Self-triggered demulsification pickering emulsion cascade catalytic system and preparation method and application thereof

Abstract

This invention relates to a self-triggered demulsification Pickering emulsion cascade catalytic system, its preparation method, and its application. The preparation method includes: preparing Fe3O4 magnetic nanoparticles by co-precipitation; coating their surface with a SiO2 shell using a modified Stöber method to obtain Fe3O4@SiO2 core-shell nanoparticles; simultaneously grafting a triamine silane coupling agent and octyltrimethoxysilane onto the Fe3O4@SiO2 surface using a one-pot method to obtain magnetic amphiphilic nanoparticles; dividing the magnetic amphiphilic nanoparticles into two batches, and immobilizing glucose oxidase and Candida antarcticis lipase B respectively via EDC / NHS-mediated amide coupling reaction to obtain nanoparticle catalysts; using the nanoparticle catalysts as solid emulsifiers in Pickering emulsions to construct an oil-water two-phase cascade catalytic system, realizing a cascade catalytic reaction of glucose oxidation – peracidification – olefin epoxidation; utilizing the accumulation of gluconic acid during the reaction to lower the pH, triggering emulsion demulsification, achieving product separation and magnetic recovery of the nanoparticle catalysts.

Description

Technical Field

[0001] This invention belongs to the field of nanocatalytic materials and enzyme immobilization technology, specifically relating to a self-triggered demulsification Pickering emulsion cascade catalytic system, its preparation method, and its application. Background Technology

[0002] Pickering emulsions are emulsion systems stabilized by solid particles, whose stability stems from the irreversible adsorption of particles at the oil-water interface. In recent years, Pickering emulsions have attracted widespread attention as a heterogeneous catalytic reaction platform. Their advantages include: the oil-water two-phase system provides a natural reaction separation environment, facilitating mass transfer and distribution of substrates and products between different phases; and the solid particle emulsifier can also serve as a carrier for enzymes or chemical catalysts, enabling the directional distribution of catalysts at the interface.

[0003] In the field of enzyme catalysis, many industrially relevant substrates (such as olefins and fatty acid esters) are poorly soluble in water, while enzymes typically need to function in an aqueous phase. Traditional single-phase catalytic systems struggle to address this contradiction. Pickering emulsions, by providing a large oil-water interface area, can significantly improve the contact efficiency between water-soluble enzymes and hydrophobic substrates, and are considered an ideal platform for realizing enzyme-catalyzed oil-water two-phase reactions.

[0004] However, the existing Pickering emulsion catalysis system has the following shortcomings: (1) The separation of products after the reaction requires the destruction of the emulsion structure, which usually relies on external stimulation (such as adding acid or base to adjust pH, heating, applying an electric field, etc.) or high-energy-consuming operations such as centrifugation, which increases the process cost and complexity; (2) The catalyst is difficult to recover. Traditional nanoparticle catalysts need to be centrifuged and washed multiple times to recover, which inevitably causes losses in the process; (3) In the construction of multi-enzyme cascade catalysis system, different enzymes often have different requirements for environmental conditions (pH, temperature, etc.). How to achieve the synergistic work of multiple enzymes in the same system is still a challenge (Rodriguez, AMB; Binks, BP Catalysis in Pickering Emulsions. SoftMatter, 2020, 16(45), 10221–10243. DOI: 10.1039 / D0SM01636E). Therefore, developing a Pickering emulsion cascade catalytic system that can spontaneously achieve demulsification and separation after the catalytic reaction is completed, and whose nanoparticle catalyst can be easily recovered, has significant academic and application value. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing a self-triggered demulsification Pickering emulsion cascade catalytic system.

[0006] Another object of the present invention is to provide a self-triggered demulsification Pickering emulsion cascade catalytic system prepared by the above method.

[0007] Another object of the present invention is to provide the application of the above-described self-triggered demulsification Pickering emulsion cascade catalytic system.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing a self-triggered demulsification Pickering emulsion cascade catalytic system, comprising the following steps: (1) Fe3O4 magnetic nanoparticles were prepared by coprecipitation method, and a SiO2 shell was coated on the surface of Fe3O4 magnetic nanoparticles by modified Stöber method to obtain Fe3O4@SiO2 core-shell nanoparticles. (2) By grafting a triamine silane coupling agent and an octyltrimethoxysilane onto the surface of Fe3O4@SiO2 core-shell nanoparticles in a one-pot method, bifunctional modification was carried out to obtain magnetic amphiphilic nanoparticles; wherein, the triamine silane coupling agent provides hydrophilic amine groups and the octyltrimethoxysilane provides hydrophobic alkyl chains. (3) The magnetic amphiphilic nanoparticles were divided into two batches. Batch A was immobilized with glucose oxidase (GOx) by EDC / NHS-mediated amide coupling reaction to obtain GOx-particle A; Batch B was immobilized with Candida antarcticis lipase B (CALB) by the same method to obtain CALB-particle B; GOx-particle A and CALB-particle B were nanoparticle catalysts. (4) Two nanoparticle catalysts were co-dispersed in an aqueous phase containing glucose, and an oil phase containing olefin substrate was added. After homogenization and emulsification, a Pickering emulsion was formed, and a cascade catalytic reaction was carried out under continuous oxygen supply. During the reaction, the continuous accumulation of gluconic acid caused the pH of the aqueous phase to spontaneously decrease to the critical value. The protonation of the triamine groups on the particle surface led to a sudden change in wettability, which triggered the demulsification of the Pickering emulsion and achieved automatic separation of the products. The nanoparticle catalyst was recovered by means of an external magnetic field.

[0009] Preferably, in step (1), the co-precipitation method involves mixing FeCl3·6H2O and FeCl2·4H2O in the order Fe... 3+ with Fe 2+ The Fe3O4 magnetic nanoparticles were obtained by dissolving the Fe3O4 in deionized water at a molar ratio of 1.8-2.0:1, adding 6-9 ml of ammonia, and stirring continuously at 70-90°C for 20-40 min. The product was collected by magnetic separation, washed, and dried to obtain Fe3O4 magnetic nanoparticles. Preferably, in step (1), the modified Stöber method involves dispersing Fe3O4 nanoparticles in a mixed solvent of ethanol and water, ultrasonically treating them, adding 3-7 ml of ammonia, and then adding tetraethyl orthosilicate (TEOS) dropwise at a rate of 0.05-0.2 mL / min using a micro-injection pump. The mixture is stirred at room temperature for 4-8 hours. The product is collected by magnetic separation, washed, and dried to obtain Fe3O4@SiO2 core-shell nanoparticles.

[0010] Preferably, in step (2), the triamine silane coupling agent is N-[3-(trimethoxysilyl)propyl]diethylenetriamine; the octyltrimethoxysilane is n-octyltrimethoxysilane; and the molar ratio of the triamine silane coupling agent to the octyltrimethoxysilane is 1:1-1:24.

[0011] More preferably, the molar ratio of the triamine silane coupling agent to octyltrimethoxysilane is 1:4 to 1:9.

[0012] Preferably, in step (2), the bifunctional modification is carried out in anhydrous toluene under nitrogen protection, with triethylamine as the condensation catalyst, and refluxed at 100-120°C for 2-6 hours.

[0013] Preferably, in step (3), the EDC / NHS-mediated amide coupling reaction is specifically as follows: the enzyme is dissolved in a buffer solution, EDC·HCl and NHS are added, and the enzyme is activated at room temperature for 20-40 min to activate the carboxyl groups (–COOH of aspartic acid / glutamic acid residues) on the enzyme surface, forming a stable active ester intermediate via NHS; the activated enzyme solution is mixed with a magnetic amphiphilic nanoparticle suspension and incubated with shaking at 20-30°C and 100-250 rpm; after incubation, the particles are collected by magnetic separation, and the residual active sites are blocked with glycine solution; Preferably, in step (3), when immobilizing GOx, the buffer used is 30-80mM PBS buffer with pH 5.5-6.5, and the incubation time is 8-16h.

[0014] Preferably, in step (3), the buffer used for immobilizing CALB is 30-80mM PBS buffer with pH 6.5-7.5, and the incubation time is 1-5h.

[0015] Preferably, in step (4), the aqueous phase is a PBS buffer containing 100-300 mM D-(+)-glucose (100-300 mM, pH 6.5-7.5); the oil phase is a fatty acid containing 50-200 mM olefin substrate; the volume ratio of the aqueous phase to the oil phase is 2:1-4:1; the mass ratio of GOx-particle A to CALB-particle B is 0.5:1-2:1; and the critical value is pH 4.5-5.5.

[0016] In a further preferred embodiment, the fatty acid in the oil phase is octanoic acid, and the olefin substrate is cis-cyclooctene; Preferably, in step (4), the cascade catalytic reaction includes: GOx catalyzes the oxidation of glucose in the aqueous phase to generate gluconic acid and H2O2, CALB catalyzes the reaction of fatty acids with H2O2 at the oil-water interface to generate peracid, and the peracid undergoes a Prilezhaev epoxidation reaction with the olefin substrate in the oil phase to generate epoxide, while regenerating fatty acids to achieve recycling.

[0017] The present invention also provides a self-triggered demulsification Pickering emulsion cascade catalytic system prepared by the above method.

[0018] This invention also provides the application of the above-mentioned self-triggered demulsification Pickering emulsion cascade catalytic system in the fields of olefin epoxidation, biocatalysis, or fine chemical synthesis.

[0019] The cascade reaction mechanism of this invention is as follows: In the system, GOx catalyzes the aerobic oxidation of glucose in the aqueous phase, generating gluconic acid and H2O2; H2O2 diffuses from the aqueous phase to the oil-water interface, where it is catalyzed by CALB to react with octanoic acid to generate peroctanoic acid; peroctanoic acid, acting as an electrophilic oxidant, undergoes a Prilezhaev epoxidation reaction with cis-cyclooctene in the oil phase to generate cyclooctane oxide, while simultaneously regenerating octanoic acid to achieve a catalytic cycle. Throughout the process, the gluconic acid continuously generated by GOx accumulates in the aqueous phase, causing the pH to gradually decrease. When the pH drops to approximately 5.0, the triamine groups on the particle surface undergo protonation (–NH2 becomes –NH3). + The surface charge and wettability undergo abrupt changes, causing particles to desorb from the oil-water interface. The Pickering emulsion becomes unstable and spontaneously breaks down into two transparent oil-water layers. At this point, the epoxidation products remain in the upper oil phase and can be directly removed for post-processing; the nanoparticle catalyst is adsorbed onto the container wall by an external magnetic field, enabling rapid recovery.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Self-triggered demulsification: The gluconic acid produced by the enzyme catalytic reaction itself is used as the endogenous driving force for pH reduction. The Pickering emulsion can be spontaneously demulsified without the addition of external acid, alkali or other stimuli. This truly realizes the integrated closed loop of reaction completion – automatic demulsification – product separation, which greatly simplifies the post-processing process.

[0021] (2) Rapid magnetic recovery: The Fe3O4 magnetic core endows the nanoparticle catalyst with magnetic responsiveness. After demulsification, only an external magnetic field is needed to completely recover the nanoparticle catalyst from the system within a few minutes, avoiding the cumbersome operation and material loss of traditional centrifugal separation, which is conducive to the multiple recycling of nanoparticle catalyst.

[0022] (3) Highly efficient cascade catalysis: GOx and CALB were fixed on magnetic amphiphilic nanoparticles in different batches. The two enzyme particles stabilized the Pickering emulsion together and worked synergistically to realize a three-step one-pot cascade reaction of glucose oxidation → peracid generation → olefin epoxidation, avoiding the separation and transfer of intermediate products and improving reaction efficiency.

[0023] (4) Simple preparation and good controllability: Fe3O4@SiO2 core-shell particles are prepared by mature co-precipitation and Stöber method; surface bifunctionalization is completed by one-pot method to simultaneously perform hydrophilic and hydrophobic modification; by adjusting the ratio of two silane coupling agents, the amphiphilic balance and demulsification response characteristics of the particles can be flexibly controlled. Attached Figure Description

[0024] Figure 1 Optical microscopy image (50 μm scale) of the Pickering emulsion stabilized by Fe3O4@SiO2 core-shell nanoparticles obtained in Example 1.

[0025] Figure 2 The images show confocal microscopy images of the Fe3O4@SiO2 core-shell nanoparticle-stabilized octanoic acid / water emulsion obtained in Example 1 (a: confocal microscopy image of the octanoic acid / water emulsion with FITC staining in the aqueous phase; b: confocal microscopy image of the octanoic acid / water emulsion with Nile Red staining in the oil phase; c: confocal microscopy image of the overlapping octanoic acid / water emulsion).

[0026] Figure 3 The image shows the water contact angle of the Fe3O4@SiO2 core-shell nanoparticles obtained in Example 1. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. All raw materials involved in the present invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be referred to.

[0028] The ammonia solution used in the examples was analytical grade concentrated ammonia solution.

[0029] Example 1 (1) Preparation of Fe3O4 magnetic nanoparticles: FeCl3·6H2O (10.4 g, 38.5 mmol) and FeCl2·4H2O (4.0 g, 20.1 mmol) were dissolved together in 50 mL of deionized water (Fe 3+ with Fe 2+The molar ratio was approximately 1.9:1. 9 mL of ammonia was added to the above solution, and the system was heated to 80°C and maintained for 30 min under continuous vigorous stirring. After the reaction was complete, the mixture was allowed to cool naturally to room temperature. The product was separated from the reaction solution using an external permanent magnet, washed three times with anhydrous ethanol, and dried under reduced pressure at 40°C to obtain black Fe3O4 powder, which is the Fe3O4 magnetic nanoparticle.

[0030] (2) Preparation of Fe3O4@SiO2 core-shell nanoparticles: 500 mg of Fe3O4 magnetic nanoparticles were dispersed in a mixed solvent of 100 mL anhydrous ethanol and 20 mL deionized water and sonicated for 30 min. 5 mL of ammonia water was added under mechanical stirring (400 rpm) and stirring was continued for 15 min. 0.5 mL of TEOS was added dropwise at a rate of 0.1 mL / min using a micro-injection pump and stirred at room temperature for 6 h. After magnetic separation, the product was washed three times with anhydrous ethanol and twice with deionized water, and then vacuum dried at 45°C for 12 h to obtain Fe3O4@SiO2 core-shell nanoparticles.

[0031] Figure 1 An optical microscope image (50 μm scale) of the Pickering emulsion stabilized by Fe3O4@SiO2 core-shell nanoparticles obtained in Example 1 is shown.

[0032] Figure 2 A confocal microscopy image of the octanoic acid / water emulsion stabilized by Fe3O4@SiO2 core-shell nanoparticles obtained in Example 1 is shown (the aqueous phase was stained with FITC, and the oil phase was stained with Nile Red).

[0033] Figure 3 The water contact angle diagram of the Fe3O4@SiO2 core-shell nanoparticles obtained in Example 1 is shown.

[0034] Depend on Figure 1 It can be seen that the bifunctionalized Fe3O4@SiO2 core-shell nanoparticles can effectively stabilize the octanoic acid / water system to form Pickering emulsions. The emulsion droplets are spherical, and the size of the dispersed phase droplets is mainly in the range of 20-100 μm, indicating that the nanoparticles have good emulsification ability.

[0035] Depend on Figure 2 Confocal fluorescence microscopy images show that the FITC-labeled aqueous phase (green) is a continuous phase, while the Nile Red-labeled oil phase (red) is dispersed within it in the form of spherical droplets. Dual-channel overlay images further confirm that the prepared Pickering emulsion is an oil-in-water (O / W) type. This emulsion type is consistent with the slightly hydrophilic amphiphilic properties of the nanoparticles after triamine / octyl bifunctionalization modification.

[0036] Depend on Figure 3It is known that the water contact angle of the bifunctionalized Fe3O4@SiO2 nanoparticles is approximately 86.5°-87.2°, close to but slightly below 90°, indicating that the particle surface has moderate amphiphilicity, leaning towards weak hydrophilicity. According to Pickering emulsion theory, particles with a contact angle close to 90° have the strongest interfacial adsorption energy, enabling them to firmly anchor at the oil-water interface, which is beneficial for forming stable O / W type Pickering emulsions. Simultaneously, this contact angle value also indicates that when the surface triamine groups undergo protonation under low pH conditions, the hydrophilicity of the particles will be further enhanced, the deviation of the contact angle from 90° will increase, and the interfacial adsorption energy will significantly decrease, thereby triggering particle desorption from the interface and emulsion demulsification, providing a theoretical basis for the self-triggered demulsification mechanism of this invention.

[0037] (3) Triamine / octyl bifunctionalization modification: 400 mg of Fe3O4@SiO2 core-shell nanoparticles were vacuum dried at 110°C for 2 h and dispersed in 5 mL of anhydrous toluene and sonicated for 30 min. Under N2 protection, 0.30 mmol of triamine silane coupling agent and 1.20 mmol of octyltrimethoxysilane (molar ratio 1:4) were added simultaneously, along with 5 mmol (0.7 mL) of triethylamine, and the mixture was heated to 110°C and refluxed for 4 h. The product was magnetically separated, washed three times with toluene, three times with ethanol, and twice with deionized water, and then vacuum dried at 80°C to obtain magnetic amphiphilic nanoparticles.

[0038] (4) Immobilization of glucose oxidase GOx: 250 mg of magnetic amphiphilic nanoparticles (batch A) were dispersed in 10 mL of PBS buffer (50 mM, pH 6.0) and sonicated for 10 min. 30 mg of GOx was dissolved in 10 mL of PBS buffer (50 mM, pH 6.0), and EDC·HCl (0.1 g) and NHS (0.04 g) were added. The mixture was activated at room temperature for 30 min to activate the carboxyl groups on the GOx surface. The GOx solution was mixed with the magnetic amphiphilic nanoparticle suspension and incubated at 25°C and 200 rpm for 12 h. After magnetic separation, the mixture was washed three times with PBS buffer (50 mM, pH 6.0), blocked with glycine solution (50 mM, pH 7.0) for 30 min, washed again, and then dispersed in PBS buffer (50 mM, pH 6.0) at 4°C for storage to obtain GOx-particles A, which served as the nanoparticle catalyst.

[0039] (5) Immobilization of Candida antarcticis lipase B CALB: 250 mg of magnetic amphiphilic nanoparticles (batch B) were dispersed in 10 mL of PBS buffer (50 mM, pH 7.0). Another 10 mg of CALB was dissolved in 10 mL of PBS buffer (50 mM, pH 7.0), and EDC·HCl (0.1 g) and NHS (0.04 g) were added. The mixture was activated at room temperature for 30 min to activate the carboxyl groups on the CALB surface. The CALB solution was mixed with the magnetic amphiphilic nanoparticle suspension and incubated at 25°C and 200 rpm for 3 h with shaking. After magnetic separation, the mixture was washed three times with PBS buffer (50 mM, pH 7.0), blocked with glycine solution (50 mM, pH 7.0) for 30 min, washed again, and then dispersed in PBS buffer (50 mM, pH 7.0) and stored at 4°C to obtain CALB-particle B, which served as a nanoparticle catalyst.

[0040] (6) Pickering emulsion cascade catalytic reaction: 108 mg D-(+)-glucose was dissolved in 3 mL PBS buffer (200 mM, pH 7.0) and placed in a 10 mL glass reaction flask. 40 mg GOx-particles A and 40 mg CALB-particles B were added and dispersed by sonication for 5 min. cis-cyclooctene was dissolved in octanoic acid to prepare a 100 mM solution, and 1 mL was added as the oil phase above the aqueous phase. The mixture was emulsified at 12000 rpm for 2 min using a high-speed homogenizer to form an O / W type Pickering emulsion. The headspace was purged with pure O2 for 1 min, then sealed and connected to an O2 balloon. The mixture was placed in a constant temperature shaker at 30°C and 200 rpm for reaction.

[0041] After approximately 10 hours of reaction, the pH of the aqueous phase decreased to about 5.0, and the emulsion spontaneously transformed from milky white to a transparent oil-water bilayer. The reaction flask was placed on a magnet for 5 minutes, and GOx-particles A and CALB-particles B were adsorbed onto the flask wall. The upper oil phase was extracted and analyzed by gas chromatography (GC), showing a cyclooctane oxide conversion rate of 29%. The recovered GOx-particles A and CALB-particles B were washed twice with PBS buffer of the corresponding concentration and pH from the immobilization step before being used in the next round of reaction.

[0042] Conversion rate (%) = (Amount of epicyclooctane produced / Amount of cis-cyclooctene initially added) × 100% Example 2 (1) Preparation of Fe3O4 magnetic nanoparticles: FeCl3·6H2O (10.9 g, 40.2 mmol) and FeCl2·4H2O (4.0 g, 20.1 mmol) were dissolved together in 50 mL of deionized water (Fe 3+ with Fe 2+The molar ratio was approximately 2.0:1. 6 mL of ammonia was added to the above solution, and the system was heated to 70°C and maintained for 20 min under continuous vigorous stirring. After the reaction was complete, the mixture was allowed to cool naturally to room temperature. The product was separated from the reaction solution using an external permanent magnet, washed three times with anhydrous ethanol, and dried under reduced pressure at 40°C to obtain black Fe3O4 powder, which is the Fe3O4 magnetic nanoparticle.

[0043] (2) Preparation of Fe3O4@SiO2 core-shell nanoparticles: 500 mg of Fe3O4 magnetic nanoparticles were dispersed in a mixed solvent of 100 mL anhydrous ethanol and 20 mL deionized water and sonicated for 20 min. 3 mL of ammonia water was added under mechanical stirring (400 rpm) and stirring was continued for 15 min. 0.5 mL of TEOS was added dropwise at a rate of 0.05 mL / min using a micro-injection pump and stirred at room temperature for 8 h. After magnetic separation, the product was washed three times with anhydrous ethanol and twice with deionized water, and then vacuum dried at 45°C for 12 h to obtain Fe3O4@SiO2 core-shell nanoparticles.

[0044] In step (3), the molar ratio of the triamine silane coupling agent to octyltrimethoxysilane is changed to 1:9 (i.e., 0.15 mmol of triamine silane coupling agent and 1.35 mmol of octyltrimethoxysilane), and the mixture is heated to 120°C and refluxed for 6 hours. The other conditions are the same as in Example 1.

[0045] (4) Immobilization of glucose oxidase GOx: 250 mg of magnetic amphiphilic nanoparticles (batch A) were dispersed in 10 mL of PBS buffer (30 mM, pH 5.5) and sonicated for 10 min. Separately, 30 mg of GOx was dissolved in 10 mL of PBS buffer (30 mM, pH 5.5), and EDC·HCl (0.1 g) and NHS (0.04 g) were added. Activation was performed at room temperature for 20 min to activate the carboxyl groups on the GOx surface. The GOx solution was mixed with the magnetic amphiphilic nanoparticle suspension and incubated at 20°C and 100 rpm for 8 h. After magnetic separation, the nanoparticles were washed three times with PBS buffer (30 mM, pH 5.5), blocked with glycine solution (50 mM, pH 7.0) for 30 min, washed again, and then dispersed in PBS buffer (30 mM, pH 5.5) and stored at 4°C to obtain GOx-particles A, which served as the nanoparticle catalyst.

[0046] (5) Immobilization of Candida antarcticis lipase B CALB: 250 mg of magnetic amphiphilic nanoparticles (batch B) were dispersed in 10 mL of PBS buffer (30 mM, pH 6.5). Another 10 mg of CALB was dissolved in 10 mL of PBS buffer (30 mM, pH 6.5), and EDC·HCl (0.1 g) and NHS (0.04 g) were added. The mixture was activated at room temperature for 20 min to activate the carboxyl groups on the CALB surface. The CALB solution was mixed with the magnetic amphiphilic nanoparticle suspension and incubated at 20°C and 100 rpm for 5 h. After magnetic separation, the mixture was washed three times with PBS buffer (30 mM, pH 6.5), blocked with glycine solution (50 mM, pH 7.0) for 30 min, washed again, and then dispersed in PBS buffer (30 mM, pH 6.5) and stored at 4°C to obtain CALB-particle B, which served as a nanoparticle catalyst.

[0047] (6) Pickering emulsion cascade catalytic reaction: 36 mg D-(+)-glucose was dissolved in 2 mL PBS buffer (100 mM, pH 7.5) and placed in a 10 mL glass reaction flask. 20 mg GOx-particles A and 40 mg CALB-particles B were added and dispersed by sonication for 5 min. cis-cyclooctene was dissolved in octanoic acid to prepare a 50 mM solution, and 1 mL was added as the oil phase above the aqueous phase. The mixture was emulsified at 12000 rpm for 2 min using a high-speed homogenizer to form an O / W type Pickering emulsion. The headspace was purged with pure O2 for 1 min and then sealed, and an O2 balloon was connected. The mixture was placed in a constant temperature shaker at 30°C and 200 rpm for reaction.

[0048] Results: After approximately 11 hours, the pH of the aqueous phase dropped to approximately 4.7, and spontaneous demulsification of the emulsion occurred. The conversion rate of cyclooctane oxide was 32%.

[0049] Example 3 (1) Preparation of Fe3O4 magnetic nanoparticles: FeCl3·6H2O (9.8 g, 36.2 mmol) and FeCl2·4H2O (4.0 g, 20.1 mmol) were dissolved together in 50 mL of deionized water (Fe 3+ with Fe 2+ The molar ratio was approximately 1.8:1. 7 mL of ammonia was added to the above solution, and the system was heated to 90°C and maintained for 40 min under continuous vigorous stirring. After the reaction was complete, the mixture was allowed to cool naturally to room temperature. The product was separated from the reaction solution using an external permanent magnet, washed three times with anhydrous ethanol, and dried under reduced pressure at 40°C to obtain black Fe3O4 powder, which is the Fe3O4 magnetic nanoparticle.

[0050] (2) Preparation of Fe3O4@SiO2 core-shell nanoparticles: 500 mg of Fe3O4 magnetic nanoparticles were dispersed in a mixed solvent of 100 mL anhydrous ethanol and 20 mL deionized water and sonicated for 20 min. 7 mL of ammonia water was added under mechanical stirring (400 rpm) and stirring was continued for 15 min. 0.5 mL of TEOS was added dropwise at a rate of 0.2 mL / min using a micro-injection pump and stirred at room temperature for 4 h. After magnetic separation, the product was washed three times with anhydrous ethanol and twice with deionized water, and then vacuum dried at 45°C for 12 h to obtain Fe3O4@SiO2 core-shell nanoparticles.

[0051] In step (3), the molar ratio of the triamine silane coupling agent to octyltrimethoxysilane is changed to 1:1 (i.e., 0.75 mmol of triamine silane coupling agent and 0.75 mmol of octyltrimethoxysilane), and the mixture is heated to 100°C and refluxed for 2 hours. The other conditions are the same as in Example 1.

[0052] (4) Immobilization of glucose oxidase GOx: 250 mg of magnetic amphiphilic nanoparticles (batch A) were dispersed in 10 mL of PBS buffer (80 mM, pH 6.5) and sonicated for 10 min. 30 mg of GOx was dissolved in 10 mL of PBS buffer (80 mM, pH 6.5), and EDC·HCl (0.1 g) and NHS (0.04 g) were added. The mixture was activated at room temperature for 40 min to activate the carboxyl groups on the GOx surface. The GOx solution was mixed with the magnetic amphiphilic nanoparticle suspension and incubated at 30°C and 250 rpm for 16 h. After magnetic separation, the mixture was washed three times with PBS buffer (80 mM, pH 6.5), blocked with glycine solution (50 mM, pH 7.0) for 30 min, washed again, and then dispersed in PBS buffer (80 mM, pH 6.5) at 4°C to obtain GOx-particles A, which served as the nanoparticle catalyst.

[0053] (5) Immobilization of Candida antarcticis lipase B CALB: 250 mg of magnetic amphiphilic nanoparticles (batch B) were dispersed in 10 mL of PBS buffer (80 mM, pH 7.5). Another 10 mg of CALB was dissolved in 10 mL of PBS buffer (80 mM, pH 7.5), and EDC·HCl (0.1 g) and NHS (0.04 g) were added. The mixture was activated at room temperature for 40 min to activate the carboxyl groups on the CALB surface. The CALB solution was mixed with the magnetic amphiphilic nanoparticle suspension and incubated at 30°C and 250 rpm for 1 h with shaking. After magnetic separation, the mixture was washed three times with PBS buffer (80 mM, pH 7.5), blocked with glycine solution (50 mM, pH 7.0) for 30 min, washed again, and then dispersed in PBS buffer (80 mM, pH 7.5) and stored at 4°C to obtain CALB-particle B, which served as a nanoparticle catalyst.

[0054] (6) Pickering emulsion cascade catalytic reaction: 216 mg D-(+)-glucose was dissolved in 4 mL PBS buffer (300 mM, pH 6.5) and placed in a 10 mL glass reaction flask. 80 mg GOx-particles A and 40 mg CALB-particles B were added and dispersed by sonication for 5 min. cis-cyclooctene was dissolved in octanoic acid to prepare a 200 mM solution, and 1 mL was added as the oil phase above the aqueous phase. The mixture was emulsified at 12000 rpm for 2 min using a high-speed homogenizer to form an O / W type Pickering emulsion. The headspace was purged with pure O2 for 1 min and then sealed, and an O2 balloon was connected. The mixture was placed in a constant temperature shaker at 30°C and 200 rpm for reaction.

[0055] Results: After approximately 8 hours, the pH of the aqueous phase dropped to about 5.3, and the emulsion spontaneously demulsified. However, due to the excessive hydrophilicity of the particles, the initial emulsion stability was poor, the droplets were not evenly dispersed, and the epoxy octane conversion rate was only 22%.

[0056] Example 4 Steps (1)-(2) are the same as in Example 1.

[0057] In step (3), the molar ratio of the triamine silane coupling agent to octyltrimethoxysilane is changed to 1:24 (i.e., 0.06 mmol of triamine silane coupling agent and 1.44 mmol of octyltrimethoxysilane), and the other conditions remain the same.

[0058] Steps (4)-(6) are the same as in Example 1.

[0059] Results: After 16 hours of reaction, the pH of the aqueous phase dropped to approximately 4.5, but no significant demulsification was observed. The reason for this was that the density of triamine groups on the particle surface was too low, and the change in wettability caused by protonation was insufficient to trigger demulsification. The conversion rate of cyclooctane oxide was 42%.

[0060] Comparative Example 1 Steps (1)-(5) are the same as in Example 1.

[0061] In step (6), D-(+)-glucose is not added, and the other conditions are the same.

[0062] Results: Due to the lack of GOx substrate, no gluconic acid was generated in the system, and the pH of the aqueous phase remained between 6.8 and 7.0 throughout the 24-hour reaction period, maintaining emulsion stability without demulsification. Simultaneously, the absence of H₂O₂ production prevented the formation of peroctanoic acid, thus inhibiting the epoxidation reaction. This comparative example demonstrates that the accumulation of gluconic acid is a key factor triggering spontaneous demulsification and verifies the necessity of the cascade catalytic route.

[0063] Comparative Example 2 Steps (1)-(2) are the same as in Example 1.

[0064] In step (3), 3-aminopropyltriethoxysilane (APTES) was used instead of the triamine silane coupling agent. The molar ratio was the same as that of the amount of triamine silane coupling agent used in Example 1 (0.30 mmol), and the other conditions were the same.

[0065] Steps (4)-(6) are the same as in Example 1.

[0066] Results: After 24 hours of reaction, the pH of the aqueous phase decreased to approximately 5.0, but the emulsion remained stable without demulsification. The reason for this is that APTES contains only one amino group (pKa approximately 9.7), while the triamine silane coupling agent contains multiple amino groups (primary and secondary amines, with a pKa gradient distribution). The latter is more sensitive to protonation near pH 5, resulting in a more significant change in wettability. This comparative example illustrates the crucial importance of selecting a triamine silane coupling agent as the pH-responsive group.

[0067] Comparative Example 3 Steps (1)-(5) are the same as in Example 1, but instead of using magnetic Fe3O4@SiO2 core-shell particles, pure SiO2 nanoparticles (synthesized by the Stöber method, with a particle size of about 350 nm) are used.

[0068] Step (6) is the same as in Example 1.

[0069] Results: The cascade reaction proceeded normally, and the emulsion spontaneously demulsified after about 10 hours. However, due to the lack of magnetism, the recovery of the nanoparticle catalyst required multiple centrifugations (8000 rpm, 10 min × 3), which was time-consuming and resulted in a loss rate of approximately 15-20% of the nanoparticle catalyst.

[0070] Loss rate (%) = (Initial nanoparticle catalyst mass - recovered nanoparticle catalyst mass) / initial nanoparticle catalyst mass × 100%.

[0071] In Example 1, the nanoparticle catalyst could be completely recovered with just 5 minutes of external magnet application, and the loss rate of the nanoparticle catalyst was less than 5%. This comparative example demonstrates the significant advantage of introducing Fe3O4 magnetic cores for the recovery of nanoparticle catalysts.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a self-triggered demulsification Pickering emulsion cascade catalytic system, characterized in that, Includes the following steps: (1) Fe3O4 magnetic nanoparticles were prepared by coprecipitation method, and a SiO2 shell was coated on the surface of Fe3O4 magnetic nanoparticles by modified Stöber method to obtain Fe3O4@SiO2 core-shell nanoparticles. (2) By grafting triamine silane coupling agent and octyltrimethoxysilane onto the surface of Fe3O4@SiO2 nanoparticles simultaneously in a one-pot method, magnetic amphiphilic nanoparticles were obtained. (3) The magnetic amphiphilic nanoparticles were divided into two batches. Batch A was immobilized with glucose oxidase GOx through an EDC / NHS-mediated amide coupling reaction to obtain GOx-particle A. Batch B was immobilized with Candida antarcticis lipase BCALB through the same method to obtain CALB-particle B. GOx-particle A and CALB-particle B are nanoparticle catalysts. (4) Two nanoparticle catalysts are dispersed together in an aqueous phase containing glucose, and an oil phase containing olefin substrate is added. The mixture is homogenized and emulsified to form a Pickering emulsion. A cascade catalytic reaction is carried out under continuous oxygen supply. During the reaction, the pH of the aqueous phase spontaneously decreases to a critical value, triggering emulsion demulsification and achieving product separation. The nanoparticle catalyst is recovered by means of an external magnetic field.

2. The preparation method according to claim 1, characterized in that, In step (1), the co-precipitation method involves mixing FeCl3·6H2O and FeCl2·4H2O according to Fe... 3+ with Fe 2+ Fe3O4 magnetic nanoparticles were obtained by dissolving the nanoparticles in deionized water at a molar ratio of 1.8-2.0:1, adding ammonia, stirring at 70-90°C for 20-40 min, and then magnetically separating, washing, and drying.

3. The preparation method according to claim 1, characterized in that, In step (1), the modified Stöber method involves dispersing Fe3O4 nanoparticles in a mixed solvent of ethanol and water, ultrasonically treating them, adding ammonia, and then adding tetraethyl orthosilicate (TEOS) dropwise at a rate of 0.05-0.2 mL / min using a micro-injection pump. The mixture is stirred and reacted at room temperature for 4-8 hours. After magnetic separation, washing, and drying, Fe3O4@SiO2 core-shell nanoparticles are obtained.

4. The preparation method according to claim 1, characterized in that, In step (2), the triamine silane coupling agent is N-[3-(trimethoxysilyl)propyl]diethylenetriamine, and the octyltrimethoxysilane is n-octyltrimethoxysilane; the bifunctional modification is carried out in anhydrous toluene under nitrogen protection, with triethylamine as the condensation catalyst, under reflux at 100-120°C for 2-6 hours.

5. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of the triamine silane coupling agent to the octyltrimethoxysilane is 1:1 to 1:

24.

6. The preparation method according to claim 1, characterized in that, In step (3), the EDC / NHS-mediated amide coupling reaction is specifically as follows: the enzyme is dissolved in a buffer solution, and EDC·HCl and NHS are added to activate the carboxyl groups on the enzyme surface for 20-40 min; the activated enzyme solution is mixed with a magnetic amphiphilic nanoparticle suspension and incubated at 20-30°C and 100-250 rpm; after incubation, the residual active sites are blocked with glycine solution.

7. The preparation method according to claim 6, characterized in that, In step (3), the buffer used for immobilizing GOx is 30-80mM PBS buffer with pH 5.5-6.5, and the incubation time is 8-16h; the buffer used for immobilizing CALB is 30-80mM PBS buffer with pH 6.5-7.5, and the incubation time is 1-5h.

8. The preparation method according to claim 1, characterized in that, In step (4), the aqueous phase is a PBS buffer containing 100-300 mM M-(+)-glucose, the PBS buffer concentration is 100-300 mM, and the pH is 6.5-7.

5. The oil phase is a fatty acid containing 50-200 mM olefin substrate. The volume ratio of the aqueous phase to the oil phase is 2:1-4:

1. The mass ratio of GOx-particle A to CALB-particle B is 0.5:1-2:

1. The critical value is pH 4.5-5.

5.

9. A self-triggered demulsification Pickering emulsion cascade catalytic system prepared by the preparation method according to any one of claims 1-8.

10. The application of the self-triggered demulsification Pickering emulsion cascade catalytic system according to claim 9 in the fields of olefin epoxidation, biocatalysis, or fine chemical synthesis.

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