Enzyme-loaded porous gel capable of being quickly reconstructed, preparation method and flowing microreactor

Abstract

The invention relates to an enzyme-loaded porous gel capable of being quickly reconstructed, a preparation method and a flow microreactor, amphiphilic nanoparticles are used as a stabilizer, bubbles are introduced into a polymer solution and polymerization crosslinking is induced to form a uniform porous gel structure with an adjustable pore diameter, the amphiphilic nanoparticles can be adsorbed on a gas-liquid interface to stabilize the bubbles, and the porous gel structure can be quickly reconstructed. Enzyme molecules can be loaded on the surface to form a porous reaction medium with catalytic activity. The obtained porous gel has good mechanical strength, controllable aperture and porosity, and can realize rapid reconstruction of a porous structure, so that the application scene of a microreactor is widened, and the effective service cycle of enzyme is remarkably prolonged when the microreactor is blocked.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, specifically to a rapidly reconfigurable enzyme-loaded porous gel, its preparation method, and a flow microreactor. Background Technology

[0002] Enzyme catalysis technology has important applications in fine chemicals, green chemistry, and biomanufacturing. To achieve continuous and efficient enzyme catalysis processes, flow microreactors based on porous gel materials have attracted widespread attention. These porous gels are typically formed into three-dimensional porous networks through physical or chemical cross-linking, immobilizing enzyme molecules on the pore wall surface to construct stable enzyme catalysis systems.

[0003] In existing technologies, to achieve stable enzyme loading, it is often necessary to introduce polymers with active functional groups to form porous gel structures that can immobilize enzymes. However, in the actual immobilization process, a large number of functional groups inside the gel matrix cannot participate in enzyme immobilization; only a small number of functional groups on the pore wall surface can be effectively utilized. This results in limited enzyme loading methods and insufficient loading capacity, thereby affecting catalytic efficiency.

[0004] Furthermore, porous gel flow microreactors are prone to clogging under continuous flow conditions. The enzymes fixed to the pore walls are difficult to recover effectively after clogging and can only be discarded with the gel as a whole, resulting in waste of enzyme resources and significantly increasing operating costs.

[0005] Furthermore, the pore size, porosity, and channel network of traditional porous gels are essentially fixed after curing, making it difficult to readjust them according to reaction requirements. This limits their applicability to reactors with specific flow rate ranges, substrate diffusion conditions, and enzyme systems. When switching between different reaction scenarios (such as from small-molecule substrates to large-molecule substrates, or operating within different flow rate and pressure ranges), the existing porous gel structure cannot be quickly reconstructed, making it difficult to meet the multi-scenario adaptability requirements of continuous flow reactions. Summary of the Invention

[0006] This invention aims to solve the problems of limited enzyme loading methods and insufficient loading capacity, difficulty in enzyme recovery and reuse when channels are blocked, and difficulty in adjusting the microreactor structure as needed in existing porous gel microreactors. It provides a rapidly reconfigurable enzyme-loading porous gel, a preparation method, and a flow microreactor to achieve multiple advantages such as high-efficiency enzyme loading, rapid recovery, and adjustable reactor structure.

[0007] As one aspect of the present invention, a rapidly reconfigurable enzyme-carrying porous gel is provided, the enzyme-carrying porous gel comprising a solid gel network and amphiphilic nanoparticles with enzymes loaded on their surfaces, the solid gel network constituting the main structure of the enzyme-carrying porous gel, and the amphiphilic nanoparticles being distributed in the solid gel network. The solid gel network includes a biodegradable or reversible sol-gel transition polymer material, which allows the solid gel network to be degraded or transformed into a solution under specific conditions. The amphiphilic nanoparticles include materials insensitive to the specific action, so that the amphiphilic nanoparticles are retained during the transformation of the solid gel network into a solution and used to reconstruct other enzyme-loaded porous gels.

[0008] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the enzyme is loaded onto the surface of the amphiphilic nanoparticles by physical adsorption or chemical coupling, and distributed on the pore walls of the solid gel network along with the amphiphilic nanoparticles, thereby realizing the enzyme catalytic function.

[0009] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the enzymes include one or more types, thereby achieving single-enzyme or multi-enzyme coupled catalytic functions.

[0010] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the pores of the solid gel network are prepared by a bubble template method, the amphiphilic nanoparticles are used to stabilize the bubble structure, and the pore size distribution and porosity of the solid gel network are controlled by adjusting the concentration of the amphiphilic nanoparticles.

[0011] As another aspect of the present invention, a flow microreactor based on a rapidly reconfigurable enzyme-loaded porous gel is provided, the flow microreactor comprising: The main body of the microreactor includes a through channel for the flow of reactants; The enzyme-carrying porous gel as described in any of the foregoing aspects is disposed within the through-channel for enzyme catalysis of the reactants.

[0012] As another aspect of the present invention, a method for preparing a rapidly reconfigurable enzyme-loaded porous gel is provided, the method comprising the following steps: (1) One or more of the enzymes are immobilized on the surface of the amphiphilic nanoparticles to obtain amphiphilic nanoparticles with enzymes loaded on the surface; (2) Mix the amphiphilic nanoparticles loaded with enzymes on the surface with one or more precursor polymers, introduce bubbles to form a dense bubble structure by bubble template method, and solidify the polymer mixture solution to obtain the enzyme-loaded porous gel. or, (1) The amphiphilic nanoparticles are mixed with one or more precursor polymers, and bubbles are introduced to form a dense bubble structure by bubble template method. The polymer mixture solution is then solidified to obtain a porous gel. (2) One or more of the enzymes are immobilized on the surface of amphiphilic nanoparticles located on the pore walls of the porous gel to obtain the enzyme-loaded porous gel.

[0013] Optionally, in combination with any of the above aspects, another implementation of this aspect further includes the following steps: (1) The enzyme-carrying porous gel is degraded or sol-gelled by the specific action to remove the solid gel network in the enzyme-carrying porous gel, wherein the specific action is a treatment method adapted to the material properties of the solid gel network, including enzyme-responsive degradation, pH-responsive degradation, temperature-responsive gel-sol transition, etc.

[0014] (2) The amphiphilic nanoparticles with enzymes loaded on their surface are separated from the degradation solution by methods such as centrifugation, dialysis or filtration; (3) The amphiphilic nanoparticles loaded with enzyme on the surface are remixed with one or more precursor polymers, and bubbles are introduced to form a dense bubble structure by bubble template method. The polymer mixture solution is then solidified to obtain the reconstructed enzyme-loaded porous gel.

[0015] Optionally, in conjunction with any of the above aspects, in another implementation of this aspect, the amphiphilic nanoparticles are organic or inorganic amphiphilic nanoparticles; wherein, the organic amphiphilic nanoparticles include block self-assembled nanoparticles, natural polymer-based amphiphilic nanoparticles, etc.; the inorganic amphiphilic nanoparticles include silicon-based amphiphilic nanoparticles, metal / metal oxide-based amphiphilic nanoparticles, carbon-based amphiphilic nanoparticles, etc.

[0016] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the precursor polymer is one or more of natural polymer groups and their modified derivatives, synthetic polymer groups, and composite polymer groups.

[0017] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the immobilization of one or more of the enzymes with the amphiphilic nanoparticles is achieved by physical adsorption or chemical coupling.

[0018] Optionally, in combination with any of the above aspects, in another implementation of this aspect, the bubble template method includes: generating bubbles in the polymer mixture solution using direct bubbling, microfluidic technology, or high-speed shearing in a homogenizer, patterning the bubble-containing polymer mixture solution to form a specific geometric structure, and then curing it.

[0019] Beneficial effects: Compared with the prior art, the present invention has at least the following advantages: 1. High-efficiency loading and catalytic efficiency enhancement of single or multiple enzymes: Amphiphilic nanoparticles provide a large specific surface area and functional groups, which can load one or more enzymes to achieve flexible multi-enzyme coupling catalysis.

[0020] 2. Synergistic effect of bubble template method and amphiphilic nanoparticles: Nanoparticles can stabilize bubbles to form uniform pores, allowing enzymes to concentrate on the pore walls, achieving efficient enzyme utilization, reducing mass transfer resistance, and improving catalytic efficiency; at the same time, by controlling the concentration of nanoparticles, the pore size distribution and porosity of porous gels can be precisely controlled.

[0021] 3. Rapid recovery of amphiphilic nanoparticles loaded with enzymes: Through the "degradation-separation-reconstruction" or "degradation-reconstruction" strategy, the rapid recovery and reuse of amphiphilic nanoparticles loaded with enzymes can be achieved, avoiding the ineffective loss of enzymes in the event of reactor blockage and reducing the cost of enzyme use.

[0022] 4. Rapid reconfiguration of microreactor structure: The gel matrix network can be rapidly reconfigured into new carriers of different shapes, pore sizes or compositions according to application requirements, improving the applicability of enzyme-loaded porous gel flow microreactors to different reaction scenarios, thereby further improving reaction efficiency. Attached Figure Description

[0023] The accompanying drawings, incorporated in and forming part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application. To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without any creative effort. These drawings and textual descriptions are not intended to limit the scope of the concept of this application in any way, but rather to illustrate the concepts of this application to those skilled in the art by referring to specific embodiments.

[0024] Figure 1 This is a schematic diagram of the structure of the enzyme-loaded porous gel flow microreactor and its carrier structure of the present invention. Figure 2 This is a particle size distribution diagram of the amphiphilic nanoparticles in Example 1 of the present invention; Figure 3 The images shown are actual pictures of the enzyme-carrying porous gel scaffolds in the embodiments of the present invention. The left image is a picture of the scaffold before degradation in Example 1, and the right image is a picture of the scaffold after degradation and reconstruction in Example 3. Figure 4 This describes the pore structure and enzyme distribution of the enzyme-carrying porous gel scaffold in Example 2 of the present invention. Figure 5 This is a comparison of the enzyme loading capacity of the porous gel in Example 2 of the present invention with and without nanoparticles; Figure 6 The effect of the degradation process on enzyme activity in Example 2 of this invention; Figure 7 This is a performance characterization of the enzyme-loaded porous gel flow microreactor prepared with or without nanoparticles in Example 4 of the present invention. Detailed Implementation

[0025] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0026] like Figure 1 As shown, the method for preparing a rapidly reconfigurable enzyme-carrying porous gel according to the present invention specifically includes the following core steps: (1) The rapidly reconfigurable enzyme-loaded porous gel is prepared from amphiphilic nanoparticles and precursor polymers. The amphiphilic nanoparticles can provide enzyme loading sites and provide a certain mechanical strength, while the polymer can form a solid gel network, thereby providing support and strength.

[0027] (2) One or more enzymes are immobilized on amphiphilic nanoparticles by physical adsorption or chemical coupling.

[0028] (3) After mixing amphiphilic nanoparticles and polymers, bubbles are generated in the solution by different methods, and the polymer solution containing bubbles is further solidified to prepare porous gel.

[0029] (4) Degrading the porous gel under different conditions; since the amphiphilic nanoparticles include materials that are not sensitive to degradation, the amphiphilic nanoparticles are retained during the degradation of the solid gel network and can be recycled to reconstruct other enzyme-loaded porous gels. For non-recyclable polymers, the enzyme-loaded amphiphilic nanoparticles on the surface are separated from the degradation liquid by methods such as centrifugation or dialysis; for recyclable polymers, separation is not necessary.

[0030] (5) For non-recyclable polymers, the amphiphilic nanoparticles with enzymes loaded on the surface of the centrifuged polymers are reconstructed with the new precursor polymers according to steps (1)-(3) to reconstruct the desired porous gel; for recyclable polymers, the reconstruction is carried out directly according to step (3).

[0031] For the above steps, the nanoparticles mentioned in step (1) are organic or inorganic amphiphilic nanoparticles; wherein, the organic amphiphilic nanoparticles include block self-assembled nanoparticles and natural polymer-based amphiphilic nanoparticles; the inorganic amphiphilic nanoparticles include silicon-based amphiphilic nanoparticles, metal / metal oxide-based amphiphilic nanoparticles, and carbon-based amphiphilic nanoparticles; the precursor polymer is one or more of natural polymer-based polymers and their modified derivatives, synthetic polymer-based polymers, and composite polymer-based polymers.

[0032] The method of loading enzymes on nanoparticles as described in step (2) can be achieved through physical adsorption or chemical coupling assisted by EDC / NHS and glutaraldehyde. It can load one or more enzymes on the surface of amphiphilic nanoparticles in different proportions according to the needs of use, so as to achieve single enzyme or multi-enzyme catalysis.

[0033] For generating bubbles in the solution as described in step (3), direct bubbling, microfluidic technology, or high-speed shearing of a homogenizer can be used to generate bubbles in the polymer solution; the polymer solution containing bubbles can be formed into a specific geometric structure by methods such as 3D printing; the curing method includes photocuring, redox curing, or other curing methods that match the material properties.

[0034] For the degradation process described in step (4), substances or conditions that trigger polymer degradation can be used, such as methacryloyl gelatin can be degraded by collagenase, and methacryloyl hyaluronic acid can be degraded by hyaluronidase; for temperature-sensitive materials, the gel state can be changed to a solution state by changing the temperature or other methods.

[0035] The detailed technical solution of the present invention will be described below with reference to specific embodiments.

[0036] Example 1: Preparation of porous gel (1) Preparation of amphiphilic nanoparticles 30 mg of PMMA-b-PMAA was dissolved in 3 mL of DMF to prepare an organic solution with a mass concentration of 10 mg / mL. This organic solution and phosphate-buffered saline (PBS) were then introduced into the organic and aqueous phase inlets of a microfluidic chip, respectively, to prepare amphiphilic nanoparticles using microfluidic mixing technology. The particle size of the resulting nanoparticles could be controlled by adjusting the initial polymer concentration in the organic solution, the flow rate ratio of the water and oil phases, or the total flow rate. The particle size distribution of the resulting nanoparticles is shown in the attached figure. Figure 2 As shown.

[0037] (2) Synthesis of gelatin methacrylamide (GelMA) 10 g of type A gelatin was added to a 250 mL round-bottom flask, followed by 100 mL of deionized water. The mixture was heated in a 50°C water bath for 2 h to dissolve the gelatin completely into a homogeneous solution. Subsequently, 6 g of methacrylic anhydride (MA) was slowly added dropwise to the solution, and the reaction was continued for 1 h to achieve methacrylylation modification of the amino groups in the gelatin molecules.

[0038] After the reaction was complete, the resulting reaction solution was diluted in 100 mL of water at 50 °C to improve the efficiency of subsequent dialysis. The diluted solution was transferred to a 50 mL centrifuge tube and centrifuged at 3500 g for 10 min. The supernatant was collected and placed in a dialysis bag for dialyzing with deionized water for 7 days to remove unreacted MA and low-molecular-weight impurities. After dialysis, the pH of the solution was adjusted to 7.0–7.4 and lyophilized to obtain GelMA solid product, which was stored at approximately 20 °C for later use.

[0039] (3) Preparation of porous gel Two mL suspensions of amphiphilic nanoparticles with concentrations of 4, 6, 8, and 10 mg / mL were taken, and 0.2 g GelMA, 0.1 g polyethylene glycol diacrylate (PEGDA), 0.02 g lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP), and 0.1 g Pluronic F127 were added sequentially. The mixtures were dissolved thoroughly at 4 °C to obtain a homogeneous and stable precursor solution. The resulting precursor solution was then placed in a high-speed homogenizer and subjected to high-speed shearing at 10,000 rpm for 5 min to form fine and uniformly distributed bubbles. The bubble-containing precursor solution was loaded into a syringe and printed using a 3D printer. After printing, it was photocured under a 495 nm light source for a total curing time of 10 min (5 min for each side). After curing, the scaffold was removed, yielding a biodegradable porous gel (see Appendix). Figure 3 (Left image).

[0040] Example 2 Preparation of enzyme-loaded porous gel The porous gel scaffold obtained in Example 1 was placed in an activation solution containing 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 8 mg / mL N-hydroxysuccinimide (NHS) and activated in a shaker at 37 °C for 12 h. After activation, the porous gel scaffold was washed three times with PBS. Then, the activated porous gel scaffold was placed in 5 mL of fluorescent lipase solution containing 1 mg / mL and reacted in a shaker at 37 °C for 6 h to obtain the enzyme-loaded porous gel scaffold. The pore structure and enzyme distribution of the enzyme-loaded porous gel scaffold are shown in the attached figure. Figure 4 As shown.

[0041] After enzyme immobilization, the concentration of residual enzyme in the solution can be determined using Coomassie Brilliant Blue solution, thereby calculating the enzyme loading on the porous gel scaffold. The effects of the presence or absence of nanoparticles and their concentration on enzyme loading are shown in the attached figure. Figure 5 As shown in the attached figure. The effect of the immobilization process on enzyme activity is as follows. Figure 6 As shown. Example 3 Degradation and Reconstruction of Enzyme-Loaded Porous Gels The enzyme-loaded porous gel scaffold obtained in Example 2 was placed in 1 mL of collagenase solution with a concentration of 1 mg / mL and reacted at 37 °C for 8 h for degradation. After degradation, the degradation solution was centrifuged at 11000 rpm for 15 min to separate the amphiphilic nanoparticles loaded with the enzyme. After discarding the supernatant, the obtained amphiphilic nanoparticles loaded with the enzyme were redispersed with ultrapure water, and the enzyme-loaded porous gel scaffold was reconstructed according to step (3) in Example 1 (see attached). Figure 3 (Right image).

[0042] Example 4: Construction and characterization of enzyme-loaded porous gel flow microreactor Construction of enzyme-loaded porous gel flow microflow reactor Enzyme-loaded porous gels were filled into glass tubes, and then rubber tubing was used to connect the glass tubes to a peristaltic pump to construct a flow microfluidic reactor.

[0043] Microreactor performance characterization 5 mL containing 0.55 mg / mL of 4-nitrophenylbutyrate ( p The reaction solution was prepared by slowly adding an ethanol solution of p-nitrophenol (NPB) to 45 mL of 0.1 M PBS buffer. Then, 5 mL of the reaction solution was pumped into a glass tube at a flow rate of 5 mL / min using a peristaltic pump, and the reaction was carried out at 25 °C. During the reaction, 200 μL samples were taken from the reaction solution at 5, 10, 20, 40, 60, 80, and 120 min, and their absorbance at 410 nm was measured to characterize the reaction product p-nitrophenol (NPB). p The concentration of -NP). The enzyme-loaded porous gel scaffolds prepared in Example 2 with and without nanoparticles were used for testing, and the reaction performance of the resulting microreactors is shown in the attached figure. Figure 7 As shown.

[0044] In this application, the descriptions of the various embodiments have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0045] The technical features of the present application can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the present application.

[0046] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A rapidly reconfigurable enzyme-carrying porous gel, characterized in that, The enzyme-loaded porous gel comprises a solid gel network and amphiphilic nanoparticles with enzymes loaded on their surfaces. The solid gel network constitutes the main structure of the enzyme-loaded porous gel, and the amphiphilic nanoparticles are distributed within the solid gel network. The solid gel network includes a biodegradable or reversible sol-gel transition polymer material, which allows the solid gel network to be degraded or transformed into a solution state under specific conditions. The amphiphilic nanoparticles include materials insensitive to the specific action, so that the amphiphilic nanoparticles are retained during the transformation of the solid gel network into a solution and used to reconstruct other enzyme-loaded porous gels.

2. The enzyme-loaded porous gel according to claim 1, characterized in that, The enzyme is loaded onto the surface of the amphiphilic nanoparticles through physical adsorption or chemical coupling, and is distributed on the pore walls of the solid gel network along with the amphiphilic nanoparticles, thereby realizing the enzyme catalytic function.

3. The enzyme-loaded porous gel according to claim 1, characterized in that, The pores of the solid gel network are prepared by the bubble template method. The amphiphilic nanoparticles are used to stabilize the bubble structure, and the pore size distribution and porosity of the solid gel network are controlled by adjusting the concentration of the amphiphilic nanoparticles.

4. A flow microreactor based on a rapidly reconfigurable enzyme-loaded porous gel, characterized in that, The flow microreactor includes: The main body of the microreactor includes a through channel for the flow of reactants; The enzyme-carrying porous gel according to any one of claims 1-3 is disposed within the through-channel for enzyme catalysis of the reactants.

5. A method for preparing a rapidly reconfigurable enzyme-carrying porous gel as described in any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) One or more of the enzymes are immobilized on the surface of the amphiphilic nanoparticles to obtain amphiphilic nanoparticles with enzymes loaded on the surface; (2) Mix the amphiphilic nanoparticles loaded with enzymes on the surface with one or more precursor polymers, introduce bubbles to form a dense bubble structure by bubble template method, and solidify the polymer mixture solution to obtain the enzyme-loaded porous gel. or, (1) The amphiphilic nanoparticles are mixed with one or more precursor polymers, and bubbles are introduced to form a dense bubble structure by bubble template method. The polymer mixture solution is then solidified to obtain a porous gel. (2) One or more of the enzymes are immobilized on the surface of amphiphilic nanoparticles located on the pore walls of the porous gel to obtain the enzyme-loaded porous gel.

6. The preparation method according to claim 5, characterized in that, It also includes the following steps: The enzyme-carrying porous gel is degraded or sol-gelled through the specific action to remove the solid gel network in the enzyme-carrying porous gel. The specific action is a treatment method adapted to the material properties of the solid gel network, including enzyme-responsive degradation, pH-responsive degradation, temperature-responsive gel-sol transition, etc. The enzyme-loaded amphiphilic nanoparticles are separated from the solution obtained in the previous step by one or more methods, including centrifugation, dialysis, or filtration. The amphiphilic nanoparticles loaded with enzymes on their surface are remixed with one or more precursor polymers, and bubbles are introduced to form a dense bubble structure using a bubble template method. The polymer mixture is then solidified to obtain the reconstructed enzyme-loaded porous gel.

7. The preparation method according to claim 5 or 6, characterized in that, The amphiphilic nanoparticles are organic or inorganic amphiphilic nanoparticles; wherein, the organic amphiphilic nanoparticles include block self-assembled nanoparticles, natural polymer-based amphiphilic nanoparticles, etc.; the inorganic amphiphilic nanoparticles include silicon-based amphiphilic nanoparticles, metal / metal oxide-based amphiphilic nanoparticles, carbon-based amphiphilic nanoparticles, etc.

8. The preparation method according to claim 5 or 6, characterized in that, The precursor polymer is one or more of the following: natural polymer groups and their modified derivatives, synthetic polymer groups, and composite polymer groups.

9. The preparation method according to claim 5 or 6, characterized in that, The immobilization of one or more of the enzymes with the amphiphilic nanoparticles is achieved through physical adsorption or chemical coupling.

10. The preparation method according to claim 5 or 6, characterized in that, The bubble template method includes generating bubbles in a polymer mixture solution using direct bubbling, microfluidic technology, or high-speed shearing in a homogenizer, patterning the bubble-containing polymer mixture solution to form a specific geometric structure, and then curing it.

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