Biomimetic enzyme functional composite membrane constructed by microfluidics and application of biomimetic enzyme functional composite membrane in removal of water pollutants

Abstract

The invention provides a biomimetic enzyme functional composite membrane constructed by microfluidics and application of the biomimetic enzyme functional composite membrane in removal of water pollutants. According to the composite membrane, lipidosome vesicles embedded with laccase molecules are prepared through a microfluidic method, a functional coating is formed by sodium alginate, and the functional coating and a porous PVDF supporting membrane are compounded to form a multi-layer membrane structure. The prepared biomimetic membrane simulates the gradient structure of a biological water channel, achieves the synergistic effect of enzyme catalytic degradation and physical filtration, has high selectivity and high permeation flux, and solves the technical problem that'flux-selectivity 'of a traditional membrane is difficult to consider. The membrane material is derived from natural renewable resources, the preparation process is green and environment-friendly, and the membrane shows efficient purification capacity in the aspect of removing heavy metal ions and organic dye pollutants and has excellent anti-pollution and self-cleaning characteristics.

Description

Technical Field

[0001] The present invention relates to the interdisciplinary field of membrane separation technology, microfluidics technology and functional materials for water treatment, and specifically to a biomimetic enzyme functional composite membrane constructed by a microfluidics method and its application in removing water pollutants (including heavy metal ions and organic dyes). Background Art

[0002] The complexity of water pollution and water resource scarcity have become major global challenges in the 21st century. Industrial emissions, agricultural runoff, and urbanization have led to increasingly severe contamination of the aquatic environment with organic pollutants, heavy metal ions, and microbial pathogens. Membrane separation technology, with its advantages of low energy consumption, high selectivity, and continuous operation, has been widely used in wastewater treatment, seawater desalination, and resource recovery. However, traditional single-layer separation membranes often suffer from uneven pore size distribution, poor anti-fouling performance, and a single function, making them inadequate for deep purification under complex water conditions. To improve the comprehensive performance of membrane materials, researchers have begun exploring new strategies for constructing multilayered, functionalized composite membranes. For example, various membrane fabrication methods, such as solvent-induced phase separation (NIPS), layer-by-layer assembly (LBL), template casting, and vapor deposition, have been used to prepare composite membranes. However, these methods suffer from limitations in membrane structure control precision, long preparation cycles, and high costs.

[0003] Inspired by the natural sand-soil-vegetation collaborative filtration structure, the design of biomimetic multilayer filtration membranes has become an important direction for improving membrane performance. In theory, multilayer composite membranes can simultaneously achieve high flux and high retention rate, increase the loading capacity of functional components, and reduce pressure loss during the filtration process. In recent years, microfluidic technology has demonstrated unique advantages in the construction of functional membranes with its ability to precisely manipulate micro- and nanoscale droplets and vesicles. Microfluidic methods can be used to efficiently prepare liposome vesicles with uniform particle size and high encapsulation efficiency, providing an ideal carrier for functional molecules such as enzymes. At the same time, biomimetic membrane separation technology has gradually become a cutting-edge research hotspot in the field of water pollution control due to its outstanding advantages in structural controllability, interface functionalization, and biocompatibility.

[0004] However, introducing active components such as enzymes into membrane material systems still faces many challenges. For example, when free enzymes are directly incorporated into the membrane, they are prone to uneven distribution and aggregation, resulting in reduced enzyme activity and possible clogging of the membrane pores. In addition, how to achieve selective removal of specific pollutants while ensuring high flux is also a bottleneck that traditional membrane technology is difficult to overcome. Therefore, there is an urgent need for a new membrane material and its preparation technology that can effectively load biological enzymes under mild conditions and construct a multi-level structure, thereby taking into account both filtration flux and selectivity, and improving the membrane's anti-fouling ability and service life. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the existing technology by providing a biomimetic enzyme-functional composite membrane constructed using microfluidic technology. This composite membrane, through a biomimetic design, organically combines the catalytic degradation function of biological enzymes with the physical interception function of porous membranes, achieving efficient removal of target pollutants while maintaining high water flux. Specifically, the present invention utilizes a microfluidic chip to precisely prepare enzyme-encapsulated liposome vesicles (enzyme vesicles), allowing the enzyme to be uniformly and stably embedded within the nano / micron-sized carrier, significantly improving the enzyme's stability and utilization within the membrane. The prepared enzyme vesicles are then dispersed in a solution of the natural polymer sodium alginate. The sodium alginate is cross-linked and solidified using Ca₂⁺ ions to form a flexible functional layer containing the enzyme vesicles. This flexible "soft layer" is then composited with a porous polyvinylidene fluoride (PVDF) support membrane prepared using a phase inversion method, creating a multilayer separation membrane system with a biomimetic "soft-hard" gradient structure. The above structure simulates the functional gradient of water channel proteins in biological membranes: the surface flexible hydrogel provides a hydrophilic interface and biocatalytic activity, and the bottom porous support membrane provides mechanical strength and mass transfer channels, thereby achieving both high selectivity and high flux.

[0006] The composite membrane preparation process of this invention is gentle and environmentally friendly. The microfluidic preparation of enzyme vesicles utilizes room temperature conditions and an aqueous system, eliminating the need for high-temperature and high-pressure treatment. The support membrane is formed using a wet phase conversion process, using water as a porogen, to avoid residual toxic organic solvents. The entire membrane-forming process is energy-efficient and environmentally friendly. The resulting biomimetic enzyme-functional composite membrane demonstrated excellent performance in actual water treatment tests: near-complete removal of typical organic dyes (such as methylene blue and malachite green) and heavy metal ions (such as Cu²⁺ and Pb²⁺), with high membrane flux and low pressure loss. Furthermore, because the enzymes are protected and immobilized within the vesicles, the membrane's anti-fouling ability and enzyme reusability are significantly enhanced, as evidenced by a flux recovery rate of nearly 100% after multiple cycles, with virtually no permanent contamination. In summary, this invention integrates biomimetic design, microfluidic nanofabrication, and green membrane-forming technology. The resulting functional membrane can be used for the efficient removal of heavy metals and recalcitrant organic matter from wastewater, possessing significant practical application value and potential for widespread adoption. Technical Solution

[0007] The specific preparation method of the microfluidic biomimetic enzyme functional composite membrane provided by the present invention is as follows: 1. Microfluidic Preparation of Enzyme Liposomes: Dissolve natural soybean lecithin in anhydrous ethanol to form an oil phase solution (e.g., approximately 4% concentration, by dissolving 0.2g of lecithin in 5mL of ethanol). Mix laccase (derived from white-rot fungi, for example), glycerol, polyethylene glycol (PEG400), and phosphate buffered saline (PBS, pH 7.2) to form an aqueous phase solution (e.g., add 0.08g of laccase and 0.4mL of glycerol to 9.6mL of PBS, followed by 20μL of PEG400 as a preservative, and disperse thoroughly by ultrasonication). Using a dual-channel microfluidic chip, the oil and aqueous phases were injected separately at room temperature, maintaining a flow rate ratio of 1:3. Stable oil-in-water emulsion droplets were generated within the converging microchamber of the microfluidic device. Microfluidic shearing enabled the continuous preparation of uniformly sized enzyme vesicle emulsions (Solution C). This emulsion was subjected to reduced-pressure rotary evaporation at approximately 40°C to remove organic solvents, such as ethanol, to obtain a high-purity enzyme-loaded liposome suspension. The obtained vesicles have a concentrated particle size distribution (average diameter is about 20 μm), and the enzyme molecules are encapsulated in the vesicles formed by the lipid bilayer, initially forming an enzyme-vesicle complex structure.

[0008] 2. Preparation of Porous PVDF Support Membrane: The porous support membrane is prepared using the non-solvent-induced phase separation (NIPS) method. A preferred formulation involves dissolving a certain amount of PVDF resin (e.g., 15 wt% PVDF, i.e., 1.5 g) and the porogen polyvinylpyrrolidone (PVP, such as K30, approximately 0.3 g) in N-methylpyrrolidone (NMP, 8.5 g). The mixture is magnetically stirred in a 70°C water bath for approximately 10 hours to fully dissolve the polymer, followed by degassing for 8 hours. The clarified casting solution is poured onto a glass plate and coated with a film applicator at a constant speed of approximately 5 mm / s to form a thin film. The glass plate is then quickly immersed in a deionized water coagulation bath to induce phase separation. PVDF precipitates in the non-solvent and forms a membrane. The resulting membrane is then rinsed in water for at least 24 hours to remove residual solvent. Finally, the membrane is dried naturally to obtain a porous PVDF support membrane with a combination of sponge-like and finger-like pore structures. The pore structure can be changed by adjusting the initial PVDF concentration. For example, at 15 wt% PVDF, the surface pores of the membrane are uniform and the internal pores are connected, which is a better support layer formula.

[0009] 3. Assembly of the Enzyme-Functional Composite Membrane: The prepared enzyme vesicle suspension (Solution C) is added to a sodium alginate (SA) solution at a specific ratio to form a casting solution for the functional layer to be coated. The preferred conditions are: 10 mL of enzyme vesicle suspension is added to every 30 mL of 2% (w / v) SA solution (volume ratio approximately 1:3). The mixture is stirred at room temperature and allowed to stand for several hours to degas to ensure a uniform, bubble-free mixture. The prepared enzyme vesicle / sodium alginate mixture is evenly coated on the surface of the PVDF support membrane by spin coating or casting to form a wet film. The wet-coated support membrane is then immersed in a 0.1 M CaCl₂ aqueous solution for approximately 30 minutes to allow ion exchange crosslinking between the sodium alginate and the Ca₂⁺, resulting in gelation into a stable calcium alginate layer. After sufficient crosslinking, the membrane is removed and gently rinsed with deionized water to remove unreacted Ca₂⁺ and soluble matter. Finally, the membrane is dried at room temperature to obtain the desired biomimetic enzyme-functional composite membrane.

[0010] In the above technical solution, the enzyme is preferably laccase, but is not limited to this. Other enzymes capable of degrading organic pollutants, such as peroxidase, can also be used. The liposome material is preferably natural lecithin to ensure biocompatibility and biodegradability. Sodium alginate is a natural polysaccharide that is highly hydrophilic and contains carboxyl groups that can chelate and crosslink with metal ions. The support membrane material is hydrophobic PVDF, whose surface becomes hydrophilic after composite coating due to the sodium alginate layer. Furthermore, a plasticizer or hydrophilic polymer (such as a small amount of PEG or PVA) can be added to the mixture of enzyme vesicles and SA as needed to further improve membrane adhesion and flexibility. The crosslinking time and ion concentration can be adjusted appropriately. For example, extending the crosslinking time to 60 minutes can produce a denser and more robust functional layer. However, excessive crosslinking may slightly reduce membrane flux, so a balance between stability and flux is crucial.

[0011] In summary, by synthesizing enzyme vesicles through microfluidics and assembling a multilayer structure, the present invention achieves efficient immobilization of enzyme molecules in a composite membrane without affecting the membrane's structural properties. The preparation method and performance of the present invention are further described below with reference to specific examples. DETAILED DESCRIPTION

[0012] The following specific examples describe the synthesis method of the present invention and the properties of the resulting PEF in detail, but the present invention is not limited to the following examples. Without departing from the spirit of the present invention, technicians can adjust the conditions according to actual needs.

[0013] Example 1: Preparation and properties of blank biomimetic membrane In this example, a blank control membrane (denoted as BBM-SA-Blank) without enzymes or vesicles was prepared. 0.9 g of sodium alginate was dissolved in 30 mL of deionized water. The solution was heated and stirred in a 60°C water bath until completely dissolved to form a uniform 2% (w / v) sodium alginate solution, which was then cooled to room temperature. The resulting solution was used directly as the casting solution without the addition of any enzymes or vesicles. The porous PVDF support membrane obtained in Implementation Example 2 was used as the substrate. The sodium alginate solution was evenly coated on the surface of the PVDF membrane by spin coating and allowed to stand for 1 minute to spread evenly. The membrane was then immersed in a 0.1 M CaCl2 solution for cross-linking for 30 minutes, allowing the sodium alginate gel to solidify and form a selective layer. The membrane was removed, rinsed with pure water, and dried to obtain a blank biomimetic membrane.

[0014] Performance Testing: Since the blank membrane lacks enzymes and vesicles, its filtration relies primarily on the physical sieving action of the sodium alginate gel layer and the complexation of heavy metal ions by the sodium alginate itself. Testing revealed a pure water permeation flux of approximately 106.9 L·m⁻²·h⁻¹·bar⁻¹, with an initial contact angle of approximately 76°, indicating increased surface hydrophilicity but low overall porosity. The membrane was used to filter a 30 mg / L methylene blue (MB) solution, achieving an initial retention rate of approximately 80%, primarily due to the barrier effect of the gel layer that partially removes dye molecules. For copper ion (Cu²⁺) solutions, the sodium alginate layer chelates Cu²⁺ to form a gel, resulting in a retention rate of approximately 60%. However, since the membrane lacks any catalytic components, it is unable to degrade organic pollutants and is susceptible to clogging with organic fouling over long-term use, resulting in a significant decrease in flux. The flux recovery rate (FRR) of the blank membrane after a single pure water rinse was approximately 95%, indicating that while no enzymatic degradation of the contaminants was possible, simple water washing could remove reversible fouling and that the membrane itself was free of fixed contaminants. This blank control confirmed that the sodium alginate layer had some interception effect on contaminants but lacked catalytic degradation, and the dense membrane structure limited water flux.

[0015] Example 2: Preparation and performance of free enzyme embedded membrane In this example, a membrane in which free laccase was directly incorporated into the sodium alginate layer (denoted as BBM-Free-Lac) was prepared to compare and evaluate the effectiveness of direct enzyme loading. First, 30 mL of a 2% (w / v) sodium alginate solution was prepared according to the method in Example 1. After the solution cooled to room temperature, 0.08 g of laccase powder was added (equivalent to a final laccase concentration of approximately 0.27% w / v, comparable to the amount of enzyme added for microfluidic vesicle encapsulation). The solution was gently stirred to fully disperse the enzyme in the sodium alginate solution. To maximize enzyme activity, stirring was performed in an ice bath and completed within 30 minutes. The resulting enzyme / sodium alginate mixture was coated onto a PVDF support membrane using the same method as in Example 1 and Ca²⁺ cross-linked for 30 minutes. The membrane was then washed and dried to obtain a free enzyme-loaded biomimetic membrane.

[0016] Performance Testing: During the membrane preparation process, the lack of a micro-nanocarrier to immobilize the enzyme caused laccase molecules to aggregate or settle within the sodium alginate colloid. SEM observation of the membrane surface revealed irregular particles approximately 1–5 μm in diameter. These are laccase crystals formed by aggregated laccase, which are unevenly distributed across the membrane surface. These aggregates obscure the membrane's pore structure, with some pores blocked. Cross-sectional observation also revealed localized accumulation and discontinuous pores. Filtration experiments showed that the free enzyme membrane achieved an initial removal efficiency of approximately 90% for dyes such as MB, slightly higher than that of a blank membrane, demonstrating that the laccase possesses a certain catalytic activity in the degradation of dyes. However, due to the lack of effective enzyme immobilization, the enzyme gradually dissipated after a period of operation, and the membrane's pollutant removal efficiency decreased significantly with the number of cycles. Furthermore, the presence of the free enzyme did not significantly improve membrane flux, with a measured pure water flux of approximately 192.4 L·m⁻²·h⁻¹·bar⁻¹, higher than that of the blank membrane but lower than that of the vesicle membrane described later. This is because some enzymes clog the membrane pores, and the macromolecular structure of the enzyme increases the mass transfer resistance. After 5 cycles of use, the FRR of the free enzyme membrane dropped to about 90%, indicating that slight irreversible contamination occurred. This may be due to the pores left after the enzyme migrated and lost during the filtration process being blocked by pollutants. This embodiment illustrates that although directly incorporating enzymes can improve the pollutant removal rate to a certain extent, enzyme stability and membrane structure are both adversely affected.

[0017] Example 3: Preparation and properties of empty liposome vesicle membrane In this example, a membrane containing only empty liposome vesicles but no enzyme (denoted as BBM-Vesicle-Empty) was prepared to investigate the effect of vesicles as structural templates on membrane pore structure and flux. Liposome vesicles were first prepared according to Technical Scheme 1, but laccase was omitted during the aqueous phase preparation. All other ingredients and conditions remained the same. This resulted in an empty vesicle suspension containing only additives such as glycerol and PEG. This empty vesicle suspension was then mixed with a 2% sodium alginate solution in a 1:3 volume ratio and stirred thoroughly to form a casting solution. This mixture was coated onto a PVDF support membrane using the same procedures as in Example 1. After Ca²⁺ cross-linking for 30 minutes, the empty vesicle biomimetic membrane was obtained.

[0018] Performance Testing: Due to the lack of enzyme involvement, the membrane only reflects the effect of vesicles on the membrane structure. Compared to the blank membrane, the surface of the empty vesicle membrane exhibits a large number of evenly distributed circular pores with uniform pore size and relatively orderly arrangement. These pores are formed by the vesicles acting as a "soft template" during the membrane formation process. After the vesicles are embedded in the sodium alginate matrix and fixed by cross-linking, they may partially collapse or escape during the drying process, leaving behind continuous micron-sized pores. This significantly increases the surface porosity of the membrane, with a cross-section exhibiting a typical finger-like pore structure and a loose, porous network. Compared to the dense cross-section of the blank membrane, the porous channels of the empty vesicle membrane are more interconnected, facilitating the transport of water molecules. Filtration tests show that the pure water flux of the empty vesicle membrane increases to approximately 250.5 L·m⁻²·h⁻¹·bar⁻¹, approximately 2.3 times higher than that of the blank membrane. This demonstrates that the introduction of vesicles effectively improves the internal porosity and surface roughness of the membrane, thereby reducing mass transfer resistance. However, due to the lack of enzymes, the empty vesicle membrane still relies primarily on physical interception to remove organic pollutants, making it difficult to degrade small molecule pollutants. In MB filtration, its retention rate is comparable to that of a blank membrane (approximately 80%), but due to its more unobstructed pores, its anti-fouling performance is improved over long periods of operation. After five cycles, the flux recovery rate of the empty vesicle membrane remained above 95%, indicating that the addition of vesicles did not introduce new sources of contamination and that the higher surface roughness reduced the adhesion of pollutants to the membrane. This example confirms the role of vesicles as microscopic "pore templates" in optimizing membrane structure.

[0019] Example 4: Preparation and properties of enzyme vesicle biomimetic membrane In this example, a functional composite membrane containing enzyme-containing liposome vesicles (denoted as BBM-V@Lac) was prepared according to the optimal solution of the present invention. Microfluidic enzyme vesicles were prepared according to Technical Solution 1, resulting in a liposome vesicle suspension with an average particle size of approximately 20 μm and encapsulated laccase. This enzyme vesicle suspension was mixed uniformly with a 2% sodium alginate solution in a volume ratio of 1:3, allowed to stand for degassing, and then coated onto a 15 wt% PVDF support membrane. After coating, the membrane was cross-linked in a 0.1 M CaCl₂ solution for 30 minutes to solidify the membrane layer. The membrane was then rinsed and dried to obtain the enzyme vesicle biomimetic membrane. The membrane surface exhibited regular honeycomb-like pores with moderate and uniform pore size. A cross-section clearly divided the membrane into a dense cortex layer, a finger-like channel layer, and a porous support layer from top to bottom. The porous channels were arranged vertically through the membrane layers in an orderly manner. This demonstrates that the enzyme vesicles can be stably embedded in the membrane structure, forming a porous interface while preventing enzyme aggregation and inactivation. In addition, fluorescence microscopy revealed that the surface of the enzyme vesicle membrane was covered with a uniform red fluorescence signal (derived from laccase labeled with rhodamine B), with no obvious fluorescence quenching or agglomeration spots, indicating that the enzyme was evenly distributed in the membrane and remained active.

[0020] In terms of filtration performance, the enzyme vesicle biomimetic membrane demonstrated outstanding performance in a variety of pollutant removal tests. For example, in filtering a 30 mg / L solution of organic dyes (MB, MG, and CV), the membrane retained approximately 98.9%, 98.8%, and 99.8%, respectively, achieving near-complete removal. This is because laccase catalyzes the degradation and polymerization of these dyes, making them easily retained or converted by the membrane. Furthermore, for 30 mg / L solutions of Cu²⁺ and Pb²⁺, the membrane retained approximately 95.3% and 90.6%, respectively. This is attributed not only to the chelation of metal ions by sodium alginate, but also to the improved pore structure of the membrane due to the introduction of the enzyme vesicles, which prevents premature clogging and allows the heavy metals to be captured and fixed in the gel layer. More importantly, the enzyme vesicle membrane maintains high water flux while maintaining high retention: under the aforementioned various pollutant filtration conditions, its permeation flux ranged from 96–108 L·m⁻²·h⁻¹·bar⁻¹, significantly higher than that of a membrane without enzyme vesicles. This demonstrates that the present invention successfully overcomes the limitation of traditional membranes that "flux and selectivity cannot be achieved at the same time."

[0021] Anti-fouling performance testing: The enzyme vesicle membrane was subjected to continuous filtration for 5 hours in MB, MG, CV, and Cu²⁺ and Pb²⁺ solutions, and the flux and retention rate were monitored. The results showed that after 5 hours, the membrane's retention rates for MB, MG, and CV remained at 98.3%, 97.9%, and 99.0%, respectively, and for Cu²⁺ and Pb²⁺ at 94.2% and 91.2%, respectively, representing a decrease of less than 1% compared to the initial values. Furthermore, the permeability of each pollutant remained at approximately 93–105 L·m⁻²·h⁻¹·bar⁻¹ (only slightly lower than the initial flux), with a flux recovery rate of 100%. This indicates that the enzyme vesicle membrane exhibited little irreversible fouling. The enzyme's catalytic activity continued throughout the filtration process, degrading organic matter in the filter cake and achieving self-cleaning properties. This excellent durability is unattainable by blank membranes or membranes containing free enzymes. The results of this example fully verify the effectiveness of the technical solution described in the present invention: through microfluidic enzyme vesicle loading, the biomimetic membrane achieves satisfactory high flux, high selectivity and high anti-fouling performance in complex wastewater treatment applications.

[0022] Example 5: Effect of vesicle content ratio on membrane performance This example investigates the effect of the enzyme vesicle addition ratio on the structure and performance of biomimetic membranes. Based on Example 4, two enzyme vesicle membranes were prepared with vesicle suspension to sodium alginate solution volume ratios of 1:1 and 1:5, respectively, and compared with an optimal membrane with a 1:3 ratio. All other preparation conditions (enzyme concentration, support membrane, cross-linking time, etc.) remained the same as in Example 4; only the vesicle content was adjusted.

[0023] Atomic force microscopy (AFM) was used to characterize the surface morphology of membranes with different vesicle ratios, revealing that vesicle content significantly affects the membrane's microstructural roughness. When the vesicle ratio was high (1:1), the membrane surface roughness reached a maximum of approximately 19.6 nm, with prominent protrusions and aggregates. Excessive vesicles may coalesce during membrane formation, causing local pore blockage, thereby compromising the membrane's mass transfer stability and structural uniformity. Conversely, when the vesicle ratio was too low (1:5), the membrane surface was very smooth, with a roughness of only approximately 12.5 nm, indicating insufficient vesicle templating, smaller membrane pores, and reduced enzyme exposure. Overall, the membrane produced at a vesicle ratio of 1:3 exhibited moderate surface roughness (approximately 14.6 nm) and a uniform, interconnected pore structure, achieving a good balance between structure and function. Performance testing also showed that the 1:3 enzyme vesicle membrane achieved the highest and most stable flux, maintaining the best retention rate. While the 1:1 ratio membrane exhibited high initial retention, its flux decreased slightly, and during circulation, excessive vesicles caused pore instability, resulting in a slightly lower flux recovery rate (approximately 95%). The 1:5 ratio membrane, due to its lower enzyme content, saw a decrease in pollutant removal efficiency of approximately 5–10%, with Cu²⁺ and Pb²⁺ retention rates falling below 90%. Therefore, it can be determined that an appropriate vesicle content is crucial for optimizing the performance of the composite membranes of this invention: excessive or insufficient vesicle content can disrupt the continuity of the membrane structure or reduce the effectiveness of the functional components. Generally, under the conditions described in this example, a 1:3 vesicle volume ratio is optimal. To further enhance the mechanical strength or durability of the membrane, the Ca²⁺ cross-linking time can be extended to 45–60 minutes to achieve a denser surface structure, but care should be taken to avoid a significant decrease in membrane flux.

[0024] In summary, a comparison of Examples 1–5 demonstrates that the present invention's microfluidic biomimetic enzyme-functional composite membrane offers significant advantages in both structure and performance. Compared to blank membranes and traditional directly enzyme-doped membranes, the enzyme vesicle composite membrane achieves both high flux and high selectivity, and exhibits superior anti-fouling and durability, demonstrating the promising application prospects of this invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Structural characterization of enzyme liposome vesicles prepared by microfluidics. (a) Optical microscopy image of empty liposome vesicles; (b) Optical microscopy image of enzyme vesicles; (c) SEM image of liposome enzyme vesicles after laccase encapsulation; (d) Fluorescence microscopy image of enzyme vesicles labeled with rhodamine B, showing uniformly distributed red fluorescence signal, indicating that laccase is successfully immobilized and well dispersed on the vesicles.

[0026] Figure 2Comparison of scanning electron microscopy (SEM) images of biomimetic membranes with different vesicle / enzyme loadings. (a), (e) Surface and cross-section of a blank sodium alginate membrane (BBM-SA-Blank); (b), (f) Surface and cross-section of a membrane incorporating empty vesicles (BBM-Vesicle-Empty); (c), (g) Surface and cross-section of a membrane incorporating free enzyme (BBM-Free-Lac); (d), (h) Surface and cross-section of an enzyme-vesicle composite membrane (BBM-V@Lac). As can be seen, (a) the surface pores are sparse and disordered, and the cross-section (e) shows a dense sponge-like structure, with the blank membrane having the lowest porosity. After adding empty vesicles, (b) uniform circular pores appear on the surface, and the cross-section (f) forms finger-like macropores and a porous network structure, with more unobstructed pores. When free enzyme is directly added, (c) enzyme particles are visible on the surface agglomerating, the pore structure is irregular, and the cross-section (g) shows partial blockage. In contrast, the surface pores of the enzyme vesicle membrane (d) are evenly distributed in a honeycomb pattern, and the cross-section (h) shows a clear multi-layer gradient structure and vertically connected pores. This shows that the introduction of enzyme vesicles not only stabilizes the loading form of the enzyme but also acts as a template to form an ideal porous structure.

[0027] Figure 3 Comparison of the filtration performance of different membranes for organic dyes and heavy metal ion pollutants. (a) Permeate flux (L·m⁻²·h⁻¹·bar⁻¹) of each membrane for methylene blue (MB), malachite green (MG), crystal violet (CV), Cu²⁺, and Pb²⁺; (b) Corresponding pollutant retention (R) (%). The colors in the figure represent: red (blank membrane) (BBM-SA-Blank), orange (free enzyme membrane) (BBM-Free-Lac), yellow (empty vesicle membrane) (BBM-V@Lac), and green (enzyme vesicle membrane) (BBM-V@Lac). It can be seen that the green enzyme vesicle membrane achieved both the highest flux and the highest removal rate for all pollutants. For example, for MB dye, the enzyme vesicle membrane achieved a flux of approximately 102 L·m⁻²·h⁻¹·bar⁻¹ and a retention of approximately 99%, significantly outperforming the other control membranes. Similar results were obtained for the heavy metals Cu²⁺ and Pb²⁺, with the enzyme vesicle membrane retaining approximately 95% and over 90%, respectively, while maintaining the highest flux. These data demonstrate the comprehensive advantages of the composite membrane in removing water pollutants: high filtration efficiency and minimal flux loss, resulting in highly efficient and stable separation performance.

Claims

1. A biomimetic enzyme functional composite membrane constructed by microfluidics, characterized by: It includes a porous supporting base membrane and a bionic functional layer arranged on the supporting base membrane; the bionic functional layer contains enzyme-loaded liposome vesicles uniformly dispersed in a sodium alginate matrix, and the matrix forms an overall composite structure with the supporting base membrane after being cross-linked and solidified by divalent metal ions.

2. The method for preparing the biomimetic enzyme functional composite membrane according to claim 1, characterized in that: The method comprises the following steps: using a microfluidic chip device to flow-aggregate an oil phase containing lecithin and an aqueous phase containing an enzyme in a volume ratio of 1:3 to generate an enzyme-encapsulated liposome vesicle solution; uniformly mixing the obtained enzyme vesicle solution with a sodium alginate solution, and coating the solution on the surface of a porous polyvinylidene fluoride (PVDF) support membrane; then immersing the membrane in a cross-linking agent calcium chloride solution for 30 minutes to gel the sodium alginate to form a functional layer loaded with enzyme vesicles; and finally washing and drying the membrane to obtain the biomimetic enzyme functional composite membrane.

3. The biomimetic enzyme functional composite membrane according to claim 1, characterized in that: The liposome vesicle is formed by natural lecithin and encapsulates laccase; the biomimetic functional layer is composed of sodium alginate and is fixed on the PVDF porous support base membrane through Ca²⁺ ion cross-linking; the composite membrane does not contain toxic organic solvent residues and has good biocompatibility.

4. The use of the biomimetic enzyme functional composite membrane in removing water pollutants according to claim 1, characterized in that: Used to remove heavy metal ions and organic dye pollutants in aqueous solutions. The heavy metal ions include Cu²⁺ and Pb²⁺, and the organic dyes include methylene blue (MB), malachite green (MG) and crystal violet (CV).

5. The biomimetic enzyme functional composite membrane according to claim 1, characterized in that: The average diameter of the enzyme-loaded liposome vesicles is 10-40 μm, and the enzyme molecules are uniformly embedded in the vesicles and distributed in the functional layer, thereby stabilizing the enzyme activity and avoiding inactivation and aggregation of the enzyme during the membrane preparation process.

6. The biomimetic enzyme functional composite membrane according to claim 1, characterized in that: The composite membrane surface has high hydrophilicity and permeation flux, its static water contact angle is less than 60°, the pure water flux reaches 250–270 L·m⁻²·h⁻¹ at a pressure of 0.1 MPa, and the water flux recovery rate after contamination is close to 100%.

7. The biomimetic enzyme functional composite membrane according to claim 1 or 5, characterized in that: The composite membrane has high filtration efficiency and anti-pollution self-cleaning properties. The retention rate of organic dyes such as methylene blue, malachite green, and crystal violet is above 98%, and the retention rate of Cu²⁺ and Pb²⁺ ions is not less than 90%, respectively. After multiple cycles of use, the membrane flux remains above 95% of the initial flux.

8. The biomimetic enzyme functional composite membrane constructed by microfluidics according to claim 1, characterized in that: The biomimetic enzyme functional composite membrane can be widely used in multiple water treatment environments, including industrial wastewater treatment, drinking water purification, medical waste liquid purification and laboratory waste liquid reuse.

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