A Pickering emulsion microreactor with enhanced enzyme catalysis and preparation method thereof

Abstract

The invention discloses a Pickering emulsion microreactor for enhanced enzyme catalysis and a preparation method thereof. The reactor comprises enzyme-carrying polymer nanoparticles, an aqueous phase and an oil phase, wherein the enzyme-carrying polymer nanoparticles are formed by grafting an enzyme onto the polymer nanoparticles; the polymer nanoparticles are self-assembled into an internally hydrophobic and externally hydrophilic structure by an amphiphilic block copolymer formed by a hydrophilic segment polymer and a hydrophobic segment polymer; the hydrophilic segment polymer is a polymer containing a carboxyl group, an aldehyde group or a hydroxyl group; the groups contained on the enzyme are covalently bonded to the carboxyl group, aldehyde group or hydroxyl group of the hydrophilic segment of the polymer nanoparticles; the Pickering emulsion microreactor for enhanced enzyme catalysis uses polymer nanoparticles to immobilize the enzyme, and can directly use the groups carried by the polymer nanoparticles for chemical coupling, so that the enzyme immobilization effect is stable, the enzyme is fully in contact with the substrate, the mass transfer resistance is small, the reaction rate is fast, and the catalytic activity and recycling rate of the enzyme are high.

Description

Technical Field

[0001] The invention relates to a Pickering emulsion microreactor and a preparation method thereof, in particular to an enzyme-enhanced Pickering emulsion microreactor and a preparation method thereof. Background Art

[0002] With the introduction of the concept of green chemistry, low-carbon and high-efficiency biochemical engineering has received more and more attention. Due to its green, mild and non-toxic characteristics, the research on using enzymes to replace traditional catalysts and perform catalytic reactions has received more and more attention.

[0003] The two-phase reaction of aqueous phase and organic phase is a common reaction in industry. The application of enzyme catalysis to such reactions can improve the reaction efficiency and selectivity, and achieve energy conservation and emission reduction in the whole process. In industrial production, the two-phase reaction is often carried out in the form of an emulsion. The dispersed phase in the emulsion is distributed in the continuous phase in the form of droplets, which greatly increases the mass transfer area and reduces the mass transfer distance, thereby increasing the interphase mass transfer rate. However, the emulsion is a thermodynamically unstable system. In order to carry out a stable reaction in it, a stabilizer must be used to improve the stability of the emulsion. The commonly used emulsion stabilizer is a surfactant, but the surfactant in the emulsion is difficult to separate, and the residual surfactant may affect the product quality, which limits the application of emulsions in food, medical and cosmetics.

[0004] Pickering emulsions use solid particles as stabilizers, and can achieve demulsification and particle separation / recovery through centrifugation and other means, which is expected to solve the problem of traditional emulsion stabilizers being difficult to recover and affecting product quality. On the one hand, the solid particles used to stabilize Pickering emulsions were clay, silica, metal hydroxides, graphite, etc., and the type of Pickering emulsion prepared (oil-in-water or water-in-oil) depends on the hydrophilicity of the particles themselves. To change the hydrophilicity of the particles, it is usually necessary to design a complex chemical modification process. On the other hand, enzyme immobilization technology is booming, and the immobilization methods can be mainly divided into physical adsorption, embedding, chemical cross-linking and chemical coupling. The physical adsorption method fixes the enzyme on the surface of the carrier by means of electrostatic adsorption and other means. The enzyme fixed by this method is easy to fall off, and has poor reusability and catalytic stability. The encapsulation method and chemical cross-linking method fix the enzyme on the surface of the carrier. The enzyme fixed by this method is easy to fall off, and has poor reusability and catalytic stability. The encapsulation method and chemical cross-linking method achieve the effect of fixing the enzyme by fixing the enzyme inside the carrier or using a cross-linking agent to form a network structure. When using this method for catalysis, it faces problems such as large mass transfer resistance and reduced enzyme catalytic efficiency. Summary of the invention

[0005] Objects of the invention: The first object of the present invention is to provide a Pickering emulsion microreactor for enhancing enzyme catalysis and improving the stability and catalytic activity of the enzyme; the second object of the present invention is to provide a method for preparing the Pickering emulsion microreactor.

[0006] Technical solution: The enzyme-catalyzed Pickering emulsion microreactor described in the present invention comprises enzyme-loaded polymer nanoparticles, an aqueous phase and an oil phase, wherein the enzyme-loaded polymer nanoparticles are formed by grafting an enzyme onto the polymer nanoparticles; the polymer nanoparticles are self-assembled from amphiphilic block copolymers formed by a hydrophilic segment polymer and a hydrophobic segment polymer to form a structure with internal hydrophobicity and external hydrophilicity; wherein the hydrophilic segment polymer is a polymer containing carboxyl groups, aldehyde groups or hydroxyl groups; the groups contained on the enzyme are covalently bonded to the carboxyl groups, aldehyde groups or hydroxyl groups of the hydrophilic segment of the polymer nanoparticles.

[0007] The use of polymer nanoparticles for enzyme immobilization allows direct use of the groups of the polymer nanoparticles for chemical coupling, eliminating the need for complex chemical modification processes. The Pickering emulsion microreactor constructed with enzyme-loaded polymer nanoparticles is used for two-phase catalytic reactions, which can enhance the mass transfer rate at the phase interface and strengthen the enzyme catalytic reaction process.

[0008] Preferably, the hydrophilic segment polymer is polymethacrylic acid, polyacrylic acid, polyacrylaldehyde, poly-4-vinylbenzaldehyde, polyvinyl alcohol or poly-2-vinyloxyethanol; polymethacrylic acid and polyacrylic acid are polymers with carboxyl groups, polyacrylaldehyde and poly-4-vinylbenzaldehyde are polymers with aldehyde groups, polyvinyl alcohol and poly-2-vinyloxyethanol are polymers with hydroxyl groups; and the hydrophilicity and hydrophobicity of the polymer nanoparticles can be controlled by changing the ratio of the hydrophilic segment polymer to the hydrophobic segment polymer.

[0009] The groups on the enzyme are covalently coupled with the groups on the polymer nanoparticles, specifically divided into: (1) covalent coupling of the amino group on the enzyme with the carboxyl group on the polymer nanoparticles; (2) covalent bonding of the amino group on the enzyme with the aldehyde group on the polymer nanoparticles; (3) covalent bonding of the carboxyl group on the enzyme with the hydroxyl group on the polymer nanoparticles.

[0010] Preferably, the hydrophobic segment polymer is polymethyl methacrylate, polyglycidyl methacrylate or polystyrene.

[0011] Preferably, the enzyme is urease, glucose oxidase, horseradish peroxidase or lipase.

[0012] The preparation method of the enzyme-catalyzed Pickering emulsion microreactor of the present invention comprises the following steps:

[0013] (1) preparing polymer nanoparticles by self-assembly of an amphiphilic block copolymer formed of a hydrophilic segment polymer and a hydrophobic segment polymer;

[0014] (2) then using a coupling agent to immobilize the enzyme on the polymer nanoparticles through chemical covalent coupling to prepare enzyme-loaded polymer nanoparticles;

[0015] (3) Finally, the buffered aqueous solution containing the enzyme-loaded polymer nanoparticles is mixed with the oil phase solution containing the reaction substrate to prepare a Pickering emulsion microreactor.

[0016] Preferably, the coupling agent described in step (2) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide or 4-dimethylaminopyridine. The groups on the surface of the polymer nanoparticles are first combined with a coupling agent to form an unstable intermediate, and then further reacted with another coupling agent to generate a semi-stable intermediate, and finally reacted with the groups on the enzyme, thereby achieving covalent coupling of the enzyme and the polymer nanoparticles.

[0017] Preferably, in step (1), the block copolymer self-assembly adopts microfluidic mixing technology, and the organic solvent containing the block copolymer and the buffered aqueous solution are mixed by a variety of microfluidic mixing chips, and the block copolymer self-assembles into polymer nanoparticles with hydrophobic interior and hydrophilic exterior under the action of nanoprecipitation; preferably, the organic solvent is acetonitrile, ethanol, N,N-dimethylformamide or tetrahydrofuran; and the buffered aqueous solution is PBS buffer, MES buffer or Tris-HCl buffer.

[0018] The microfluidic mixing technology is used, block copolymers are used as raw materials, and polymer nanoparticles with uniform particle size and controllable morphology are prepared through self-assembly of block copolymers. Preferably, the various microfluidic mixing chips include Tesla structure chips, chips with double helix structures, and fishbone chips.

[0019] Preferably, in step (3), a mixer or microfluidic technology is used to mix and prepare a Pickering emulsion microreactor; a mixer is used: the prepared buffered aqueous solution containing enzyme-carrying polymer nanoparticles is mixed with the oil phase solution in a certain proportion, and stirred under the action of the mixer, thereby constructing a Pickering emulsion microreactor; microfluidic technology: the buffered aqueous solution containing enzyme-carrying polymer nanoparticles and the oil phase solution are respectively introduced into a microfluidic droplet chip in a certain proportion, and the aqueous phase is sheared into spherical droplets by fluid shearing, thereby constructing a Pickering emulsion microreactor. The constructed Pickering emulsion microreactor is used for a two-phase catalytic reaction, which can enhance the mass transfer rate at the phase interface and strengthen the enzyme catalytic reaction process.

[0020] Preferably, the oil phase in the Pickering emulsion microreactor is an oil phase solution such as isooctyl alcohol, toluene, mineral oil, or dichloromethane.

[0021] Invention Mechanism: The present invention uses polymer nanoparticles to immobilize enzymes, and can directly use the groups of polymer nanoparticles for chemical coupling, eliminating the complicated chemical modification process. Immobilizing the enzyme on the surface of the carrier by chemical coupling can not only improve the catalytic stability, storage stability and recyclability of the enzyme, but also make the enzyme contact with the substrate more sufficient, the mass transfer resistance is smaller, and the reaction rate is faster. Combining enzyme immobilization technology with Pickering emulsion can simultaneously improve the reaction efficiency, stability and economy of the reaction process, which is of great significance to the realization of green chemical industry. Compared with the physical adsorption method, the enzyme immobilization effect of the chemical coupling method is more stable, which can improve the catalytic stability and recycling rate of the enzyme; compared with the embedding method and the cross-linking method, the chemical coupling method fixes the enzyme on the surface of the polymer nanoparticles, so that the enzyme contacts with the substrate more fully, the mass transfer resistance is smaller, and the reaction rate is faster. The method of fixing the enzyme with polymer nanoparticles as a carrier is universal and can be used to immobilize a variety of enzymes. In addition, the use of enzyme-loaded polymer nanoparticle-stabilized Pickering emulsion microreactors in enzyme-catalyzed reaction processes can greatly increase the mass transfer area and enhance the mass transfer effect between interfaces, thereby improving the reaction efficiency.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The Pickering emulsion microreactor for enhanced enzyme catalysis uses polymer nanoparticles to immobilize the enzyme, and can directly use the groups carried by the polymer nanoparticles for chemical coupling. The enzyme immobilization effect is stable, the enzyme and the substrate are fully in contact, the mass transfer resistance is small, the reaction rate is fast, and the catalytic activity and recycling rate of the enzyme are high; (2) The specific functional groups carried by the polymer nanoparticles can directly react with the functional groups in the enzyme molecules to achieve covalent coupling, thereby avoiding the complex modification process of the enzyme carrier in the previous process of enzyme immobilization by covalent coupling; (3) The preparation method can controllably synthesize block copolymers with specific composition and block ratio. By regulating the block ratio of the hydrophilic and hydrophobic segments, the hydrophilicity of the polymer nanoparticles can be controlled; (4) The preparation method is simple, and the enzyme is grafted onto the polymer nanoparticles by covalent coupling, avoiding the complex chemical modification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the polymer nanoparticle preparation, enzyme immobilization and water-oil two-phase catalysis process in the Pickering emulsion microreactor of the present invention.

[0024] Figure 2 The H NMR spectrum of the polymethyl methacrylate-b-polymethacrylic acid (PMMA-b-PMAA) diblock copolymer prepared in Example 1;

[0025] Figure 3 This is a transmission electron microscopy image of polymer nanoparticles prepared in Example 1;

[0026] Figure 4 This is a fluorescence confocal microscopy image of the lipase-loaded polymer nanoparticles prepared in Example 1;

[0027] Figure 5 The particle size and monodispersity of the polymer nanoparticles prepared at different flow rate ratios in Examples 1, 3, and 4;

[0028] Figure 6 The figure is a graph showing the effect of different nanoparticle sizes on the immobilization efficiency in Examples 1, 3, and 4;

[0029] Figure 7 The effect of different enzyme concentrations on the immobilization efficiency in Examples 1, 5, and 6 is shown;

[0030] Figure 8 The effect of different enzyme immobilization reaction times on the immobilization efficiency in Examples 1, 7, 8, and 9 is shown;

[0031] Fig. 9 This is a reaction time-conversion diagram of the hydrolysis reaction of p-nitrophenyl palmitate catalyzed by the enzyme-loaded polymer nanoparticles prepared in Example 1 in a Pickering emulsion microreactor. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with embodiments.

[0033] Example 1

[0034] The preparation method of the enzyme-catalyzed Pickering emulsion microreactor of the present invention comprises the following steps:

[0035] (1) Synthesis of polymer nanoparticles composed of PMMA-b-PMAA diblock copolymer

[0036] first step:

[0037] Synthesis of polymethyl methacrylate macro chain transfer agent (CTA-PMMA): Methyl methacrylate monomer, 2-cyano-2-propylbenzodisulfide, and azobisisobutyronitrile (AIBN) were dissolved in N,N-dimethylformamide (DMF) at a molar ratio of 2400:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6 hours. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for use.

[0038] Step 2:

[0039] Synthesis of diblock copolymer: Methacrylic acid monomer, CTA-PMMA prepared in the first step reaction, and azobisisobutyronitrile were dissolved in DMF at a molar ratio of 1200:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6 hours. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for use.

[0040] Step 3:

[0041] Preparation of polymer nanoparticles: The diblock copolymer prepared in the second step was dissolved in DMF solution to prepare an organic phase solution with a concentration of 10 mg / mL, and the aqueous phase was prepared with PBS buffer solution (pH = 7.4). Under the action of a microinjection pump, the organic phase solution and the aqueous phase solution were passed into a microfluidic chip containing a Tesla structure, with a flow rate ratio of the organic phase to the aqueous phase of 1:10 and a total flow rate of 1 mL / min. The resulting solution was centrifuged at 10,000 rpm for 20 minutes, and the precipitated nanoparticles were added to the aqueous phase solution to prepare an aqueous solution containing 1 mg / mL polymer nanoparticles.

[0042] The synthetic route of polymer nanoparticles is as follows:

[0043]

[0044] The nuclear magnetic resonance hydrogen spectrum of the diblock copolymer synthesized in the second step is as follows Figure 2 As shown, 1H NMR (400 MHz, DMSO-d6) δ 12.27 (s, 1H), 4.36 (s, 1H), 3.49–3.40 (m, 3H), 2.21–1.51 (m, 1H), 1.06 (t, J=7.0 Hz, 3H).

[0045] The polymer nanoparticles synthesized in the third step are Figure 3 As shown, the average particle size is about 280 nm, and the particle size distribution is concentrated, and there is no particle aggregation.

[0046] (2) Lipase loading

[0047] 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 40 mg of N-hydroxysuccinimide (NHS), and 60 μL of lipase (activity 2 U / μL) were added to 20 mL of the aqueous solution containing nanoparticles prepared in (1), and the mixture was reacted at 37°C for 4 h. The resulting solution was centrifuged at 10,000 rpm for 20 min, and the precipitated nanoparticles were washed with an aqueous solution, and the process was repeated three times.

[0048] The reaction scheme for lipase loading is shown below:

[0049]

[0050] (3) Preparation of Pickering emulsion microreactor

[0051] The enzyme-loaded nanoparticle aqueous solution obtained in (2) was mixed with an octanol solution containing 3.78 mg / mL p-nitrophenyl palmitate (P-NPP) at a volume ratio of 5:1 to generate water-in-oil droplets. 3 mL of the solution was homogenized at 25,000 rpm for 30 s using a homogenizer, and the resulting Pickering emulsion microreactor was placed in a 37°C constant temperature metal bath for reaction.

[0052] Example 2

[0053] The basic steps are the same as those in Example 1. The difference from Example 1 is that the reaction time of the second step synthesis in step (1) is changed to 1 h, and in step (3), the aqueous solution of the enzyme-loaded nanoparticles is mixed with an octanol solution containing 0.38 mg / mL P-NPP in a volume ratio of 1:5.

[0054] Example 3

[0055] The basic steps are the same as those in Example 1. The difference from Example 1 is that in the third step of step (1), the DMF solution and the aqueous solution are introduced into the microfluidic chip containing the Tesla structure at a flow rate ratio of 1:20.

[0056] Example 4

[0057] The basic steps are the same as those in Example 1. The difference from Example 1 is that in the third step of step (1), the DMF solution is introduced into the microfluidic chip containing the Tesla structure at a flow rate ratio of 1:30 to the aqueous solution.

[0058] Example 5

[0059] The basic steps are the same as those in Example 1. The difference from Example 1 is that in step (2), 80 μL of lipase is added.

[0060] Example 6

[0061] The basic steps are the same as those in Example 1. The difference from Example 1 is that in step (2), 100 μL of lipase is added.

[0062] Example 7

[0063] The basic steps are the same as those in Example 1, except that in step (2), the reaction time is 1 h.

[0064] Example 8

[0065] The basic steps are the same as those in Example 1, except that in step (2), the reaction time is 2 h.

[0066] Example 9

[0067] The basic steps are the same as those in Example 1, except that in step (2), the reaction time is 3 h.

[0068] Example 10

[0069] (1) Synthesis of polymer nanoparticles composed of polymethyl methacrylate-b-polyacrylic acid diblock copolymer

[0070] first step:

[0071] Synthesis of polymethyl methacrylate macro chain transfer agent (CTA-PMMA): Methacrylic acid monomer, 2-cyano-2-propylbenzodisulfide, and AIBN were dissolved in DMF at a molar ratio of 2400:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6h. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for use.

[0072] Step 2:

[0073] Synthesis of diblock copolymer: acrylic acid monomer, CTA-PMMA from the first step reaction, and AIBN were dissolved in DMF at a molar ratio of 1200:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6 hours. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for later use.

[0074] Step 3:

[0075] Preparation of polymer nanoparticles: The diblock copolymer prepared in the second step was dissolved in DMF solution to prepare an organic phase solution with a concentration of 10 mg / mL, and the aqueous phase was prepared with MES buffer solution (pH = 6.5). Under the action of a microinjection pump, the organic phase solution and the aqueous phase solution were passed into a microfluidic chip containing a double helix structure, with a flow rate ratio of the organic phase to the aqueous phase of 1:10 and a total flow rate of 1 mL / min. The resulting solution was centrifuged at 10,000 rpm for 20 minutes, and the precipitated nanoparticles were added to the aqueous phase solution to prepare an aqueous solution containing 1 mg / mL polymer nanoparticles.

[0076] (2) Loading of glucose oxidase

[0077] 37 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloric acid (EDC.HCl), 68 mg of N-hydroxysulfosuccinimide (Sulfo-NHS), and 60 μL of glucose oxidase were added to the 20 mL aqueous solution containing nanoparticles prepared in (1), and the mixture was reacted at 37°C for 4 h. The resulting solution was centrifuged at 10,000 rpm for 20 min, and the precipitated nanoparticles were washed with an aqueous solution, which was repeated three times.

[0078] (3) Preparation of Pickering emulsion microreactor

[0079] The enzyme-loaded nanoparticle aqueous solution obtained in (2) and the n-octanol solution were introduced into the microfluidic droplet chip at a volume ratio of 5:1 to generate water-in-oil droplets with a total flow rate of 100 μL / min. 3 mL of the obtained Pickering emulsion was taken, 1.89 mg of β-D-glucose was added, and the mixture was placed in a 37°C constant temperature metal bath for reaction.

[0080] Embodiment 11

[0081] (1) Synthesis of polymer nanoparticles composed of poly(glycidyl methacrylate)-b-polyacrylaldehyde diblock copolymer

[0082] first step:

[0083] Synthesis of poly(glycidyl methacrylate) macro chain transfer agent (CTA-PGMA): Glycidyl methacrylate monomer, 2-cyano-2-propylbenzodisulfide, and AIBN were dissolved in DMF at a molar ratio of 2400:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6 hours. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for use.

[0084] Step 2:

[0085] Synthesis of diblock copolymer: Acrolein monomer, CTA-PGMA from the first step reaction, and AIBN were dissolved in DMF at a molar ratio of 1200:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6 hours. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for later use.

[0086] Step 3:

[0087] Preparation of polymer nanoparticles: The diblock copolymer prepared in the second step was dissolved in DMF solution to prepare an organic phase solution with a concentration of 10 mg / mL, and the aqueous phase was a phosphate buffer solution (pH = 7.4). Under the action of a microinjection pump, the organic phase solution and the aqueous phase solution were passed into a microfluidic chip containing a fishbone structure, with a flow rate ratio of the organic phase to the aqueous phase of 1:10 and a total flow rate of 1 mL / min. The resulting solution was centrifuged at 10,000 rpm for 20 minutes, and the precipitated nanoparticles were added to the aqueous phase solution to prepare an aqueous solution containing 1 mg / mL polymer nanoparticles.

[0088] (2) Urease loading

[0089] 60 μL of urease was added to the 20 mL aqueous solution containing nanoparticles prepared in (1) and reacted at 37° C. for 4 h. The resulting solution was centrifuged at 10,000 rpm for 20 min, and the precipitated nanoparticles were washed with an aqueous solution, which was repeated three times.

[0090] (3) Preparation of Pickering emulsion microreactor

[0091] The enzyme-loaded nanoparticle aqueous solution obtained in (2) was mixed with an octanol solution containing 0.6 mg / mL urea in a ratio of 5:1 to prepare a 3 mL solution, and homogenized at 25,000 rpm for 30 s under the action of a homogenizer to generate water-in-oil droplets. The resulting Pickering emulsion microreactor was placed in a constant temperature metal bath at 37°C for reaction.

[0092] Example 12

[0093] The basic steps are the same as those in Example 11, except that: in step (1), the second step synthesizes poly(glycidyl methacrylate)-b-poly(4-vinylbenzaldehyde) diblock copolymer, and 4-vinylbenzaldehyde monomer, CTA-PGMA from the first step reaction, and AIBN are dissolved in DMF at a molar ratio of 1200:1:0.1; in step (3), the aqueous solution of the nanoparticles loaded with enzyme obtained in (2) and the n-octanol solution are passed into the microfluidic droplet chip at a volume ratio of 1:5 to generate water-in-oil droplets, with a total flow rate of 100 μL / min. Take 3 mL of the obtained Pickering emulsion, add 0.3 mg of urea, and place in a 37°C constant temperature metal bath for reaction.

[0094] Example 13

[0095] (1) Synthesis of polymer nanoparticles composed of polystyrene-b-polyvinyl alcohol diblock copolymer

[0096] first step:

[0097] Synthesis of polystyrene macro chain transfer agent (CTA-PS): Styrene monomer, 2-cyano-2-propyl-4-cyanobenzenedithiocarbonate, and AIBN were dissolved in DMF at a molar ratio of 2400:1:0.1, sealed and deoxygenated with argon, and reacted at 80°C for 6 hours. After the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for use.

[0098] Step 2:

[0099] Synthesis of diblock copolymer: Vinyl alcohol monomer, CTA-PS from the first step reaction, and AIBN were dissolved in DMF at a molar ratio of 1200:1:0.1, sealed and deoxygenated with argon, reacted at 80°C for 6 hours, and after the reaction was completed, the solution was dripped into pre-frozen ether and purified three times. The purified material was rotary evaporated overnight and stored at 4°C for later use.

[0100] Step 3:

[0101] Preparation of polymer nanoparticles: The diblock copolymer prepared in the second step was dissolved in DMF solution to prepare an organic phase solution with a concentration of 10 mg / mL, and the aqueous phase was a phosphate buffer solution (pH = 7.0). Under the action of a microinjection pump, the organic phase solution and the aqueous phase solution were passed into a microfluidic chip containing a Tesla structure, with a flow rate ratio of the organic phase to the aqueous phase of 1:10 and a total flow rate of 1 mL / min. The resulting solution was centrifuged at 10,000 rpm for 20 minutes, and the precipitated nanoparticles were redissolved with the aqueous phase solution to prepare an aqueous solution containing 1 mg / mL polymer nanoparticles.

[0102] (2) Loading of horseradish peroxidase

[0103] 37 mg of EDC.HCl, 20 mg of 4-dimethylaminopyridine, and 60 μL of horseradish peroxidase were added to the 20 mL aqueous solution containing nanoparticles prepared in (1), and the mixture was reacted at 37°C for 4 h. The resulting solution was centrifuged at 10,000 rpm for 20 min, and the precipitated nanoparticles were washed with an aqueous solution, and the process was repeated three times.

[0104] (3) Preparation of Pickering emulsion microreactor

[0105] The enzyme-loaded nanoparticle aqueous solution obtained in (2) and the n-octanol solution were passed into the microfluidic droplet chip at a ratio of 5:1 to generate water-in-oil droplets with a total flow rate of 100 μL / min. 3 mL of the obtained Pickering emulsion was taken, 0.17 mg of hydrogen peroxide was added, and the mixture was placed in a 37°C constant temperature metal bath for reaction.

[0106] Embodiment 14

[0107] The basic steps are the same as those of Example 13, except that in step (1), in the second step of synthesizing polystyrene-b-poly 2-ethyleneoxyethanol diblock copolymer, 2-ethyleneoxyethanol monomer, CTA-PS obtained in the first step reaction, and AIBN are dissolved in DMF at a molar ratio of 1200:1:0.1.

[0108] Test of factors affecting immobilization efficiency

[0109] The lipase loaded on the polymer nanoparticles was detected. The test method was as follows: 200 μM fluorescein isothiocyanate (FITC) was added to the aqueous solution containing the enzyme-loaded nanoparticles and stirred for 1 h in a dark environment. The obtained fluorescent enzyme-loaded nanoparticle aqueous solution was prepared in a Pickering emulsion microreactor according to (3), and the sample was observed under a fluorescence confocal microscope with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

[0110] The test results of the polymer nanoparticles prepared in Example 1 are as follows: Figure 4 As shown, the water-oil interface of the Pickering emulsion microreactor exhibits bright fluorescence, proving that FITC fluorescently stains the enzyme immobilized on the surface of the nanoparticles and enriches it at the water-oil interface.

[0111] The particle size and monodispersity of the polymer nanoparticles prepared at different flow rate ratios in Examples 1, 3 and 4 are shown in FIG. Figure 5 As shown, as the flow rate ratio of the aqueous phase to the organic phase increases, the particle size of the polymer nanoparticles decreases and the polydispersity stabilizes at around 0.2.

[0112] The results of the effect of the particle size of the polymer nanoparticles prepared in Examples 1, 3 and 4 on the immobilization efficiency are shown in Figure 6 As shown in the figure, with the continuous decrease of particle size, the enzyme immobilization efficiency continues to increase. This is because under the premise that the total substance remains unchanged, with the decrease of particle size, the specific surface area of ​​the particles increases, and the area in contact with the enzyme solution increases, thereby increasing the enzyme immobilization efficiency.

[0113] The effects of different enzyme concentrations on the curing efficiency of enzyme-loaded polymer nanoparticles prepared in Examples 1, 5 and 6 are shown in Figure 2. Figure 7 As shown, Examples 1, 5 and 6 added 60 μL, 80 μL and 100 μL of enzyme stock solution, respectively. The activity of the enzyme stock solution used was 2 U / μL, so the enzyme activities in the prepared solutions were 120 U, 160 U and 200 U, respectively. Figure 7 In the process, as the enzyme activity increases, the immobilization efficiency decreases because the surface groups of the polymer nanoparticles have been fully coupled with the enzyme, and the remaining enzyme molecules can no longer be effectively immobilized.

[0114] The effects of different reaction times on the curing efficiency of enzyme-loaded polymer nanoparticles prepared in Examples 1, 7, 8, and 9 are shown in Figure 2. Figure 8 As shown, with the increase of reaction time, the contact between enzyme molecules and polymer surface groups increased, thereby improving the enzyme immobilization efficiency.

[0115] Performance Characterization

[0116] Reaction time-conversion rate of the enzyme-loaded polymer nanoparticles prepared in Example 1 for catalyzing the hydrolysis of p-nitrophenyl palmitate in a Pickering emulsion microreactor Fig. 9 As shown, during 0-10 min, the catalytic efficiency increases linearly due to the large specific surface area of ​​the Pickering emulsion; during 10-60 min, the rate of increase of the catalytic conversion rate decreases gradually; during 60-120 min, the conversion rate increases by less than 10%.

Claims

1. A Pickering emulsion micro-reaction liquid with enhanced enzyme catalysis, characterized in that: The invention comprises enzyme-carrying polymer nanoparticles, an aqueous phase and an oil phase, wherein the enzyme-carrying polymer nanoparticles are formed by grafting an enzyme onto polymer nanoparticles; the polymer nanoparticles are self-assembled from an amphiphilic block copolymer formed by a hydrophilic segment polymer and a hydrophobic segment polymer to form a structure with internal hydrophobicity and external hydrophilicity; wherein the hydrophilic segment polymer is a polymer containing a carboxyl group, an aldehyde group or a hydroxyl group; the groups contained on the enzyme are covalently bonded to the carboxyl group, the aldehyde group or the hydroxyl group of the hydrophilic segment of the polymer nanoparticles; and the reaction liquid is used in a Pickering emulsion microreactor; The hydrophilic segment polymer is polymethacrylic acid, polyacrylic acid, polyacrylaldehyde, poly-4-vinylbenzaldehyde, polyvinyl alcohol or poly-2-ethyleneoxyethanol; The hydrophobic segment polymer is polymethyl methacrylate, polyglycidyl methacrylate or polystyrene; The enzyme is urease, glucose oxidase, horseradish peroxidase or lipase.

2. A method for preparing the enzyme-catalyzed Pickering emulsion micro-reaction liquid according to claim 1, characterized in that: The following steps are involved: (1) Using an amphiphilic block copolymer formed by a hydrophilic segment polymer and a hydrophobic segment polymer as a raw material, polymer nanoparticles are prepared by self-assembly of the block copolymer; (2) Then, a coupling agent is used to immobilize the enzyme on the polymer nanoparticles through chemical covalent coupling to prepare enzyme-loaded polymer nanoparticles; (3) Finally, the buffered aqueous solution containing the enzyme-loaded polymer nanoparticles is mixed with the oil phase solution containing the reaction substrate to prepare a Pickering emulsion microreaction solution.

3. The method for preparing the enzyme-catalyzed Pickering emulsion micro-reaction liquid according to claim 2, characterized in that: The coupling agent in step (2) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide or 4-dimethylaminopyridine.

4. The method for preparing the enzyme-catalyzed Pickering emulsion micro-reaction liquid according to claim 2, characterized in that: In step (1), the block copolymer self-assembly adopts microfluidic mixing technology, and the organic solvent containing the block copolymer and the buffered aqueous solution are mixed through a variety of microfluidic mixing chips. The block copolymer self-assembles into polymer nanoparticles with hydrophobic interior and hydrophilic exterior under the action of nanoprecipitation.

5. The method for preparing the enzyme-catalyzed Pickering emulsion micro-reaction liquid according to claim 4, characterized in that: The organic solvent is acetonitrile, ethanol, N,N-dimethylformamide or tetrahydrofuran; the buffered aqueous solution is PBS buffer, MES buffer or Tris-HCl buffer.

6. The method for preparing the enzyme-catalyzed Pickering emulsion micro-reaction liquid according to claim 2, characterized in that: In step (3), the Pickering emulsion micro-reaction solution is mixed and prepared by using a mixer or microfluidics technology.

7. The method for preparing the enzyme-catalyzed Pickering emulsion micro-reaction liquid according to claim 4, characterized in that: The various microfluidic hybrid chips include Tesla structure chips, chips with double helix structures and herringbone chips.

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