimethoxysilyl)propyl methacrylate (TPM) is added to 40-80 mL of deionized water and stirred vigorously for 5-6 h, and then 50-75 mL of the mixture is quickly mixed with 70-85 mL of deionized water, 20-50 μL of ammonia water (NH3·H2O) (28 wt%) and 60-150 μL of the polystyrene seed liquid obtained in step (2) to obtain a uniform mixture, and then the mixture is left to stand for 40-90 min;

[0029] (4) The milky white solution obtained in step (3) is added to a 100-150 mM sodium hydroxide solution at a volume ratio of 1:3, and after 40-60 s, a photoinitiator (Darocur 1173, 0.1 v / v%) is quickly added to the mixture, and ultraviolet irradiation is performed for 1-2 h;

[0030] (5) The solution obtained in step (4) is centrifuged at 3500-4800 rpm for 4-6 min, the supernatant is removed, and the precipitate is collected to obtain TPM-PS microcapsules, which are resuspended in tetrahydrofuran for 30-60 min to selectively etch the PS seeds, thereby obtaining a TPM microcapsule solution with single holes;

[0031] (6) The TPM single-hole microcapsule solution obtained in step (5) is centrifuged at 3500-4800 rpm for 4-6 min, the supernatant is removed, and the precipitate is collected and washed with anhydrous ethanol and water three times in turn to obtain TPM single-hole microcapsules.

[0032] Further, the step three is specifically:

[0033] (1) The TPM single-hole microcapsules obtained in step two are used as templates, and the TPM single-hole microcapsules are added to a cationic polyelectrolyte solution (composed of a polyallylamine hydrochloride solution containing sodium chloride, wherein the concentration of sodium chloride is 0.2-0.5 M, and the concentration of polyallylamine hydrochloride is 1-3 mg / mL), and after oscillation on a shaker for 15-20 min, centrifugation is performed, the centrifugate is discarded, the obtained precipitate is washed with a 0.2-0.5 M sodium chloride solution three times, and is added to an anionic polyelectrolyte solution (composed of a sodium polystyrene sulfonate solution containing sodium chloride, wherein the concentration of sodium chloride is 0.2-0.5 M, and the concentration of sodium polystyrene sulfonate is 1-3 mg / mL), and after oscillation on a shaker for 15-20 min, centrifugation is performed, the centrifugate is discarded, the obtained precipitate is washed with a 0.2-0.5 M NaCl solution three times, and the assembly of a polyelectrolyte bilayer is completed;

[0034] (2) Step (1) is repeated 30 times to obtain a polyelectrolyte microcapsule with a single-hole structure;

[0035] (3) The polyelectrolyte microcapsules obtained in step (2) are centrifuged at 5500-6800 rpm for 1-2 min, the supernatant is removed, 5-6 mg of the precipitate is added to 0.4-1 mL of an ethanol photoacid mixture, oscillation is performed for 20-40 min, centrifugation is performed, the supernatant is discarded, and the obtained precipitate is washed with deionized water three times to obtain a polyelectrolyte single-hole microcapsule loaded with a photoacid inside; the ethanol photoacid mixture is obtained by mixing a 50-200 μM photoacid aqueous solution and anhydrous ethanol at a volume ratio of 1:1.

[0036] Further, the step four is specifically: taking 0.3-0.6 mL of the polyelectrolyte single-hole microcapsule loaded with the photoacid obtained in step three, centrifuging at 5500-6800 rpm for 1-2 min, then removing the supernatant and retaining the precipitate; adding 0.5-1.5 mL of the class cystosome vesicle suspension obtained in step one into the precipitate, mixing uniformly by blowing, then oscillating in an ice bath for 30-45 min, and then washing with the class cystosome protective solution for three times to remove the excess class cystosome vesicles, so as to obtain the photoacid-driven single-hole colloidal motor.

[0037] Compared with the prior art, the present application has the following beneficial effects: the preparation process of the present application is simple and has good repeatability, and the prepared single-hole colloidal motor has both light-controlled directional movement and autonomous ATP synthesis capacity. l F o The core advantage of the present application is that the polyelectrolyte microcapsule with a single channel is used as the supporting platform of the F l F o The ATP synthase assembled in the single-hole region can be efficiently driven to rotate to drive the motor to realize submarine forward swimming. In addition, the single-hole colloidal motor can sense the ADP concentration gradient in the environment and exhibit a chemotactic autonomous navigation behavior, and continuously synthesize ATP during the movement, thereby realizing truly self-powered movement. This feature effectively overcomes the key bottleneck that the traditional enzyme-driven motor is easily disturbed by ions in the physiological medium and the propulsion force significantly decays. The present application realizes the organic integration of spatial constraint of proton flow and biological molecular motor driving through the single-hole structure, and provides a new technical solution for the ATP supply shortage problem related to mitochondrial dysfunction and other metabolic diseases, and has a broad prospect in biomedical applications. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a preparation process schematic diagram of the photoacid-driven single-hole colloidal motor according to the present application;

[0039] Figure 2 is a scanning electron microscope graph of the photoacid-driven single-hole colloidal motor according to the present application;

[0040] Figure 3 is a transmission electron microscope graph of the photoacid-driven single-hole colloidal motor according to the present application;

[0041] Figure 4 is a laser confocal fluorescence graph of the photoacid-driven single-hole colloidal motor according to the present application;

[0042] Figure 5is a self-propelled movement and movement direction diagram of the photoacid-driven single-hole colloidal motor under ultraviolet light irradiation according to the present application;

[0043] Figure 6 is a photo-phosphorylation speed diagram of the photoacid-driven single-hole colloidal motor under different light intensities according to the present application;

[0044] Figure 7 is a directional migration movement diagram of the photoacid-driven single-hole colloidal motor in the presence of a chemotactic source according to the present application. DETAILED DESCRIPTION

[0045] The technical solutions of the present application are further described below in conjunction with the drawings and examples, but are not limited thereto, and any modification or equivalent replacement of the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application shall be covered in the protection scope of the present application.

[0046] The present application utilizes a unique single-hole channel structure to realize precise regulation of photo-controlled proton release and transmembrane transport, significantly improves the proton driving efficiency, and thus efficiently activates F l F o The rotation of the ATP synthase synthesizes ATP to realize a light-responsive movement behavior, and can realize directional movement and energy metabolism regulation in a complex biological medium. The present application provides a new way for the application of colloidal motors in future biological environments, and is especially suitable for active energy regulation in precise diagnosis and treatment of diseases.

[0047] Example 1

[0048] The preparation process of the photoacid-driven single-hole colloidal motor in this embodiment is shown in Figure 1 and includes the following steps:

[0049] Step one, extract thylakoid vesicles;

[0050] a, prepare a buffer solution, wherein the chloroplast buffer solution is composed of 68.56 g of sucrose, 0.3727 g of potassium chloride (KCl), 0.88995 g of disodium hydrogen phosphate (Na2HPO4), 2.04135 g of potassium dihydrogen phosphate (KH2PO4), and 0.2033 g of magnesium chloride (MgCl2) dissolved in water and made up to 500 mL; the chloroplast lysis solution is composed of 10 mL of 10 mM Hepes-NaOH (pH=7.5), 0.50825 g of magnesium chloride (MgCl2) dissolved in water and made up to 500 mL; the thylakoid protection solution is composed of 2.5 mL of 10 mM Hepes-NaOH (pH=7.5), 0.254125 g of magnesium chloride (MgCl2), and 1.86375 g of potassium chloride (KCl) dissolved in water and made up to 500 mL, and is stored in a 4℃ refrigerator for standby;

[0051] b. Wash 500g of fresh spinach leaves, remove the stems, store them at 4℃ overnight, add 500mL of chloroplast buffer, mix and homogenize, crush to break down the leaf mesophyll and separate the chloroplasts.

[0052] c. Filter the homogenate obtained in step b through 8 layers of gauze to collect the filtrate, centrifuge at 4000 rpm for 5 min to collect the precipitate, and resuspend it in 20 mL of chloroplast buffer.

[0053] d. Add 8-12 mL of 60% (w / v) sucrose, 8-12 mL of 40% (w / v) sucrose, and 8-12 mL of 20% (w / v) sucrose to the centrifuge tube from bottom to top. Then add the mixture obtained in step (3) above the 20% (w / v) sucrose and centrifuge at 3500-4800 rpm for 4-6 min at 4℃. Take the dark green solution between the 20% (w / v) and 40% (w / v) sucrose solutions.

[0054] e. Wash the solution obtained in step d with chloroplast buffer at 4000 rpm for 5 min at 4℃, remove the supernatant, collect the precipitate, and repeat the washing three times to obtain purified chloroplasts.

[0055] f. Centrifuge the purified chloroplasts obtained in step e at 5000 rpm for 5 min at 4℃, remove the supernatant, keep the precipitate, add 30 mL of chloroplast lysis buffer, mix well, and place in a refrigerator at 4℃ for 1~2 h.

[0056] g. Centrifuge the mixture obtained in step f at 5000 rpm for 5 min at 4 °C, remove the supernatant, retain the precipitate to obtain thylakoids, and re-disperse them in 10 mL of thylakoid protection solution.

[0057] h. Pour the suspension obtained in step g into a mortar, add ultrafine quartz sand, the quartz sand should not exceed the surface of the suspension, mix evenly and grind for 30 min, centrifuge at 1000 rpm for 2 min at 4℃, remove the bottom quartz sand precipitate, take the supernatant and centrifuge at 8000 rpm for 5 min, remove the precipitate, and recover the supernatant to obtain thylakoid membrane fragment fluid.

[0058] i. The thylakoid membrane fragment solution obtained in step h is extruded sequentially through a cell membrane extruder using polycarbonate membranes with 1 μm, 800 nm, 400 nm, 200 nm and 100 nm pore sizes, from large pore size to small pore size. Each pore size requires 21 cycles of extrusion to obtain a thylakoid vesicle suspension.

[0059] Step 2: Single-pore microcapsules are obtained using heterogeneous nucleation, self-expansion, and selective etching methods;

[0060] j. The purified styrene was repeatedly washed with 10 wt% sodium hydroxide in a separatory funnel, followed by washing the purified styrene with deionized water to remove the excess sodium hydroxide until the solution was neutral, and finally mixed with anhydrous sodium sulfate or anhydrous magnesium sulfate for 30 min to remove the excess deionized water, and the upper clear light yellow purified styrene was removed for use;

[0061] k. The 13 mL purified styrene obtained in step j was added to 120 mL deionized water, and the nitrogen gas was continuously purged for 30 min, and then gradually heated to 70°C, and 0.3 g of potassium persulfate was added, and the reaction was stirred for 24 h to obtain a 350 nm polystyrene seed, which was centrifuged and recovered, and washed with deionized water for 3 times, and then the polystyrene seed was dispersed in deionized water and freeze-dried for 24 h to form a 6-10 wt% polystyrene seed solution, which was stored at room temperature for use;

[0062] l. 6 mL of 3-(trimethoxysilyl)propyl methacrylate (TPM) was added to 60 mL of deionized water and stirred vigorously for 5-6 h, and then 60 mL of the mixture was quickly mixed with 80 mL of deionized water, 30 μL of ammonia water (NH3·H2O) (28 wt%), and 100 μL of the polystyrene seed solution obtained in step k to form a uniform mixture, and then the mixture was allowed to stand for 60 min;

[0063] m. The milky white solution obtained in step l was added to 110 mM sodium hydroxide solution at a volume ratio of 1:3, and then 0.1 v / v% of a photoinitiator (Darocur 1173) was quickly added to the mixture after 40 s, and then the mixture was subjected to ultraviolet irradiation for 1-2 h;

[0064] n. The solution obtained in step m was centrifuged at 4000 rpm for 5 min, and the supernatant was removed, and the precipitate was collected to obtain TPM-PS microcapsules, which were resuspended in tetrahydrofuran for 30 min to selectively etch the PS seed, and then a TPM microcapsule solution with a single hole was obtained;

[0065] o. The TPM single-hole microcapsule solution obtained in step n was centrifuged at 4000 rpm for 5 min, and the supernatant was removed, and the precipitate was collected and washed with anhydrous ethanol and water for 3 times to obtain TPM single-hole microcapsules;

[0066] Step three, a template-assisted layer-by-layer self-assembly method was used to alternately assemble cationic polyelectrolyte and anionic polyelectrolyte on the single-hole microcapsule particles to obtain polyelectrolyte microcapsules with a single hole;

[0067] p. Using the TPM single-hole microcapsule obtained in step o as a template, the TPM single-hole microcapsule was added to a cationic polyelectrolyte solution (consisting of a polyallylamine hydrochloride solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2 M and the concentration of polyallylamine hydrochloride was 2 mg / mL), and after oscillation on a shaker for 15 min, centrifugation was performed, the supernatant was discarded, and the obtained precipitate was washed with 0.2 M sodium chloride solution three times by centrifugation. The obtained precipitate was added to an anionic polyelectrolyte solution (consisting of a sodium polystyrene sulfonate solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2 M and the concentration of sodium polystyrene sulfonate was 2 mg / mL), and after oscillation on a shaker for 15 min, centrifugation was performed, the supernatant was discarded, and the obtained precipitate was washed with 0.5 M NaCl solution three times, thereby completing the assembly of a polyelectrolyte bilayer;

[0068] q. The step p was repeated for 30 times, thereby obtaining a polyelectrolyte microcapsule with a single-hole structure.

[0069] r. After centrifugation of the polyelectrolyte microcapsule obtained in step q at 6000 rpm for 2 min, the supernatant was removed, 5.5 mg of the precipitate was added to 0.5 mL of an ethanol photoacid mixture, oscillation was performed for 30 min, centrifugation was performed, the supernatant was discarded, and the obtained precipitate was washed with deionized water three times, thereby obtaining a polyelectrolyte single-hole microcapsule loaded with photoacid inside. The ethanol photoacid mixture was obtained by mixing 150-200 μM photoacid aqueous solution and anhydrous ethanol at a volume ratio of 1:1.

[0070] Step four, the cochleate-like vesicle described in step one was mixed with the polyelectrolyte single-hole microcapsule loaded with photoacid described in step three by the method of vesicle fusion, thereby obtaining a photoacid-driven single-hole colloidal motor.

[0071] s. 0.5 mL of the polyelectrolyte single-hole microcapsule loaded with photoacid obtained in step r was centrifuged at 6000 rpm for 2 min, and the supernatant was removed. 1 mL of the cochleate-like vesicle suspension obtained in step i was added to the precipitate, and after mixing by blowing, oscillation was performed in an ice bath for 30 min, and then the cochleate-like vesicle protection solution was used for washing three times to remove excess cochleate-like vesicles, thereby obtaining a photoacid-driven single-hole colloidal motor.

[0072] The photoacid-driven single-hole colloidal motor obtained in Example 1 was dispersed on a hydrophilic silicon wafer, and after freeze-drying treatment, the morphology under a scanning electron microscope was as shown in Figure 2 The diameter of the single-hole colloidal motor was about 2.4 μm, the surface roughness was increased, the surface structure was complete, the same spherical structure as the TPM microcapsule was maintained, and after assembly of the polyelectrolyte layer and the cochleate-like vesicle, only a shallow surface depression was present at the original single-hole position, indicating that the polyelectrolyte layer and the cochleate-like vesicle were successfully assembled on the surface of the single-hole colloidal motor. l F o- The thylakoid membrane, functionalized with ATP synthase, completely covered the single pore. The single-pore colloidal motor driven by the biomolecular motor obtained in Example 1 was stained with 0.1% phosphotungstic acid for two minutes and then observed under a transmission electron microscope. Figure 3 As shown, the single-pore colloidal motor retains the original hollow cavity and single-pore channel. Magnified boundaries reveal a clear TPM microcapsule shell-polyelectrolyte layer-thylakoid membrane sandwich structure, embedded with F... l F o The thylakoid membrane thickness of ATP synthase is approximately 5 nm, indicating the presence of F... l F o -Thylakoid vesicles of ATP synthase were successfully assembled on the surface of the colloidal motor.

[0073] The laser confocal scanning image of the photoacid-driven single-pore colloidal motor obtained in Example 1 is shown below. Figure 4 As shown, the polyelectrolyte membrane was labeled with carbon quantum dots (QCDs), the thylakoid membrane was labeled with the lipophilic dye Dil, and the colloidal motor lumen was loaded with 6-carboxyfluorescein (6-CF). The merged fluorescence images showed the colocalization of all components, confirming the presence of F-embedded fluorescein. l F o The thylakoid membrane of ATP synthase uniformly covers the surface of the colloidal motor. Furthermore, localized changes in fluorescence intensity on the colloidal motor surface can identify single-pore sites, further confirming precise, pore-specific membrane assembly. In summary, these results confirm the presence of F-embedded... l F o - The thylakoid membrane of ATP synthase was successfully integrated into the single-pore surface of TPM polyelectrolyte microcapsules, resulting in well-defined, functionally partitioned single-pore colloidal motors.

[0074] The photoacid-driven single-well colloidal motor obtained in Example 1 was placed in ADP buffer (pH 8.0, 10 mM Mricine-NaOH, 5 mM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2, and 30 mM NaCl). 0.5 h before the start of the reaction, DTT was added to the single-well colloidal motor to bring its final concentration to 50 mM to reduce and activate F. l F o -ATP synthase. Use 15 W / m 2 Irradiate the object with LED light of wavelength 365nm and observe its movement under a fluorescence microscope. The results are as follows: Figure 5 As shown, blue fluorescence was observed in the encapsulated photoacid 2-NBA after UV irradiation of the product 2-NBS. Notably, the brighter spots observed at the edges correspond to the single pores of the monopore colloidal motor, due to the accumulation of negatively charged 2-NBS within the charged polyelectrolyte film through the single-pore channels. This observation confirms that the monopore colloidal motor exhibits high fluorescence intensity at F...l F o Driven by ATP synthase, it moves in the opposite direction to the single pore, similar to the forward propulsion of a micro-submarine driven by a nanopropeller.

[0075] Take 3 ml of the photoacid-driven single-well colloidal motor obtained in Example 1 and mix it with 3 ml of ADP buffer. The ADP buffer consists of 10 mM Tricine-NaOH (pH 8.0), 2 mM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2, and 30 mM NaCl. Use a 15 W / m... 2 The device is irradiated with LED light of a specific wavelength, and samples are taken at regular intervals. The ATP content is detected by high-performance liquid chromatography (HPLC), and the ATP production rate is calculated. Figure 6 As shown, ATP production increases rapidly in the first 10 minutes, then gradually plateaus at approximately 3.74 μM in 2 mM ADP. This indicates that the single-pore colloidal motor possesses photophosphorylation activity, and its self-propelled motion is driven by the photophosphorylation process.

[0076] A cylindrical agarose gel was pre-soaked in 10 mM OH solution. - Incubate overnight in (pH 8.0) and 7.5 mM ADP solution, and place on the left edge of a culture dish filled with PBS solution to establish an ADP / OH mixture. - Concentration gradient. The photoacid-driven single-well colloidal motor obtained in Example 1 was placed on the right side of the culture dish, and the directional chemotactic motion of the colloidal motor was observed under an optical microscope. Figure 7 As shown, these single-pore colloidal motors can sense localized ADP / OH levels. - Gradient, and along the concentration gradient towards ADP / OH-rich areas. - The regions are arranged and move forward, accompanied by the production of ATP.

[0077] The preparation process of this invention is simple, and the prepared single-pore colloidal motor has good biocompatibility. It can carry out photophosphorylation reaction under ultraviolet light. The energy ATP is produced through the rotation of the biomolecular motor, and the single-pore colloidal motor can achieve self-driven and directional migration. It has broad application prospects in biomedical fields such as biosynthesis, drug loading, and regulation of cellular energy metabolism.

[0078] The raw materials exemplified in this invention, as well as the upper and lower limits and ranges of the raw materials, and the upper and lower limits and ranges of the process parameters (such as temperature, time, etc.) can all realize this invention. Examples will not be listed one by one here.

[0079] The above descriptions are only the preferable embodiments of the present application, which are only illustrative but not restrictive. It is understood by those skilled in the art that the present application can be changed, modified or even equivalent within the spirit and scope defined by the claims of the present application, and all of them will fall into the protection scope of the present application.

Claims

1. A photoacid-driven single-hole colloidal motor, characterized by: The colloidal motor takes a polyelectrolyte single-hole microcapsule loaded with a photoacid as a core, and a thylakoid-like vesicle membrane is assembled on the surface of the core.

2. The photoacid-driven single-hole colloidal motor of claim 1, wherein: The photoacid is a photo-induced acid generator 2-hydroxybenzaldehyde (2-NBA) loaded in the single-hole microcapsule; the single-hole microcapsule has a single-hole structure, a particle size of 2-3 μm, and a hole size of 300-350 nm.

3. The photoacid-driven single-hole colloidal motor of claim 1, wherein: The polyelectrolyte is alternately assembled by a cationic polyelectrolyte polyallylamine hydrochloride and an anionic polyelectrolyte sodium polystyrene sulfonate, and has a thickness of 50-150 nm.

4. The photoacid-driven single-hole colloidal motor of claim 1, wherein: The polyelectrolyte microcapsule is alternately assembled by 30 layers of cationic polyelectrolyte and 30 layers of anionic polyelectrolyte on the single-hole microcapsule.

5. The photoacid-driven single-hole colloidal motor of claim 1, wherein: The thylakoid-like vesicle is extracted from a thylakoid membrane of a chloroplast, has a size of 100±10 nm, and has a membrane thickness of 3-7 nm after assembly.

6. A method for preparing the photoacid-driven single-hole colloidal motor according to any one of claims 1 to 5, characterized in that: The method comprises the following preparation steps: Step one: extract thylakoid-like vesicles; Step two: obtain single-hole microcapsules by using a heterogeneous nucleation, self-expansion and selective etching method; Step three: alternately assemble a cationic polyelectrolyte and an anionic polyelectrolyte on a single-hole microcapsule particle by using a template-assisted layer-by-layer self-assembly method to obtain a polyelectrolyte single-hole microcapsule with a single-hole channel in the interior, and load a photoacid into the polyelectrolyte single-hole microcapsule by using a solvent exchange method; Step four: mix the thylakoid-like vesicles obtained in step one with the polyelectrolyte single-hole microcapsule loaded with the photoacid obtained in step three by using a vesicle fusion method to obtain a single-hole colloidal motor driven by the photoacid.

7. The method of claim 6, wherein: The step one specifically comprises: (1) prepare three buffers: a chloroplast buffer, a chloroplast lysis buffer and a thylakoid protection buffer; wherein the chloroplast buffer is prepared by dissolving 50-80 g of sucrose, 0.2-0.5 g of potassium chloride (KCl), 0.5-1.2 g of sodium phosphate dibasic (Na2HPO4), 1.5-2.8 g of potassium phosphate monobasic (KH2PO4) and 0.15-0.34 g of magnesium chloride (MgCl2) in water and making up to 500 mL; the chloroplast lysis buffer is prepared by dissolving 8-12 mL of 10 mM Hepes-NaOH (pH=7.5) and 0.3-0.6 g of magnesium chloride (MgCl2) in water and making up to 500 mL; and the thylakoid protection buffer is prepared by dissolving 1.5-3.6 mL of 10 mM Hepes-NaOH (pH=7.5), 0.16-0.36 g of magnesium chloride (MgCl2) and 1.2-2.4 g of potassium chloride (KCl) in water and making up to 500 mL, and is stored in a 4℃ refrigerator for standby; (2) wash 500 g of fresh spinach leaves to remove stems, and then store them in a 4℃ refrigerator overnight, add 300-600 mL of the chloroplast buffer, homogenize and crush, destroy the leaf pulp, and separate the chloroplasts; (3) filter the homogenate liquid obtained in step (2) with 8 layers of gauze, centrifuge the filtrate at 3500-4500 rpm for 3-6 min to collect the precipitate, and resuspend it in 10-30 mL of the chloroplast buffer; (4) In the centrifuge tube from bottom to top, add 8-12 mL of 60% (w / v) sucrose, 8-12 mL of 40% (w / v) sucrose, 8-12 mL of 20% (w / v) sucrose, then add the mixture obtained in step (3) above 20% (w / v) sucrose, centrifuge at 4°C at 3500-4800 rpm for 4-6 min, take the dark green solution between 20% (w / v) and 40% (w / v) sucrose solution; (5) The solution obtained in step (4) is washed with chloroplast buffer at 4°C at 3500-4800 rpm for 4-6 min, the supernatant is removed, the precipitate is collected, and the washing is repeated three times to obtain purified chloroplasts; (6) The purified chloroplasts obtained in step (5) are centrifuged at 4°C at 4500-5800 rpm for 4-6 min, the supernatant is removed, and the precipitate is left. Add 25-45 mL of chloroplast lysis solution, mix well and place in a 4°C refrigerator for 1-2 h; (7) The mixture obtained in step (6) is centrifuged at 4°C at 4500-5800 rpm for 4-6 min, the supernatant is removed, and the precipitate is obtained. Resuspend and disperse in 6-12 mL of thylakoid protection solution; (8) Pour the suspension obtained in step (7) into a mortar, add ultra-fine quartz sand, the quartz sand should not exceed the surface of the suspension, mix evenly, and grind for 30-60 min. Centrifuge at 4°C at 1000-1500 rpm for 2-3 min, remove the bottom quartz sand precipitate, centrifuge the supernatant at 7000-8500 rpm for 4-6 min, remove the precipitate, and recover the supernatant to obtain the thylakoid membrane fragment solution; (9) The thylakoid membrane fragment solution obtained in step (8) is sequentially extruded through a cell membrane extruder using 1 μm, 800 nm, 400 nm, 200 nm, and 100 nm polycarbonate membranes from large to small pore size. Each pore size requires 21-42 cycles of extrusion to obtain a thylakoid vesicle suspension.

8. The method of claim 6, wherein: The step two is specifically: (1) The styrene (PS) is repeatedly washed with 8-15 wt% sodium hydroxide (NaOH) in a separatory funnel, then the washed styrene is mixed with deionized water to remove excess sodium hydroxide, and the solution is washed to neutral. Finally, mix with anhydrous sodium sulfate (Na2SO4) or anhydrous magnesium sulfate (MgSO4) and stand for 30-60 min to remove excess deionized water. Remove the upper clear light yellow purified styrene for use; (2) Take 10-15 mL of purified styrene obtained in step (1) and add 100-150 mL of deionized water. Purify with nitrogen gas for 20-40 min. Gradually heat to 70°C, then add 0.15-0.4 g of potassium persulfate (KPS). Stir for 24 h to obtain 300-350 nm polystyrene seeds. Centrifuge and wash the precipitate with deionized water 3 times. Then disperse the polystyrene seeds in deionized water and freeze-dry for 24 h. Resuspend in deionized water to form a 6-10 wt% polystyrene seed solution. Store at room temperature for future use. (3) 4-8 mL of 3-(trimethoxysilyl)propyl methacrylate (TPM) was added into 40-80 mL of deionized water and stirred vigorously for 5-6 h, 50-75 mL of the mixed solution was quickly mixed with 70-85 mL of deionized water, 20-50 μL of ammonia (NH3·H2O) (28 wt%) and 60-150 μL of the polystyrene seed solution obtained in step (2) above to be uniform, and then was left to stand for 40-90 min; (4) The milky white solution obtained in step (3) was added into 100-150 mM sodium hydroxide solution at a volume ratio of 1:3, and then a photoinitiator (Darocur 1173, 0.1 v / v%) was quickly added into the mixed solution after 40-60 s, and then was subjected to ultraviolet irradiation for 1-2 h; (5) The solution obtained in step (4) was centrifuged at 3500-4800 rpm for 4-6 min, the supernatant was removed, and the precipitate was collected to obtain TPM-PS microcapsules, which were resuspended in tetrahydrofuran for 30-60 min to selectively etch the PS seed, thereby obtaining a TPM microcapsule solution with a single hole; (6) The TPM single-hole microcapsule solution obtained in step (5) was centrifuged at 3500-4800 rpm for 4-6 min, the supernatant was removed, and the precipitate was collected, which was washed with anhydrous ethanol and water for three times in turn, thereby obtaining TPM single-hole microcapsules.

9. The method of claim 6, wherein: The step three is specifically: (1) The TPM single-hole microcapsules obtained in step two were used as a template, and the TPM single-hole microcapsules were added into a cationic polyelectrolyte solution (consisting of a polyallylamine hydrochloride solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2-0.5 M, and the concentration of polyallylamine hydrochloride was 1-3 mg / mL), and then was oscillated on a shaker for 15-20 min and centrifuged, and then the centrifugal liquid was discarded, and the obtained precipitate was washed with 0.2-0.5 M sodium chloride solution for three times, and then was added into an anionic polyelectrolyte solution (consisting of a sodium polystyrene sulfonate solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2-0.5 M, and the concentration of sodium polystyrene sulfonate was 1-3 mg / mL), and then was oscillated on a shaker for 15-20 min and centrifuged, and then the centrifugal liquid was discarded, and the obtained precipitate was washed with 0.2-0.5 M NaCl solution for three times, thereby completing the assembly of a polyelectrolyte bilayer; (2) Step (1) was repeated for 30 times, thereby obtaining polyelectrolyte microcapsules with a single-hole structure; (3) The polyelectrolyte microcapsules obtained in step (2) were centrifuged at 5500-6800 rpm for 1-2 min, the supernatant was removed, 5-6 mg of the precipitate was added into 0.4-1 mL of an ethanol photoacid mixture, and then was oscillated for 20-40 min and centrifuged, and then the supernatant was discarded, and the obtained precipitate was washed with deionized water for three times, thereby obtaining polyelectrolyte single-hole microcapsules loaded with photoacid inside; the ethanol photoacid mixture was obtained by mixing 50-200 μM photoacid aqueous solution and anhydrous ethanol at a volume ratio of 1:

1.

10. The method of claim 6, wherein: The step four is specifically: taking 0.3-0.6 mL of the polyelectrolyte single-hole microcapsule loaded with the photoacid obtained in step three, centrifuging at 5500-6800 rpm for 1-2 min, removing the supernatant and retaining the precipitate; adding 0.5-1.5 mL of the class cystoid vesicle suspension obtained in step one to the precipitate, mixing uniformly by blowing, oscillating in an ice bath for 30-45 min, and then washing with the class cystoid protection solution for three times to remove the excess class cystoid vesicles, so as to obtain the photoacid-driven single-hole colloidal motor.

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