A method for modifying an anion exchange membrane with lysozyme and THPS

Abstract

The application discloses a method for modifying an anion exchange membrane by using lysozyme and THPS, which comprises the following steps: step 1, pretreating a commercial anion exchange membrane; step 2, adding a reaction solution 1 containing levodopamine and lysozyme into a feed liquid chamber of a modification device, and making one side of the pretreated original membrane contact with the reaction solution 1; after stirring reaction is completed at room temperature, the reaction solution is poured out, and the obtained membrane is washed with deionized water to obtain an E-Lys membrane; step 3, adding a reaction solution 2 containing THPS into the feed liquid chamber, and making the modified side of the E-Lys membrane contact with the reaction solution 2 to generate a Mannich-type reaction; after continuous reaction is completed at room temperature, the reaction solution is poured out, and the modified membrane is washed with deionized water to obtain an E-Lys@Thps membrane. The application obtains a green and environment-friendly anion exchange membrane with good anti-biological pollution performance, bacteriostatic performance and separation performance.

Description

Technical Field

[0001] This invention relates to a method for modifying anion exchange membranes. Technical Background

[0002] In recent years, various research and support policies for seawater desalination have gradually alleviated the freshwater crisis globally. Among them, membrane-based technologies, due to their high efficiency and low energy consumption, have played a significant role in promoting seawater desalination, especially electrodialysis (ED) technology, which now accounts for 6% of the world's saltwater desalination capacity.

[0003] With the widespread application of electrodialysis (ED) technology in various fields, the problem of biofouling has become increasingly prominent. Bacterial-mediated biofouling, in particular, tends to grow and multiply on membrane surfaces, forming dense biofilms that severely impact membrane separation efficiency and lifespan. In the field of antibacterial membranes, the main research focus is on constructing antibacterial functional layers on membrane surfaces. However, in the process of surface modification research, the biosafety and environmental protection of antibacterial agents (such as heavy metal ions and their oxides, antibiotics, etc.) are often overlooked. Therefore, it is necessary to study a green and environmentally friendly method for combating biofouling in electrodialysis systems to improve the performance of anion exchange membranes. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for modifying anion exchange membrane with lysozyme and tetramethyl hydroxysulfate (THPS) to obtain an anion exchange membrane that is green and environmentally friendly and has good anti-biofouling, antibacterial and separation performance.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for modifying anion exchange membranes with lysozyme and THPS, wherein THPS refers to tetramethylolphosphine sulfate, the method comprising the following steps:

[0007] Step 1: Immerse the commercial anion exchange membrane sequentially in sodium hydroxide solution and sodium chloride solution, then wash and dry it with deionized water to complete the pretreatment of the original membrane;

[0008] Step 2: At room temperature, the original membrane treated in Step 1 is installed in the modification device, and reaction solution ① is added to the feed chamber of the modification device so that one side of the original membrane is in contact with reaction solution ①. The reaction solution ① is a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution with a pH between 8.0 and 9.0 containing 9.00-11.00 mM levonorgestrel and 0.5-1 mg / mL lysozyme. The mixture is stirred at room temperature and reacted for 10-14 hours. After the reaction is completed, the reaction solution is poured off and thoroughly washed with deionized water. The resulting membrane is named E-Lys membrane.

[0009] Step 3: At room temperature, add reaction solution ② to the feed chamber and bring the modified surface of the E-Lys membrane into contact with the reaction solution ② to carry out a Mannich-type reaction. The reaction solution ② is an aqueous solution of 1.0-1.5 mM THPS. The reaction is carried out continuously at room temperature for 6-8 hours. After the reaction is complete, pour out the reaction solution, take out the modified membrane, rinse it with deionized water, and name it E-Lys@Thps membrane.

[0010] Preferably, in step 1, the commercial anion exchange membrane is sequentially immersed in 0.2 M sodium hydroxide solution and sodium chloride solution for 30 minutes, then the membrane is removed and rinsed with deionized water, and dried for at least 24 hours.

[0011] Preferably, in step 2, the pH of the tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution is controlled between 8.3 and 8.7.

[0012] Preferably, in step 2, the reaction time is controlled at 12 hours.

[0013] Preferably, in step 2, the content of L-dopamine in the reaction solution ① is 9.00-11.00 mM, and most preferably 10.01 mM.

[0014] Preferably, in step 2, the concentration of lysozyme in the reaction solution ① is 0.7 mg / mL.

[0015] Preferably, in step 3, the reaction time is controlled at 7 hours.

[0016] Preferably, in step 3, the THPS content in the reaction solution ② is between 1.0 and 1.5 mM, with an optimal value of 1.31 mM.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses lysozyme and THPS to modify the anion exchange membrane to improve its resistance to biofouling, and prepares a green and environmentally friendly anion exchange membrane with resistance to biofouling. The modified anion exchange membrane significantly improves the membrane's resistance to biofilm formation and antibacterial properties without affecting the separation performance, and is also green and environmentally friendly. Attached Figure Description

[0018] Figure 1 The following diagram illustrates the surface modification process of the anion exchange membrane of the present invention: (a) co-deposition of levodopamine and lysozyme under alkaline conditions; (b) Mannich reaction between THPS and lysozyme; (c) schematic diagram of THPS and lysozyme synergistically hydrolyzing bacterial cytokerate; and (d) schematic diagram of surface modification of the anion exchange membrane to achieve "synergistic antibacterial" effect.

[0019] Figure 2 SEM images of the cross-sectional morphology (ac) and surface morphology (df) of the original membrane, E-Lys membrane, and E-Lys@Thps membrane.

[0020] Figure 3 AFM images of the original membrane, E-Lys membrane, and E-Lys@Thps membrane.

[0021] Figure 4 : The antibacterial activity of lysozyme and lysozyme@THPS against Escherichia coli and Staphylococcus aureus solutions.

[0022] Figure 5 (a) OD values ​​(detected by ELISA reader) of the control group, original membrane, E-Lys and E-Lys@Thps membrane after soaking in Escherichia coli and Staphylococcus aureus solutions for 0, 12, 24 and 36 hours at 37°C; (bg) SEM images of the original membrane, E-Lys and E-Lys@Thps membrane (soaked in 2.5% glutaraldehyde at 4°C for 3 hours) after soaking for 36 hours.

[0023] Figure 6 Survival rates of Staphylococcus aureus and Escherichia coli biofilms in CLSM and 3D images after 16 hours of incubation at 37°C using the original membrane, E-Lys membrane, and E-Lys@Thps membrane.

[0024] Figure 7 (a) Antimicrobial test of Escherichia coli and Staphylococcus aureus after 15, 60 and 720 min of incubation (LB plate method); (b) Antimicrobial test of Escherichia coli and Staphylococcus aureus after 1 hour of incubation on the original, E-Lys and E-Lys@Thps membranes at 37°C (fluorescence microscopy method).

[0025] Figure 8 (a) Conductivity, energy consumption, and current efficiency of the original membrane, E-Lys membrane, and E-Lys@Thps membrane after (a) primary ED desalination, (b) secondary ED desalination, and (c) tertiary ED desalination in the dilution and concentration chambers; (d) Photographs of the original membrane, E-Lys membrane, and E-Lys@Thps membrane after primary, secondary, and tertiary ED desalination; (e) Desalination rates of the original membrane, E-Lys membrane, and E-Lys@Thps membrane after primary, secondary, and tertiary ED desalination. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited:

[0027] The sodium dihydrogen phosphate, disodium hydrogen phosphate, tris(hydroxymethyl)aminomethane, levonorgestrel, sodium chloride, and sodium sulfate used in the embodiments of this invention were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd. and Shanghai Aladdin Reagent Co., Ltd.; the base membrane was a homogeneous Type-II commercial anion exchange membrane manufactured by Fuji Corporation. The bacterial strains used in this invention were Escherichia coli (K88) and Staphylococcus aureus (ATCC6538).

[0028] Example 1

[0029] The commercial anion exchange membrane was first rinsed with deionized water, and then successively immersed in sodium hydroxide solution (0.2M) and sodium chloride solution (0.2M), each for 30 minutes. Afterward, it was rinsed with deionized water and dried for 24 hours to obtain the original membrane (the modification principle diagram can be found in the reference: Y. Yao, J. Mu, J. Liao, et al. Imparting antibacterial adhesion property to anion exchange membrane by constructing negatively charged functional layer [J]. Separation and Purification Technology 2022, 120628).

[0030] At room temperature, reaction solution ① was added to the feed chamber containing the original membrane, with one side of the original membrane in contact with reaction solution ①. Reaction solution ① was a Tris-HCl buffer solution with a pH controlled at 8.5 containing 10.01 mM levonorgestrel and 0.7 mg / mL lysozyme. The mixture was stirred at room temperature and reacted for 12 hours. After the reaction was complete, the reaction solution was removed from the feed chamber and thoroughly washed with deionized water. The resulting membrane was named the E-Lys membrane.

[0031] At room temperature, reaction solution ② was added to the feed chamber, and the modified surface of the E-Lys membrane was brought into contact with reaction solution ② to initiate the Mannich reaction. Reaction solution ② was an aqueous solution containing 1.31 mM THPS. The reaction was carried out continuously at room temperature for 7 hours. After the reaction was complete, the reaction solution was poured off, the modified membrane was removed, and it was rinsed thoroughly with deionized water. This membrane was named the E-Lys@Thps membrane. The specific modification process is as follows: Figure 1 As shown. The surface and cross-sectional morphology of the original membrane, E-Lys membrane, and E-Lys@Thps membrane were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Specific results are shown in the figure. Figure 2 , Figure 3As shown in the figure, the results indicate that, compared to the original membrane, the E-Lys membrane formed a distinct modified layer on both its surface and cross-section. This suggests that lysozyme can form a co-deposited layer on the membrane surface through oxidative self-polymerization with L-DOPA. Furthermore, compared to the E-Lys membrane, the lysozyme surface on the E-Lys@Thps membrane exhibited significant changes, further demonstrating that THPS reacted with the lysozyme on the membrane surface via a Mannich reaction, thereby altering the membrane's surface morphology.

[0032] Example 2

[0033] To test the synergistic antibacterial ability of lysozyme and THPS, Escherichia coli and Staphylococcus aureus were selected as test strains for antibacterial testing.

[0034] Lysozyme@Thps solution was prepared by continuously reacting 100 mL of an aqueous solution containing 0.7 mg / mL lysozyme and 1.31 mM THPS for 7 hours at room temperature. Then, 1 mL of this solution was added dropwise to a glass slide and dried in a vacuum oven at 40°C for 24 hours. After drying, 20 μL of bacterial culture (including OD) was... 600 =0.01 Escherichia coli and Staphylococcus aureus were added to glass slides and air-dried. Then, they were soaked in 2.5wt% glutaraldehyde at 2-8 °C for 4 hours and dried in a vacuum drying oven at 40 °C for 24 hours.

[0035] As a comparison, at room temperature, 1 mL of an aqueous solution containing 0.7 mg / mL lysozyme was added dropwise onto a glass slide and dried in a vacuum drying oven at 40 °C for 24 hours. After drying, 20 μL of bacterial suspension (including OD) was... 600 =0.01 Escherichia coli and Staphylococcus aureus were added to glass slides and air-dried. Then, they were soaked in 2.5wt% glutaraldehyde at 2-8 °C for 4 hours and dried in a vacuum drying oven at 40 °C for 24 hours.

[0036] Its antibacterial properties were examined using scanning electron microscopy (SEM), and the results are as follows: Figure 4 As shown in the figure, both lysozyme and Lysozyme@Thps exhibited good antibacterial properties against Staphylococcus aureus. However, due to the protective lipopolysaccharide in its cell wall, lysozyme was insensitive to Escherichia coli. In contrast, Lysozyme@Thps showed a significantly enhanced antibacterial effect against Escherichia coli. This indicates that THPS can effectively compensate for the insensitivity of lysozyme to Escherichia coli, achieving a synergistic antibacterial effect.

[0037] Example 3

[0038] To evaluate the antimicrobial growth ability of the E-Lys@Thps membrane prepared in Example 1, the optical density (OD) value after contact between the bacterial solution and the membrane was tested and monitored by SEM images. All test membranes were cut into 1.5 cm × 1.5 cm squares, and all samples were completely sterilized. All experiments were performed on a sterile workbench to prevent interference from external bacteria. *Escherichia coli* and *Staphylococcus aureus* were used as test bacteria, and their OD values ​​(Synergy LX, BioTek, USA, UV at 600 nm) were diluted to 0.1 with LB buffer. After thorough mixing, the supernatant was collected, and its OD value was measured. Each sample was immersed in a 12-well plate containing 1 mL of bacterial solution, and its OD value was measured at 12, 24, and 36 hours. The results are shown below. Figure 5 As shown in Figure a. After bacterial culture for 36 hours, the test membrane was removed and immersed in glutaraldehyde (2.5 wt%) buffer at 4°C for 3 hours, followed by drying in a vacuum oven at 40°C for 24 hours. The morphology of biofilms on pristine, E-Lys, and E-Lys@Thps membranes was monitored using SEM images (SU8100, Hitachi, Japan). The results are as follows. Figure 5 b-5g, E-Lys@Thps not only exhibits a perfect anti-biofilm effect, but also does not cause biofouling of the membrane.

[0039] Example 4

[0040] To evaluate the anti-biofilm ability of the E-Lys@Thps membrane prepared in Example 1, confocal laser scanning microscopy (CLSM, Axioscope 5, Zeiss, American) was used. All test membranes were cut into 1.5 cm × 1.5 cm squares and were completely sterilized. *Escherichia coli* and *Staphylococcus aureus* were used as test bacteria, and their OD values ​​were diluted to 0.1 with LB solution. At 37 °C, 2000.0 μL of *E. coli* and *Staphylococcus aureus* were respectively applied to the surface of the test membranes and allowed to remain in contact for 16 hours. Then, SYTO9 and PI dye were added to the *Staphylococcus aureus* and *E. coli* system, and staining was performed in the dark for approximately 30 minutes. After preparing the mixed solution samples, the three-dimensional morphology of the biofilm on the membrane surface was measured using CLSM. The results are as follows: Figure 6 As shown, lysozyme and THPS have good synergistic anti-biofilm properties.

[0041] Example 5

[0042] To determine the antibacterial rate of the E-Lys@Thps membrane prepared in Example 1, fluorescence microscopy (Axioscope 5, Zeiss, USA) was used. *Escherichia coli* and *Staphylococcus aureus* were selected as the test bacteria, and their OD values ​​were diluted to 0.1 with PBS. At 37 °C, 100 μL of the diluted *E. coli* and *S. aureus* were evenly spread on the membrane surface using the same procedure as the LB plate method, allowing for sufficient contact for 1 hour. Then, SYTO9 and PI dye were added to the *S. aureus* and *E. coli* system, and staining was performed in the dark for approximately 10 minutes. After the mixed solution sample was prepared, live and dead bacteria were distinguished using fluorescence microscopy. The results are as follows: Figure 7 As shown, the prepared E-Lys@Thps membrane has good antibacterial properties.

[0043] Example 6

[0044] To test the separation performance of the E-Lys@Thps membrane prepared in Example 1, electrodialysis (ED) desalting was performed on a laboratory-made apparatus. The laboratory-made apparatus, its schematic diagram, and experimental procedures have been described in previous studies, specifically in the reference [J. Liao, J. Zhu, S. Yang, N. Pan, X. Yu, C. Wang, J. Li, Jiangnan Shen. Long-side-chain type imidazolium-functionalized fluoro-methyl poly(aryleneether ketone) anion exchange membranes with superior electrodialysis performance. Journal of Membrane Science. 574, 2019, 181-195.]. 80 mL of 0.5 M sodium chloride solution was injected into each of the two dilution chambers and the concentration chamber of the homemade apparatus, and 0.3 M sodium sulfate solution was pumped into the electrode cell. Circulation was maintained during desalting, and the conductivity of the dilution and concentration chambers was measured every 10 min using a conductivity meter. After the ED process was completed, the remaining solution volumes in the concentration and dilution chambers were measured. The electrodes at both ends of the device are connected to an external power supply, and a constant current of 0.30 A (constant current density of 15.28 mA / cm²) is applied. 2 The result is as follows: Figure 8 As shown, the modified E-Lys@Thps membrane exhibits excellent desalination performance.

Claims

1. A method for modifying anion exchange membranes with lysozyme and THPS, wherein THPS refers to tetramethylolphosphine sulfate, characterized in that: The method includes the following steps: Step 1: Immerse the commercial anion exchange membrane sequentially in sodium hydroxide solution and sodium chloride solution, then wash and dry it with deionized water to complete the pretreatment of the original membrane; Step 2: At room temperature, the original membrane treated in Step 1 is installed in the modification device, and reaction solution ① is added to the feed chamber of the modification device so that one side of the original membrane is in contact with reaction solution ①. The reaction solution ① is composed of tris(hydroxymethyl)aminomethane hydrochloride buffer containing 9.00-11.00 mM levonorgestrel and 0.5-1 mg / mL lysozyme. The pH of the tris(hydroxymethyl)aminomethane hydrochloride buffer is between 8.0 and 9.

0. The mixture is stirred at room temperature and reacted for 10-14 hours. After the reaction is completed, the reaction solution is poured off and thoroughly washed with deionized water. The resulting membrane is named E-Lys membrane. Step 3: At room temperature, add reaction solution ② to the feed chamber and bring the modified surface of the E-Lys membrane into contact with the reaction solution ② to carry out a Mannich-type reaction. The reaction solution ② is an aqueous solution of 1.0-1.5 mM THPS. The reaction is carried out continuously at room temperature for 6-8 hours. After the reaction is complete, pour out the reaction solution, take out the modified membrane, rinse it with deionized water, and name it E-Lys@Thps membrane.

2. The method for modifying anion exchange membranes with lysozyme and THPS as described in claim 1, characterized in that: In step 2, the pH of the tris(hydroxymethyl)aminomethane hydrochloride buffer solution is controlled between 8.3 and 8.

7.

3. The method for modifying anion exchange membranes with lysozyme and THPS as described in claim 1, characterized in that: In step 2, the reaction time is controlled at 12 hours.

4. The method for modifying anion exchange membranes with lysozyme and THPS as described in claim 1, characterized in that: In step 2, the content of L-dopamine in the reaction solution ① is 10.01 mM.

5. The method for modifying anion exchange membranes with lysozyme and THPS as described in claim 1, characterized in that: In step 2, the concentration of lysozyme in the reaction solution ① is 0.7 mg / mL.

6. The method for modifying anion exchange membranes with lysozyme and THPS as described in claim 1, characterized in that: In step 3, the reaction time is controlled at 7 hours.

7. The method for modifying anion exchange membranes with lysozyme and THPS as described in claim 1, characterized in that: In step 3, the THPS content in the reaction solution ② is 1.31 mM.

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