A photoacid-driven single-hole colloidal motor and a preparation method thereof

Abstract

The application relates to a light-acid-driven single-hole colloidal motor and a preparation method thereof, and belongs to the field of colloidal motors. The colloidal motor takes a polyelectrolyte single-hole microcapsule loaded with a light acid as a core, and a thylakoid vesicle membrane is assembled on the surface. The method is as follows: a biomolecular motor F l F o l F o ATPase is embedded through the method of homogenate, gradient centrifugation and extrusion; secondly, a single-hole microcapsule is prepared through a method of heterogeneous nucleation, self-swelling and selective etching; thirdly, polyelectrolyte single-hole microcapsules are prepared through layer-by-layer self-assembly with the single-hole microcapsule as a template; and fourthly, thylakoid vesicles are assembled on the surface of the polyelectrolyte single-hole microcapsule. The application realizes precise regulation of light-controlled proton release and transmembrane transport by utilizing a unique single-hole channel structure, significantly improves the proton driving efficiency, and thus efficiently activates the rotation of the F l F o ATPase to synthesize ATP, realizes light-responsive motion behavior, and can realize directional motion and energy metabolism regulation in a complex biological medium.

Description

Technical Field

[0001] This invention belongs to the field of colloidal motors, specifically relating to a photo-acid driven single-hole colloidal motor and its preparation method. Background Technology

[0002] Chemically driven colloidal motors can convert chemical energy into directed motion at the microscopic scale, showing great potential in the construction of non-physical equilibrium models and biomedical nanorobots. However, the motion performance of such colloidal motors is often severely limited in complex biological media such as lesion sites. For example, enzyme-driven colloidal motors mostly rely on self-diffusion or self-electrophoresis for propulsion, but 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 in living organisms (such as rotational F-type motors)... l F o ATP synthase (F-ATP synthase) can function efficiently in physiological environments. In recent years, researchers have attempted to synthesize F-ATP synthase... l F o While ATP synthase has been introduced into synthetic systems to construct colloidal motors that respond to light or chemical gradients, effective means of regulating proton transmembrane transport remain lacking, limiting their controllability and integration of biological functions. Therefore, developing a colloidal motor system capable of precisely regulating proton flow and possessing both motion control and ATP synthesis capabilities has become a critical issue requiring breakthroughs in this field. Summary of the Invention

[0003] To address the aforementioned technical challenges, this invention provides a photo-acid-driven monopore colloidal motor and its preparation method. The method of this invention is simple and widely applicable. The prepared monopore colloidal motor has a unique structure, enabling the integrated control of light-controlled motion and ATP synthesis. It exhibits excellent motion control capabilities and biocompatibility in complex biological environments.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A photoacid-driven single-pore colloidal motor, the colloidal motor having a core of a polyelectrolyte single-pore microcapsule loaded with photoacid, and a vesicle-like membrane assembled on its surface, wherein F is distributed on the membrane. l F o -ATP synthase.

[0006] Furthermore, the photoacid is a photoacid-producing agent, 2-hydroxybenzaldehyde (2-NBA), which is loaded inside a single-pore microcapsule. The photoacid has the property of releasing protons in response to ultraviolet light. The single-pore microcapsule has a single-pore structure with a particle size of 2μm~3μm, preferably 2.2μm, and a pore size of 300~350nm. The single-pore channel is used to realize the directional release and transmembrane transport of light-controlled protons.

[0007] Furthermore, the polyelectrolyte is assembled by alternating cationic polyelectrolyte polyallylamine hydrochloride and anionic polyelectrolyte sodium polystyrene sulfonate, with a thickness of 50~150 nm.

[0008] Furthermore, the polyelectrolyte microcapsules are composed of 30 layers of cationic polyelectrolytes and 30 layers of anionic polyelectrolytes alternately assembled on a single-pore microcapsule.

[0009] Furthermore, the thylakoid vesicles are extracted from the thylakoid membranes of chloroplasts from spinach, kale, celery, or romaine lettuce, with a thylakoid vesicle size of 100±10 nm and an assembled membrane thickness of 3~7 nm.

[0010] A method for preparing the above-mentioned photo-acid driven single-pore colloidal motor, the method comprising the following preparation steps:

[0011] Step 1: Extract thylakoid vesicles;

[0012] Step 2: Single-pore microcapsules are obtained using heterogeneous nucleation, self-expansion, and selective etching methods. The heterogeneous nucleation uses polystyrene with a size of 300nm~400nm (preferably 350nm) as heterogeneous seed, the self-expansion occurs in a 110mM sodium hydroxide solution, and the etching solvent is tetrahydrofuran.

[0013] Step 3: Using a template-assisted layer-by-layer self-assembly method, cationic and anionic polyelectrolytes are alternately assembled onto single-pore microcapsule particles to obtain polyelectrolyte single-pore microcapsules with internal single-pore channels; photoacid is loaded into the polyelectrolyte single-pore microcapsules using a solvent exchange method.

[0014] Step 4: By using the vesicle fusion method, the vesicle-like vesicles obtained in Step 1 are mixed with the polyelectrolyte single-pore microcapsules loaded with photoacid obtained in Step 3 to obtain a photoacid-driven single-pore colloidal motor.

[0015] Furthermore, step one specifically includes:

[0016] (1) Prepare three buffer solutions: chloroplast buffer, chloroplast lysis buffer, and thylakoid protection solution; wherein, the chloroplast buffer solution is composed of 50-80g sucrose, 0.2-0.5g potassium chloride (KCl), 0.5-1.2g disodium hydrogen phosphate (Na2HPO4), 1.5-2.8g potassium dihydrogen phosphate (KH2PO4), and 0.15-0.34g magnesium chloride (MgCl2) dissolved in water and brought to a final volume of 500mL; the chloroplast lysis buffer solution is composed of 8-12mL of 10mM Hepes-NaOH (pH=7.5), 0.3-0.6g magnesium chloride (MgCl2) dissolved in water and brought to a final volume of 500mL; the thylakoid protection solution is composed of 1.5-3.6mL of 10mM Hepes-NaOH (pH=7.5), dissolved in water and brought to a final volume of 500mL; Hepes-NaOH (pH=7.5), 0.16~0.36g magnesium chloride (MgCl2), 1.2~2.4g potassium chloride (KCl) dissolved in water and diluted to 500mL, is stored in a refrigerator at 4℃ for later use;

[0017] (2) Wash 500g of fresh spinach leaves, remove the stems, and store them at 4℃ overnight. Add 300~600mL of chloroplast buffer, mix and homogenize, crush to break down the leaf mesophyll and separate the chloroplasts.

[0018] (3) Filter the homogenate obtained in step (2) through 8 layers of gauze to collect the filtrate, centrifuge at 3500~4500rpm for 3~6min to collect the precipitate, and resuspend it in 10~30mL of chloroplast buffer.

[0019] (4) 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.

[0020] (5) Wash the solution obtained in step (4) with chloroplast buffer at 3500~4800 rpm for 4~6 min at 4℃, remove the supernatant, collect the precipitate, and repeat the washing three times to obtain purified chloroplasts;

[0021] (6) Centrifuge the purified chloroplasts obtained in step (5) at 4℃ at 4500~5800rpm for 4~6min, remove the supernatant, keep the precipitate, add 25~45mL of chloroplast lysis buffer, mix well and place in a 4℃ refrigerator for 1~2h.

[0022] (7) Centrifuge the mixture obtained in step (6) at 4°C at 4500~5800 rpm for 4~6 min, remove the supernatant, retain the precipitate to obtain thylakoids, and resuspend and disperse them in 6~12 mL of thylakoid protection solution;

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

[0024] (9) The thylakoid membrane fragments obtained in step (8) are extruded sequentially through a cell membrane extruder using polycarbonate membranes of 1 μm, 800 nm, 400 nm, 200 nm and 100 nm, from large pore size to small pore size. Each pore size requires 21 to 42 cycles of extrusion to obtain a thylakoid vesicle suspension.

[0025] Furthermore, step two specifically involves:

[0026] (1) Wash styrene (PS) repeatedly with 8-15 wt% sodium hydroxide (NaOH) in a separatory funnel. Then wash the styrene mixture with deionized water to remove excess sodium hydroxide until the solution is neutral. Finally, mix with anhydrous sodium sulfate (Na2SO4) or anhydrous magnesium sulfate (MgSO4) and let stand for 30-60 minutes to remove excess deionized water. Transfer the clear, pale yellow purified styrene from the upper layer for later use.

[0027] (2) Take 10-15 mL of the purified styrene obtained in step (1) and add it to 100-150 mL of deionized water. Purify with nitrogen for 20-40 min. Gradually raise the temperature to 70°C and add 0.15-0.4 g of potassium persulfate (KPS). Stir and react for 24 h to obtain polystyrene seeds of 300-350 nm. Centrifuge to recover the precipitate, wash it three times with deionized water, disperse the polystyrene seeds in deionized water and freeze-dry for 24 h. Resuspend the seeds in deionized water to form a 6-10 wt% polystyrene seed solution and store it at room temperature for later use.

[0028] (3) Add 4-8 mL of 3-(trimethoxysilyl)propyl methacrylate (TPM) to 40-80 mL of deionized water and stir vigorously for 5-6 h. Then, quickly mix 50-75 mL of this mixture 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, and let it stand for 40-90 min.

[0029] (4) Add the milky white solution obtained in step (3) to 100~150mM sodium hydroxide solution at a volume ratio of 1:3. After 40~60s, quickly add the photoinitiator (Darocur 1173, 0.1v / v%) to the mixture and irradiate with ultraviolet light for 1~2h.

[0030] (5) Centrifuge the solution obtained in step (4) at 3500~4800 rpm for 4~6 min, remove the supernatant, collect the precipitate to obtain TPM-PS microcapsules, resuspend in tetrahydrofuran for 30~60 min to selectively etch PS seeds, and obtain a TPM microcapsule solution with a single pore.

[0031] (6) Centrifuge the TPM single-well microcapsule solution obtained in step (5) at 3500~4800 rpm for 4~6 min, remove the supernatant, collect the precipitate, and wash it three times with anhydrous ethanol and water in sequence to obtain TPM single-well microcapsules.

[0032] Furthermore, step three specifically includes:

[0033] (1) Using the TPM single-well microcapsules obtained in step 2 as templates, the TPM single-well microcapsules were added to a cationic polyelectrolyte solution (composed of a polyallylamine hydrochloride solution containing sodium chloride, wherein the concentration of sodium chloride is 0.2~0.5M and the concentration of polyallylamine hydrochloride is 1~3mg / mL). After shaking on a shaker for 15~20min, the microcapsules were centrifuged and the centrifuged liquid was discarded. The resulting precipitate was washed three times by centrifugation with 0.2~0.5M sodium chloride solution. The microcapsules were then added to anionic polyelectrolyte solution (composed of a sodium polystyrene sulfonate solution containing sodium chloride, wherein the concentration of sodium chloride is 0.2~0.5M and the concentration of sodium polystyrene sulfonate is 1~3mg / mL). After shaking on a shaker for 15~20min, the microcapsules were centrifuged and the centrifuged liquid was discarded. The resulting precipitate was washed three times by centrifugation with 0.2~0.5M NaCl solution to complete the assembly of a polyelectrolyte bilayer.

[0034] (2) Repeat step (1) 30 times to obtain polyelectrolyte microcapsules with a single-pore structure;

[0035] (3) After centrifuging the polyelectrolyte microcapsules obtained in step (2) at 5500~6800rpm for 1~2min, remove the supernatant, take 5~6mg of precipitate and add 0.4~1mL of ethanol-photonic acid mixture, shake for 20~40min and centrifuge, discard the supernatant, and wash the obtained precipitate three times with deionized water to obtain polyelectrolyte single-pore microcapsules loaded with photonic acid; the ethanol-photonic acid mixture is obtained by mixing 50~200μM photonic acid aqueous solution and anhydrous ethanol at a volume ratio of 1:1.

[0036] Further, step four specifically involves: taking 0.3-0.6 mL of polyelectrolyte single-pore microcapsules containing 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 thylakoid vesicle suspension obtained in step one to the precipitate, mixing by pipetting, shaking in an ice bath for 30-45 min, and then washing three times with thylakoid preservation solution to remove excess thylakoid vesicles, thereby obtaining a photoacid-driven single-pore colloidal motor.

[0037] Compared with existing technologies, this invention has the following advantages: the preparation process is simple and reproducible, and the prepared single-pore colloidal motor possesses both light-controlled directional motion and autonomous ATP synthesis capabilities. Its core advantage lies in using polyelectrolyte microcapsules with a single channel as the F... l F o -A supporting platform for ATP synthase. This monopore structure allows for precise regulation of proton flow pathways: under UV excitation, protons are released only from photoacids within the cavity and forced to exit through a single channel, thus creating a localized, high-intensity proton gradient near the pore opening. This mechanism ensures that only F124 protons assembled in the monopore region are released. l F o -ATP synthase can be efficiently driven to rotate, propelling the motor to achieve submarine-like forward movement. Furthermore, this monopore colloidal motor can sense the ADP concentration gradient in the environment, exhibiting chemotactic autonomous navigation behavior and continuously synthesizing ATP during movement, achieving truly self-powered motion. This characteristic effectively overcomes the key bottleneck of traditional enzyme-driven motors, which are susceptible to ion interference and experience significant propulsion attenuation in physiological media. This invention achieves the organic integration of proton flow spatial constraint and biomolecular motor drive through a monopore structure, providing a new technical solution to the problem of insufficient ATP supply associated with mitochondrial dysfunction and other metabolic diseases, and has broad prospects for biomedical applications. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation process of the photo-acid driven single-pore colloidal motor described in this invention;

[0039] Figure 2 This is a scanning electron microscope image of the photoacid-driven single-pore colloidal motor described in this invention.

[0040] Figure 3 This is a transmission electron microscope image of the photoacid-driven single-pore colloidal motor described in this invention.

[0041] Figure 4 This is a laser confocal fluorescence image of the photoacid-driven single-pore colloidal motor described in this invention;

[0042] Figure 5This is a diagram showing the self-propulsion motion and motion direction of the photo-acid driven single-hole colloidal motor described in this invention under ultraviolet light irradiation.

[0043] Figure 6 This is a photophosphorylation rate diagram of the photoacid-driven single-pore colloidal motor described in this invention under different light intensities;

[0044] Figure 7 This is a diagram of the directional migration motion of the photoacid-driven single-pore colloidal motor described in this invention in the presence of a chemotactic source. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0046] This invention utilizes a unique single-pore channel structure to achieve precise control of optically controlled proton release and transmembrane transport, significantly improving proton-driven efficiency and thus efficiently activating F. l F o The rotation of ATP synthase to synthesize ATP enables light-responsive motor behavior, allowing for directed movement and energy metabolism regulation in complex biological media. This invention provides a new approach for the application of colloidal motors in future biological environments, particularly suitable for active energy regulation in precision disease diagnosis and treatment.

[0047] Example 1

[0048] The preparation process of the photo-acid driven single-pore colloidal motor in this embodiment is as follows: Figure 1 As shown, it includes the following steps:

[0049] Step 1: Extract thylakoid vesicles;

[0050] a. Prepare the buffer solutions: the chloroplast buffer consists of 68.56g sucrose, 0.3727g potassium chloride (KCl), 0.88995g disodium hydrogen phosphate (Na2HPO4), 2.04135g potassium dihydrogen phosphate (KH2PO4), and 0.2033g magnesium chloride (MgCl2) dissolved in water and brought to a final volume of 500mL; the chloroplast lysis buffer consists of 10mL of 10mM Hepes-NaOH (pH=7.5) and 0.50825g magnesium chloride (MgCl2) dissolved in water and brought to a final volume of 500mL; the thylakoid protection solution consists of 2.5mL of 10mM Hepes-NaOH (pH=7.5), 0.254125g magnesium chloride (MgCl2), and 1.86375g potassium chloride (KCl) dissolved in water and brought to a final volume of 500mL. Store at 4℃ for later use.

[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. Wash styrene (PS) repeatedly with 10wt% sodium hydroxide in a separatory funnel. Then wash the styrene mixture with deionized water to remove excess sodium hydroxide until the solution is neutral. Finally, mix with anhydrous sodium sulfate or anhydrous magnesium sulfate and let stand for 30 minutes to remove excess deionized water. Transfer the clear, pale yellow purified styrene from the upper layer for later use.

[0061] k. Add 13 mL of purified styrene obtained in step j to 120 mL of deionized water, continuously purify with nitrogen for 30 min, gradually raise the temperature to 70 °C, add 0.3 g of potassium persulfate, stir and react for 24 h to obtain 350 nm polystyrene seeds, centrifuge to recover the precipitate, wash with deionized water three times, disperse the polystyrene seeds in deionized water, freeze dry for 24 h, resuspend in deionized water to form a 6-10 wt% polystyrene seed solution, and store at room temperature for later use.

[0062] 1. Add 6 mL of 3-(trimethoxysilyl)propyl methacrylate (TPM) to 60 mL of deionized water and stir vigorously for 5-6 h. Then, quickly mix 60 mL of this mixture with 80 mL of deionized water, 30 μL of ammonia (NH3·H2O) (28 wt%) and 100 μL of the polystyrene seed solution obtained in step k above, and let it stand for 60 min.

[0063] m. Add the milky white solution obtained in step l to 110mM sodium hydroxide solution at a volume ratio of 1:3. After 40 seconds, quickly add the photoinitiator (Darocur 1173, 0.1 v / v%) to the mixture and irradiate with ultraviolet light for 1-2 hours.

[0064] n. Centrifuge the solution obtained in step m at 4000 rpm for 5 min, remove the supernatant, collect the precipitate to obtain TPM-PS microcapsules, resuspend in tetrahydrofuran for 30 min to selectively etch PS seeds, and obtain a TPM microcapsule solution with single pores.

[0065] o. Centrifuge the TPM single-well microcapsule solution obtained in step n at 4000 rpm for 5 min, remove the supernatant, collect the precipitate, and wash it three times with anhydrous ethanol and water to obtain TPM single-well microcapsules.

[0066] Step 3: Using a template-assisted layer-by-layer self-assembly method, cationic polyelectrolytes and anionic polyelectrolytes are alternately assembled onto single-pore microcapsule particles to obtain polyelectrolyte microcapsules with single pores.

[0067] p. Using the TPM single-well microcapsules obtained in step o as a template, the TPM single-well microcapsules were added to a cationic polyelectrolyte solution (composed of a polyallylamine hydrochloride solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2M and the concentration of polyallylamine hydrochloride was 2mg / mL). After shaking on a shaker for 15min, the microcapsules were centrifuged and the centrifuged liquid was discarded. The resulting precipitate was washed three times with 0.2M sodium chloride solution by centrifugation. The precipitate was then added to anionic polyelectrolyte solution (composed of a sodium polystyrene sulfonate solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2M and the concentration of sodium polystyrene sulfonate was 2mg / mL). After shaking on a shaker for 15min, the microcapsules were centrifuged and the centrifuged liquid was discarded. The resulting precipitate was washed three times with 0.5M NaCl solution by centrifugation to complete the assembly of a single polyelectrolyte bilayer.

[0068] q. Repeat step p 30 times to assemble polyelectrolyte microcapsules with a single-pore structure;

[0069] r. After centrifuging the polyelectrolyte microcapsules 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 ethanol-photonic acid mixture, shaken for 30 min, centrifuged again, and the supernatant was discarded. The resulting precipitate was washed three times with deionized water to obtain polyelectrolyte single-pore microcapsules loaded with photonic acid. The ethanol-photonic acid mixture was obtained by mixing 150~200 μM photonic acid aqueous solution and anhydrous ethanol at a volume ratio of 1:1.

[0070] Step 4: Using a vesicle fusion method, the vesicle-like vesicles described in Step 1 are mixed with the polyelectrolyte single-pore microcapsules loaded with photoacid described in Step 3 to obtain a photoacid-driven single-pore colloidal motor.

[0071] s. Take 0.5 mL of polyelectrolyte single-pore microcapsules containing photoacid obtained in step r, centrifuge at 6000 rpm for 2 min, remove the supernatant and keep the precipitate; add 1 mL of thylakoid vesicle suspension obtained in step i to the precipitate, mix by pipetting, shake in an ice bath for 30 min, and then wash three times with thylakoid preservation solution to remove excess thylakoid vesicles, and obtain photoacid-driven single-pore colloidal motor.

[0072] The photoacid-driven monoporous colloidal motor obtained in Example 1 was dispersed on a hydrophilic silicon wafer and freeze-dried. Its morphology under a scanning electron microscope is shown below. Figure 2 As shown, the diameter of the single-pore colloidal motor is approximately 2.4 μm. The surface roughness is increased, but the surface structure remains intact, maintaining the same spherical structure as the TPM microcapsules. Furthermore, after the assembly of the polyelectrolyte layer and thylakoid vesicles, only a shallow surface depression remains at the original single-pore location, indicating that the polyelectrolyte layer and F... 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 description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that necessary changes, modifications, and even equivalents may be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A photo-acid driven single-hole colloidal motor, characterized in that: The colloidal motor uses a polyelectrolyte single-pore microcapsule loaded with photoacid as its core, with a vesicle-like membrane assembled on its surface, wherein F is distributed on the membrane. l F o -ATP synthase; the photoacid is the photoacid-producing agent 2-hydroxybenzaldehyde, which is loaded inside a single-pore microcapsule; the polyelectrolyte is composed of alternating cationic polyelectrolyte polyallylamine hydrochloride and anionic polyelectrolyte sodium polystyrene sulfonate, and the polyelectrolyte microcapsule is composed of 30 layers of cationic polyelectrolyte and 30 layers of anionic polyelectrolyte alternatingly assembled on a single-pore microcapsule.

2. The photo-acid driven single-hole colloidal motor according to claim 1, characterized in that: The single-pore microcapsules have a single-pore structure, a particle size of 2μm~3μm, and a pore size of 300~350nm.

3. The photo-acid driven single-hole colloidal motor according to claim 1, characterized in that: The thickness of the polyelectrolyte is 50~150nm.

4. The photo-acid driven single-hole colloidal motor according to claim 1, characterized in that: The thylakoid vesicles are extracted from the thylakoid membrane of chloroplasts, and the size of the thylakoid vesicles is 100±10nm. The thickness of the assembled membrane is 3~7nm.

5. A method for preparing a photo-acid-driven single-pore colloidal motor according to any one of claims 1 to 4, characterized in that: The method includes the following preparation steps: Step 1: Extract thylakoid vesicles; Step 2: Single-pore microcapsules are obtained using heterogeneous nucleation, self-expansion, and selective etching methods; Step 3: Using a template-assisted layer-by-layer self-assembly method, cationic and anionic polyelectrolytes are alternately assembled onto single-pore microcapsule particles to obtain polyelectrolyte single-pore microcapsules with internal single-pore channels; photoacid is loaded into the polyelectrolyte single-pore microcapsules using a solvent exchange method. Step 4: By using the vesicle fusion method, the vesicle-like vesicles obtained in Step 1 are mixed with the polyelectrolyte single-pore microcapsules loaded with photoacid obtained in Step 3 to obtain a photoacid-driven single-pore colloidal motor.

6. The preparation method according to claim 5, characterized in that: Step one specifically involves: (1) Prepare three buffer solutions: chloroplast buffer, chloroplast lysis buffer, and thylakoid protection solution; wherein, the chloroplast buffer solution is composed of 50~80g sucrose, 0.2~0.5g potassium chloride, 0.5~1.2g disodium hydrogen phosphate, 1.5~2.8g potassium dihydrogen phosphate, and 0.15~0.34g magnesium chloride dissolved in water and brought to a final volume of 500mL; the chloroplast lysis buffer solution is composed of 8~12mL of 10mM Hepes-NaOH, pH=7.5, and 0.3~0.6g magnesium chloride dissolved in water and brought to a final volume of 500mL; the thylakoid protection solution is composed of 1.5~3.6mL of 10mM Hepes-NaOH, pH=7.5, 0.16~0.36g magnesium chloride, and 1.2~2.4g potassium chloride dissolved in water and brought to a final volume of 500mL, and stored in a refrigerator at 4℃ for later use; (2) Wash 500g of fresh spinach leaves, remove the stems, and store them at 4℃ overnight. Add 300~600mL of chloroplast buffer, mix and homogenize, crush to break down the leaf mesophyll and separate the chloroplasts. (3) Filter the homogenate obtained in step (2) through 8 layers of gauze to collect the filtrate, centrifuge at 3500~4500rpm for 3~6min to collect the precipitate, and resuspend it in 10~30mL of chloroplast buffer. (4) Add 8-12 mL of 60% sucrose, 8-12 mL of 40% sucrose, and 8-12 mL of 20% sucrose to the centrifuge tube from bottom to top. Then add the mixture obtained in step (3) above the 20% sucrose and centrifuge at 3500-4800 rpm for 4-6 min at 4℃. Take the dark green solution between the 20% and 40% sucrose solutions. (5) Wash the solution obtained in step (4) with chloroplast buffer at 3500~4800 rpm for 4~6 min at 4℃, remove the supernatant, collect the precipitate, and repeat the washing three times to obtain purified chloroplasts; (6) Centrifuge the purified chloroplasts obtained in step (5) at 4℃ at 4500~5800rpm for 4~6min, remove the supernatant, keep the precipitate, add 25~45mL of chloroplast lysis buffer, mix well and place in a 4℃ refrigerator for 1~2h. (7) Centrifuge the mixture obtained in step (6) at 4°C at 4500~5800 rpm for 4~6 min, remove the supernatant, retain the precipitate to obtain thylakoids, and resuspend and disperse them in 6~12 mL of thylakoid protection solution; (8) Pour the suspension obtained in step (7) into a mortar, add ultrafine quartz sand, the quartz sand should not exceed the surface of the suspension, mix evenly and grind for 30~60 min, centrifuge at 1000~1500 rpm for 2~3 min at 4℃, remove the bottom quartz sand precipitate, take the supernatant and centrifuge at 7000~8500 rpm for 4~6 min, remove the precipitate, and recover the supernatant to obtain thylakoid membrane fragment liquid; (9) The thylakoid membrane fragments obtained in step (8) are extruded sequentially through a cell membrane extruder using polycarbonate membranes of 1 μm, 800 nm, 400 nm, 200 nm and 100 nm, from large pore size to small pore size. Each pore size requires 21 to 42 cycles of extrusion to obtain a thylakoid vesicle suspension.

7. The preparation method according to claim 5, characterized in that: Step two specifically involves: (1) Wash styrene repeatedly with 8-15 wt% sodium hydroxide in a separatory funnel, then wash the styrene after washing with deionized water to remove excess sodium hydroxide until the solution is neutral. Finally, mix with anhydrous sodium sulfate or anhydrous magnesium sulfate and let stand for 30-60 minutes to remove excess deionized water. Transfer the clear, pale yellow purified styrene from the upper layer for later use. (2) Take 10-15 mL of the purified styrene obtained in step (1) and add it to 100-150 mL of deionized water. Purify with nitrogen for 20-40 min. Gradually raise the temperature to 70℃ and add 0.15-0.4 g of potassium persulfate. Stir and react for 24 h to obtain polystyrene seeds of 300-350 nm. Centrifuge to recover the precipitate, wash it three times with deionized water, 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 and store at room temperature for later use. (3) Add 4-8 mL of 3-(trimethoxysilyl)propyl methacrylate to 40-80 mL of deionized water and stir vigorously for 5-6 h. Then, quickly mix 50-75 mL of this mixture with 70-85 mL of deionized water, 20-50 μL of 28 wt% ammonia water and 60-150 μL of the polystyrene seed solution obtained in step (2) above, and let it stand for 40-90 min. (4) Add the milky white solution obtained in step (3) to 100~150mM sodium hydroxide solution at a volume ratio of 1:

3. After 40~60s, quickly add the photoinitiator to the mixture and irradiate with ultraviolet light for 1~2h. (5) Centrifuge the solution obtained in step (4) at 3500~4800 rpm for 4~6 min, remove the supernatant, collect the precipitate to obtain TPM-PS microcapsules, resuspend in tetrahydrofuran for 30~60 min to selectively etch PS seeds, and obtain a TPM microcapsule solution with a single pore. (6) Centrifuge the TPM single-well microcapsule solution obtained in step (5) at 3500~4800 rpm for 4~6 min, remove the supernatant, collect the precipitate, and wash it three times with anhydrous ethanol and water in sequence to obtain TPM single-well microcapsules.

8. The preparation method according to claim 5, characterized in that: Step three specifically involves: (1) Using the TPM single-well microcapsules obtained in step two as templates, the TPM single-well microcapsules were added to a cationic polyelectrolyte solution. The cationic polyelectrolyte solution was composed of a polyallylamine hydrochloride solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2~0.5M and the concentration of polyallylamine hydrochloride was 1~3mg / mL. After shaking on a shaker for 15~20min, the mixture was centrifuged and the centrifuged liquid was discarded. The resulting precipitate was washed three times by centrifugation with 0.2~0.5M sodium chloride solution and added to anionic polyelectrolyte solution. The anionic polyelectrolyte solution was composed of a sodium polystyrene sulfonate solution containing sodium chloride, wherein the concentration of sodium chloride was 0.2~0.5M and the concentration of sodium polystyrene sulfonate was 1~3mg / mL. After shaking on a shaker for 15~20min, the mixture was centrifuged and the centrifuged liquid was discarded. The resulting precipitate was washed three times by centrifugation with 0.2~0.5M NaCl solution to complete the assembly of a single polyelectrolyte bilayer. (2) Repeat step (1) 30 times to obtain polyelectrolyte microcapsules with a single-pore structure; (3) After centrifuging the polyelectrolyte microcapsules obtained in step (2) at 5500~6800rpm for 1~2min, remove the supernatant, take 5~6mg of precipitate and add 0.4~1mL of ethanol-photonic acid mixture, shake for 20~40min and centrifuge, discard the supernatant, and wash the obtained precipitate three times with deionized water to obtain polyelectrolyte single-pore microcapsules loaded with photonic acid; the ethanol-photonic acid mixture is obtained by mixing 50~200μM photonic acid aqueous solution and anhydrous ethanol at a volume ratio of 1:

1.

9. The preparation method according to claim 5, characterized in that: Step four specifically involves: taking 0.3-0.6 mL of polyelectrolyte single-pore microcapsules containing photoacid obtained in step three, centrifuging at 5500-6800 rpm for 1-2 min, discarding the supernatant and retaining the precipitate; adding 0.5-1.5 mL of thylakoid vesicle suspension obtained in step one to the precipitate, mixing by pipetting, shaking in an ice bath for 30-45 min, and then washing three times with thylakoid preservation solution to remove excess thylakoid vesicles, thereby obtaining a photoacid-driven single-pore colloidal motor.

Images