Aaquaporin bionic nanofiltration membrane as well as preparation method and application thereof

Abstract

The invention relates to an aquaporin bionic nanofiltration membrane and a preparation method and application thereof.The aquaporin bionic nanofiltration membrane is composed of a cell sheet rich in aquaporin and a porous base membrane, and the cell sheet rich in aquaporin serves as a functional unit; through a vacuum filtration method, layer-by-layer assembly and cross-linking curing, the bonding firmness in the active layer is improved. The preparation method disclosed by the invention is simple in operation process, the embedding amount of the aquaporin in the nanofiltration membrane is increased, and the biological activity of the aquaporin is ensured. The aquaporin bionic nanofiltration membrane disclosed by the invention is high in stability and excellent in permeation selectivity, and has a wide application prospect in the field of membrane separation.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaporin biomimetic nanofiltration membranes, and particularly relates to an aquaporin biomimetic nanofiltration membrane, a preparation method thereof, and an application thereof. A cell membrane sheet rich in aquaporins is used as a functional separation unit for separation and concentration in the fields of sewage treatment, biology, medicine, electronics, food, etc. Background Art

[0002] Aquaporin is a transmembrane protein with specific water molecule transport channels, which can enable rapid water molecule transport while completely excluding other molecules, ions, and protons. In recent years, researchers have carried out a large amount of work on the development of aquaporin biomimetic membranes. Generally, aquaporins are embedded in lipids or block copolymer vesicles to simulate the cell membrane microenvironment required for the functional activity of aquaporin membrane proteins. Then, vesicles rich in aquaporins are spread after adsorption, rupture, and fusion, or vesicles rich in aquaporins are encapsulated on the surface of the substrate membrane to form an aquaporin functional layer, thereby preparing an aquaporin biomimetic separation membrane. See the literature Advanced Science, 2021, 8(20): e2101882; Journal of Membrane Science, 2024, 697: 122578; Journal of Membrane Science, 2016, 508: 94 - 103; Journal of Materials Chemistry A, 2015, 3(40): 20118 - 20126; Environmental Science & Technology, 2015, 49(6): 3761 - 3768 and patents CN201880010493.3, CN201780010287.8, CN202022853770.4, CN201880068439.4, CN201511008292.4. However, there are still problems such as difficult to guarantee the biological activity of aquaporins and limited loading amount, resulting in the water permeability and selectivity of the membrane being difficult to reach the ideal expectation.

[0003] Currently, using the cell membrane sheet of Escherichia coli with inherent nanochannels as the membrane material, a cell membrane sheet biomimetic nanofiltration membrane with high dye and antibiotic rejection rates and high water permeability coefficients can be prepared. See the literature Journal of Membrane Science, 2024, 697: 122578. However, the content of functional proteins on the cell membrane sheet of Escherichia coli is relatively low, resulting in challenges in significantly improving the permeation selectivity of the biomimetic nanofiltration membrane. Summary of the Invention

[0004] To solve the problems existing in the above-mentioned prior art, the present invention provides a new method for preparing an aquaporin biomimetic nanofiltration membrane, that is, a biomimetic nanofiltration membrane is prepared by using a cell membrane sheet rich in aquaporin and a porous substrate membrane. The cell membrane sheet serves as a functional unit, and its binding firmness in the active layer is improved through layer-by-layer assembly and cross-linking curing. The operation process of the preparation method is simple, which increases the embedding amount of aquaporin in the nanofiltration membrane and ensures its biological activity. The nanofiltration membrane has good permeation selectivity and high stability, and has broad application prospects in the field of membrane separation.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: A method for preparing an aquaporin biomimetic nanofiltration membrane, comprising the following steps: (1) Escherichia coli cells rich in aquaporin are ultrasonically lysed using an ultrasonic cell disruptor, and then the cell membrane sheet rich in aquaporin is obtained after centrifugal purification; (2) A porous substrate membrane with a positively charged surface is prepared by using the mussel biomimetic co-deposition method; (3) The cell membrane sheet rich in aquaporin is layer-by-layer assembled and cross-linked and cured on the surface of the porous substrate membrane by vacuum filtration to obtain an aquaporin biomimetic nanofiltration membrane.

[0006] Further, the working conditions of the ultrasonic cell disruptor in step (1) are as follows: the ultrasonic intensity is 100 - 200 W, 1 s of ultrasonic treatment and 3 s of pause form a cycle, and Escherichia coli cells rich in aquaporin are lysed for 10 - 40 min. During this process, the cells are kept in an ice bath. Subsequently, centrifugation is carried out at 5000 rpm and 4 °C for 10 - 30 min, and the Escherichia coli cell membrane sheet is recovered from the supernatant. Finally, the cell membrane sheet is centrifugally purified using a microfiltration substrate membrane with a pore size in the range of 0.1 - 10.0 microns to obtain cell membrane sheets rich in aquaporin with different sizes; all preparation processes are carried out at 4 °C or on ice.

[0007] Further, the preparation method of Escherichia coli cells rich in aquaporin in step (1) is to express aquaporin on the Escherichia coli cell membrane sheet. Expressing aquaporin on the Escherichia coli cell membrane sheet requires four steps: transformation, picking colonies, inoculation, and induction culture. (a) Transformation: First, add 2 μL of pET-52b(+)-His-AQP plasmid (Ampicillin sodium Amp resistance) solution to 100 μL of competent cells, mix well, and then perform 7 min of ice bath, 70 s of heat shock in a 42 °C water bath, and 7 min of ice bath in sequence. Subsequently, add it to 200 μL of LB liquid medium and incubate and activate it in an oscillator at a temperature of 37 °C and a rotation speed of 150 rpm for 1 h. Then, in a medium containing 50 μg mL -1Plate on the LB solid medium of Amp and incubate overnight in an electrothermal constant temperature incubator at 37°C to prepare monoclonal antibodies against aquaporin. Among them, the competent cells include BL21(DE3), Rosetta(DE3), C43(DE3), and C41(DE3). The composition of the LB liquid medium is 5 g of yeast extract powder, 10 g of tryptone, and 10 g of NaCl in 1 L. The composition of the LB solid medium is 5 g of yeast extract powder, 10 g of tryptone, 10 g of NaCl, and 15 g of agar in 1 L. (b) Pick colonies: Pick colonies and place them in 20 mL of LB liquid medium containing 50 μg / mL -1 Amp and grow in a shaker at 37°C for 12 h. (c) Inoculation: Dilute the above bacterial solution 100-fold in 2 L of TB liquid medium containing 50 μg / mL -1 Amp and continue to grow in a shaker at 37°C for 2 - 3 h until the cell density OD 600 nm = 0.6 - 1.0. Among them, the composition of the TB liquid medium is 47 g of TB medium and 4 mL of glycerol in 1 L. (d) Induction culture: Add IPTG (0.1 - 1.0 mM) to the above solution and continue to culture in a shaker to obtain Escherichia coli cells rich in aquaporin. The culture conditions are 37°C for 4 h or 16°C for 12 h.

[0008] Further, the mussel-inspired co-deposition method in step (2) is to mix an aqueous solution of tea polyphenol derivatives and an aqueous solution of polyethyleneimine at a volume ratio of 1:1 and co-deposit them on the surface of the microfiltration membrane at room temperature for 0.5 - 2 h. Among them, the tea polyphenol derivatives and polyethyleneimine form a complex through Michael addition and Schiff base reactions, and migrate and deposit towards the gas / liquid or liquid / solid interface. Then, thoroughly wash the membrane surface with deionized water to remove unbound reagents, and obtain a positively charged porous microfiltration membrane. -1 Mix an aqueous solution of tea polyphenol derivatives and an aqueous solution of polyethyleneimine at a concentration of 0.5 - 2.0 mg / mL -1 at a volume ratio of 1:1 and co-deposit them on the surface of the microfiltration membrane at room temperature for 0.5 - 2 h. Among them, the tea polyphenol derivatives and polyethyleneimine form a complex through Michael addition and Schiff base reactions, and migrate and deposit towards the gas / liquid or liquid / solid interface. Then, thoroughly wash the membrane surface with deionized water to remove unbound reagents, and obtain a positively charged porous microfiltration membrane.

[0009] Further, the molecular weight of the polyethyleneimine is 600 - 10000 Da. The tea polyphenol derivatives are organic molecules containing a dihydroxyphenyl moiety and its derivatives, including anthocyanins, dopamine, flavanols, tannic acid, gallic acid, flavonols, flavonoids, flavanones, isoflavones, chalcones, resveratrol, gallic acid, lignans, ferulic acid, xanthones.

[0010] Further, the microfiltration base membrane in the step (2) includes cellulose acetate membrane, nitrocellulose membrane, mixed ester membrane, regenerated cellulose membrane, polyethersulfone membrane, polytetrafluoroethylene membrane, polyvinylidene fluoride membrane, polyvinylidene fluoride membrane, nylon membrane, and ceramic membrane.

[0011] Further, in the step (3), by the vacuum filtration method, a solution of 0.32 - 1.60 mg of cell membrane sheets rich in aquaporin is deposited on the surface of the porous base membrane prepared in the step (2) under a pressure of 0.1 - 1.0 bar, and then the cell membrane sheets rich in aquaporin are cross-linked for 25 min with a cross-linking agent of 0.5 - 2.0 wt.%, to obtain an aquaporin biomimetic nanofiltration membrane.

[0012] Further, the cross-linking agent used in the step (3) is a molecule containing no less than two functional groups, and the two or more functional groups are the same or different functional groups, and the functional groups are amino group, carboxyl group, hydroxyl group, acyl chloride, aldehyde group, N-hydroxysuccinimide ester, maleic anhydride, epoxy group, and siloxane; common cross-linking agents include glutaraldehyde, paraformaldehyde, N-[α-maleimidylacetoxy] succinimide ester, bis-[2-(succinimidyloxycarbonyloxy)ethyl] sulfone, 5(bis-N-succinimidyl[pentylene glycol] ester), disuccinimidyl glutarate, disuccinimidyl suberate, and γ-glycidoxypropyltrimethoxysilane.

[0013] The present invention also provides an aquaporin biomimetic nanofiltration membrane prepared by the preparation method described above.

[0014] The present invention also provides an application of the aquaporin biomimetic nanofiltration membrane. The aquaporin biomimetic nanofiltration membrane is widely used in the separation and concentration fields of sewage treatment, biology, medicine, electronics, and food, and can efficiently permeate water molecules and efficiently retain salt ions and dye molecules.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. By using genetic engineering means, the present invention expresses aquaporin on Escherichia coli cells, greatly increasing the expression level of aquaporin.

[0016] 2. During the assembly process, directly using cell membrane sheets rich in aquaporin as the membrane-making material is expected to simplify the membrane-making process, and is beneficial to maintaining the biological activity of aquaporin and increasing the loading amount of aquaporin on the membrane.

[0017] 3. During the assembly process, cross-linking improves the binding degree and stability between the cell membrane sheets rich in functional proteins and the porous base membrane.

[0018] 4. The interlayer nanochannels formed by stacked cell membranes and the specific water transport channels inherent in the cell membranes rich in aquaporins endow the membrane with excellent permeation selectivity.

[0019] 5. The method of the present invention uses the layer-by-layer assembly method. The process of introducing the assembly units is simple and is not restricted by the substrate material, expanding the selection of the substrate membrane materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 Measuring the size and Zeta potential of the cell membranes rich in aquaporins dispersed in PBS buffer using a nano-particle size and Zeta potential analyzer (a); Atomic force microscope image of the cell membranes rich in aquaporins (b) and the corresponding membrane height profile (c).

[0022] Figure 2 Scanning electron microscope images of the surface (a) and cross-section (b) of the base membrane prepared by mussel-inspired co-deposition and the aquaporin-based biomimetic nanofiltration membrane.

[0023] Figure 3 Attenuated total reflection-Fourier transform infrared spectra (a) and Zeta potential (b) of the base membrane prepared by mussel-inspired co-deposition and the aquaporin-based biomimetic nanofiltration membrane.

[0024] Figure 4 Pure water permeability coefficient and rejection rates for different salts of the aquaporin-based biomimetic nanofiltration membrane prepared in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The following will further illustrate the present invention in combination with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the above invention.

[0026] Example 1 This example is for the preparation of Escherichia coli cells rich in aquaporins, which is specifically as follows: The preparation method of Escherichia coli cells rich in aquaporin is to express aquaporin on the Escherichia coli cell membrane sheet. Expressing aquaporin on the Escherichia coli cell membrane sheet requires four steps: transformation, picking clones, inoculation, and induction culture. (a) Transformation: First, add 2 μL of pET-52b(+)-His-AQP plasmid (Ampicillin sodium Amp resistance) solution to 100 μL of competent cells (including but not limited to BL21(DE3), Rosetta(DE3), C43(DE3), C41(DE3), etc.). After mixing, perform 7 min ice bath, 42 °C water bath heat shock for 70 s, and 7 min ice bath in sequence. Then add it to 200 μL of LB liquid medium (1 L: yeast extract powder 5 g, tryptone 10 g, and NaCl 10 g) and incubate and activate it in an oscillator at a temperature of 37 °C and a rotation speed of 150 rpm for 1 h. Then plate it on LB solid medium (1 L: yeast extract powder 5 g, tryptone 10 g, NaCl 10 g, and agar 15 g) containing 50 μg mL -1 Amp and culture it overnight in a 37 °C electrothermal constant temperature incubator to prepare monoclonal antibodies of aquaporin. (b) Picking clones: Pick the clones and place them in 20 mL of LB liquid medium containing 50 μgmL -1 Amp and grow them in a 37 °C oscillator for 12 h. (c) Inoculation: Dilute the above bacterial solution 100-fold in 2 L of TB liquid medium (1 L: TB medium 47 g and 4 mL glycerol) containing 50 μg mL -1 Amp and continue to grow it in a 37 °C oscillator for 2 - 3 h until the cell density OD 600 nm = 0.6 - 1.0. (d) Induction culture: Add IPTG (0.1 - 1.0 mM) to the above solution and continue to culture it in an oscillator (37 °C, 4 h or 16 °C, 12 h) to obtain Escherichia coli cells rich in aquaporin.

[0027] Example 2 This example is a preparation method of an aquaporin biomimetic nanofiltration membrane, which is specifically as follows: (1) Using the genetic engineering technology in Example 1, the gene of aquaporin was introduced into the cell membrane of Escherichia coli to increase the expression level of aquaporin. Among them, the competent cells used were BL21(DE3), the IPTG concentration was 0.5 mM, and the IPTG induction culture condition was incubation at 37 °C for 4 h. Other methods were the same as in Example 1. Subsequently, the Escherichia coli rich in aquaporin was broken by an ultrasonic crusher. The working conditions of the ultrasonic cell crusher were an ultrasonic intensity of 100 W and a cell lysis time of 10 min. Subsequently, centrifugation was carried out at 5000 rpm and 4 °C for 10 min, and Escherichia coli cell membrane sheets with a size of 1100 nm were recovered from the supernatant. As Figure 1 shown in the size, atomic force microscope image and the corresponding membrane height profile of the cell membrane sheet rich in aquaporin, it can be observed that the size of the cell membrane sheet rich in aquaporin is 1081 nm ( Figure 1 a) and the thickness is 5 nm ( Figure 1 b and 1c); all preparation processes were carried out at 4 °C or on ice.

[0028] (2) A 2.0 mg mL -1 aqueous dopamine solution and a 2.0 mg mL -1 aqueous polyethyleneimine solution were mixed at a volume ratio of 1:1 and co-deposited on the surface of a 0.45 μm polyethersulfone microfiltration substrate membrane at room temperature for 2 h. Dopamine and polyethyleneimine reacted to form a complex through Michael addition and Schiff base reactions and migrated and deposited to the gas / liquid or liquid / solid interface; then the membrane surface was thoroughly washed with deionized water to remove unbound reagents, and a positively charged porous polyethersulfone microfiltration substrate membrane was prepared. It can be seen from the characterization results of the scanning electron microscope that the positively charged porous polyethersulfone microfiltration substrate membrane presents a macroporous structure ( Figure 2 a). Due to the co-deposition of dopamine and polyethyleneimine on the surface of the polyethersulfone microfiltration substrate membrane, the potential of the prepared substrate membrane is positive at pH = 7 ( Figure 3 b).

[0029] (3) The 0.56 mg Escherichia coli cell membrane sheets collected in step (1) were redispersed in PBS buffer and vacuum filtered onto the porous substrate membrane at a pressure of 0.5 bar. At the same time, a 1.0 wt.% glutaraldehyde solution was used for cross-linking fixation for 25 min to increase the binding firmness between cell membrane sheets and between cell membrane sheets and the porous substrate membrane, and an aquaporin biomimetic nanofiltration membrane was obtained. Among them, the equation for the Schiff base reaction between the primary amino group in aquaporin and the glutaraldehyde aqueous solution is as follows: P-NH2+OHC-R→P-N=CH-R+H2O. It can be seen from the characterization results of the scanning electron microscope that the surface of the biomimetic membrane loaded with cell membrane sheets rich in aquaporin is uniform and has no obvious defects ( Figure 2b). In addition, strong absorption peaks of -C=O- and -NH- groups were observed at 1660 cm -1 and 1540 cm -1 in the attenuated total reflection-Fourier transform infrared spectrum of the membrane ( Figure 3 a). In addition, as the negatively charged Escherichia coli cell membrane rich in aquaporin was deposited on the surface of the positively charged polyethersulfone microfiltration substrate membrane, the potential of the aquaporin biomimetic nanofiltration membrane changed from positive to negative ( Figure 3 b). In summary, the Escherichia coli cell membrane rich in aquaporin was successfully deposited on the surface of the positively charged porous polyethersulfone microfiltration substrate membrane.

[0030] When the composite membrane prepared in this example was used to treat an aqueous sodium sulfate solution containing 1000 ppm, the experimental pressure was 1 bar, the rejection rate of sodium sulfate was greater than 76%, and the water flux was greater than 68 LMH bar -1 ( Figure 4 ). In addition, from the test results of the separation performance of the aquaporin biomimetic nanofiltration membrane, the rejection rate order of the membrane for different inorganic salts is as follows: sodium sulfate (75.5%) > magnesium sulfate (45.7%) > sodium chloride (38.9%) > magnesium chloride (30.2%). This is a typical separation characteristic of a negatively charged nanofiltration membrane based on the synergistic effect of size sieving and Donnan effect. Among them, the rejection rate of the membrane for NaCl is higher than that for MgCl2 because the positive charge density of Mg 2+ is high, making the negatively charged membrane more attractive to divalent cations, resulting in divalent cations being more likely to permeate through the membrane.

[0031] Example 3 This example is a preparation method of an aquaporin biomimetic nanofiltration membrane, which is specifically as follows: (1) Using the genetic engineering technology in Example 1, the gene of aquaporin was introduced into the Escherichia coli cell membrane to increase the expression level of aquaporin. Among them, the competent cells used were C43(DE3), the IPTG concentration was 0.5 mM, and the IPTG induction culture condition was incubation at 16 °C for 12 h. Other methods were the same as in Example 1. Subsequently, the Escherichia coli rich in aquaporin was broken by an ultrasonic crusher. The working conditions of the ultrasonic cell crusher were an ultrasonic intensity of 100 W and a cell lysis time of 20 min. Subsequently, centrifugation was carried out at 5000 rpm and 4 °C for 10 min, and Escherichia coli cell membrane sheets with a size of 540 nanometers were recovered from the supernatant.

[0032] (2) Using a 2.0 mg mL -1 tannic acid aqueous solution and a 2.0 mg mL -1Polyethyleneimine dopamine was mixed at a volume ratio of 1:1 and co-deposited on the surface of a 0.22-μm polyethersulfone microfiltration substrate membrane at room temperature for 1 h to prepare a positively charged porous polyethersulfone microfiltration substrate membrane.

[0033] (3)The 0.48 mg of Escherichia coli cell membrane sheets collected in step (1) were redispersed in PBS buffer and vacuum filtered onto the porous polyethersulfone microfiltration substrate membrane under a pressure of 0.4 bar. Meanwhile, a 1.0 wt.% paraformaldehyde solution was used for crosslinking fixation for 25 min to increase the binding firmness between the cell membrane sheets and between the cell membrane sheets and the porous substrate membrane, obtaining an aquaporin biomimetic nanofiltration membrane.

[0034] The aquaporin biomimetic nanofiltration membrane prepared in this example was used to treat an aqueous solution containing 100 ppm of dye (brilliant blue R). The experimental pressure was 1 bar, and the rejection rate of brilliant blue R was greater than 99.8%, and the water flux was greater than 142 LMH bar -1 .

[0035] Example 4 This example is a preparation method of an aquaporin biomimetic nanofiltration membrane, and the specific method is as follows: (1)The gene of aquaporin was introduced into the Escherichia coli cell membrane by using the genetic engineering technology in Example 1 to increase the expression level of aquaporin. Among them, the competent cells used were Rosetta (DE3), the IPTG concentration was 0.3 mM, and the IPTG induction culture condition was incubation at 16 °C for 12 h. Other methods were the same as in Example 1. Subsequently, the Escherichia coli rich in aquaporin was broken by an ultrasonic crusher. The working conditions of the ultrasonic cell crusher were an ultrasonic intensity of 150 W and a cell lysis time of 20 min. Subsequently, centrifugation was carried out at 5000 rpm and 4 °C for 20 min, and Escherichia coli cell membrane sheets with a size of 200 nm were recovered from the supernatant.

[0036] (2)A 2.0 mg mL -1 aqueous solution of gallic acid and a 1.0 mg mL -1 aqueous solution of polyethyleneimine were mixed at a volume ratio of 1:1 and co-deposited on the surface of a 0.10-μm polyvinylidene fluoride microfiltration substrate membrane at room temperature for 0.5 h to prepare a positively charged porous polyvinylidene fluoride microfiltration substrate membrane.

[0037] (3)The collected 0.80 mg of cell sheets were redispersed in PBS buffer and vacuum filtered onto the porous polyvinylidene fluoride microfiltration substrate membrane under a pressure of 0.7 bar. Meanwhile, a 2.0 wt.% glutaraldehyde solution was used for crosslinking fixation for 25 min to increase the binding firmness between the cell membrane sheets and between the cell membrane sheets and the porous substrate membrane, obtaining an aquaporin biomimetic nanofiltration membrane.

[0038] The prepared nanofiltration membrane was used to treat an aqueous solution containing 1000 ppm of sodium sulfate. The experimental pressure was 1 bar, the rejection rate of sodium sulfate was greater than 72%, and the water flux was greater than 34 LMH bar -1 。

[0039] Example 5 This example is a preparation method of an aquaporin biomimetic nanofiltration membrane, which is specifically as follows: (1)Using the genetic engineering technology in Example 1, the gene of aquaporin was introduced into the cell membrane of Escherichia coli to increase the expression level of aquaporin. Among them, the competent cells used were Rosetta (DE3), the IPTG concentration was 0.7 mM, and the IPTG induction culture condition was incubation at 37 °C for 4 h. Other methods were the same as in Example 1. Subsequently, the Escherichia coli rich in aquaporin was broken by an ultrasonic crusher. The working conditions of the ultrasonic cell crusher were an ultrasonic intensity of 100 W and a cell lysis time of 20 min. Subsequently, centrifugation was carried out at 5000 rpm and 4 °C for 10 min, and Escherichia coli cell membrane sheets with a size of 540 nanometers were recovered from the supernatant.

[0040] (2)A dopamine aqueous solution of 2.0 mg mL -1 and a polyethyleneimine aqueous solution of 1.0 mg mL -1 were mixed at a volume ratio of 1:1 and co-deposited on the surface of a 0.22-micron cellulose acetate microfiltration substrate membrane at room temperature for 1.5 h to prepare a positively charged porous cellulose acetate microfiltration substrate membrane.

[0041] (3)The 0.32 mg of Escherichia coli cell membrane sheets collected in step (1) were redispersed in PBS buffer and vacuum filtered onto the porous cellulose acetate microfiltration substrate membrane under a pressure of 0.5 bar. At the same time, a solution of bis-[2-(succinimidyloxycarbonyloxy)ethyl] sulfone with a concentration of 1.0% was used for cross-linking and fixation for 25 min to increase the binding firmness between the cell membrane sheets and between the cell membrane sheets and the porous substrate membrane, and an aquaporin biomimetic nanofiltration membrane was obtained.

[0042] The aquaporin biomimetic nanofiltration membrane prepared in this example was used to treat an aqueous solution containing 100 ppm of dye (Congo red). The experimental pressure was 1 bar, the rejection rate of Congo red was greater than 99.8%, and the water flux was greater than 117 LMH bar -1 。

[0043] Example 6 This example is a preparation method of an aquaporin biomimetic nanofiltration membrane, which is specifically as follows: (1) Using the genetic engineering technology in Example 1, the gene of aquaporin was introduced into the cell membrane of Escherichia coli to increase the expression level of aquaporin. Among them, the competent cells used were BL21(DE3), the IPTG concentration was 0.5 mM, and the IPTG induction culture condition was incubation at 16 °C for 12 h. Other methods were the same as in Example 1. Subsequently, the Escherichia coli rich in aquaporin was broken by an ultrasonic crusher. The working conditions of the ultrasonic cell crusher were an ultrasonic intensity of 200 W and a cell lysis time of 10 min. Subsequently, centrifugation was carried out at 5000 rpm and 4 °C for 10 min, and Escherichia coli cell membrane sheets with a size of 540 nm were recovered from the supernatant. (2) Using 1.0 mg mL -1 tannic acid and 2.0 mg mL -1 polyethylenimine were mixed at a volume ratio of 1:1 and co-deposited on the surface of a 0.10-μm nylon microfiltration substrate membrane at room temperature for 2.0 h to prepare a positively charged porous nylon microfiltration substrate membrane.

[0044] (3) The 0.64 mg of Escherichia coli cell membrane sheets collected in step (1) were redispersed in PBS buffer and vacuum filtered onto the porous nylon microfiltration substrate membrane under a pressure of 1.0 bar. At the same time, cross-linking fixation was carried out for 25 min using bis[succinimidyl]suberate with a concentration of 1.0 wt.%, to increase the binding firmness between cells and between cells and the porous substrate membrane, and an aquaporin biomimetic nanofiltration membrane was obtained.

[0045] Using the aquaporin biomimetic nanofiltration membrane prepared in this example to treat an aqueous solution containing 100 ppm of dye (Congo red), the experimental pressure was 1 bar, the rejection rate of Congo red was greater than 99.8%, and the water flux was greater than 51 LMH bar -1 .

[0046] Example 7 This example is a preparation method of an aquaporin biomimetic nanofiltration membrane, which is specifically as follows: (1) Using the genetic engineering technology in Example 1, the gene of aquaporin was introduced into the cell membrane of Escherichia coli to increase the expression level of aquaporin. Among them, the competent cells used were C41(DE3), the IPTG concentration was 0.7 mM, and the IPTG induction culture condition was incubation at 37 °C for 4 h. Other methods were the same as in Example 1. Subsequently, the Escherichia coli rich in aquaporin was broken by an ultrasonic crusher. The working conditions of the ultrasonic cell crusher were an ultrasonic intensity of 100 W and a cell lysis time of 30 min. Subsequently, centrifugation was carried out at 5000 rpm and 4 °C for 30 min, and Escherichia coli cell membrane sheets with a size of 300 nm were recovered from the supernatant.

[0047] (2) Using 1.0 mg mL-1 The dopamine aqueous solution and 2.0 mg mL -1 The positively charged porous ceramic microfiltration substrate membrane was prepared by co-depositing the dopamine aqueous solution and the polyethyleneimine aqueous solution at a volume ratio of 1:1 on the surface of a 0.20-μm ceramic microfiltration substrate membrane at room temperature for 1.5 h.

[0048] (3) The 1.60 mg of Escherichia coli cell membrane sheets collected in step (1) were redispersed in PBS buffer and vacuum filtered onto the porous ceramic microfiltration substrate membrane under a pressure of 0.3 bar. Meanwhile, a glutaraldehyde solution with a concentration of 1.5 wt.% was used for cross-linking and fixation for 25 min to increase the binding firmness between the cell membrane sheets and between the cell membrane sheets and the porous substrate membrane, obtaining an aquaporin biomimetic nanofiltration membrane.

[0049] The aquaporin biomimetic nanofiltration membrane prepared in this example was used to treat an aqueous solution containing 1000 ppm of sodium sulfate. The experimental pressure was 1 bar, the rejection rate of sodium sulfate was greater than 70%, and the water flux was greater than 68 LMH bar -1 .

[0050] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification are only used to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a water channel protein biomimetic nanofiltration membrane, characterized in that: The following steps are involved: (1) Escherichia coli cells rich in aquaporins were ultrasonically lysed using an ultrasonic cell disruptor, and then centrifuged and purified to obtain cell membrane sheets rich in aquaporins; (2) Preparation of porous substrate membranes with positive surface charge using mussel biomimetic co-deposition method; (3) Cell membrane sheets rich in water channel proteins were assembled layer by layer and cross-linked and solidified on the surface of the porous basement membrane by vacuum filtration to produce a water channel protein biomimetic nanofiltration membrane.

2. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 1, wherein: In the step (1), the ultrasonic cell pulverizer operates under the ultrasonic intensity of 100-200 W, with a 1 s ultrasonic cycle followed by a 3 s pause, and the aquaporin-rich Escherichia coli cells are lysed for 10-40 min. During this process, the cells are kept in an ice bath, and then centrifuged at 5000 rpm and 4°C for 10-30 min to recover the Escherichia coli cell membrane sheets from the supernatant. Finally, the cell membrane sheets are centrifuged and purified using a microfiltration base membrane with a pore size in the range of 0.1-10.0 μm to obtain aquaporin-rich cell membrane sheets of different sizes. All preparation processes are performed at 4°C or on ice.

3. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 2, wherein: The preparation method of the Escherichia coli cells rich in aquaporin in step (1) is to express aquaporin on the Escherichia coli cell membrane. The expression of aquaporin on the Escherichia coli cell membrane requires four steps: transformation, cloning, inoculation and induction culture. (a) Transformation: First, 2 μL of pET-52b(+)-His-AQP plasmid solution was added to 100 μL of competent cells, mixed and then subjected to ice bath for 7 min, heat shock in 42°C water bath for 70 s and ice bath for 7 min, then added to 200 μL of LB liquid culture medium and cultured in an oscillator at 37°C and 150 rpm for 1 h for incubation activation, and then incubated in a 50 μg / mL -1 Amp was plated on LB solid medium and cultured overnight in a 37°C electric constant temperature incubator to prepare monoclonal antibodies against aquaporin. Competent cells included BL21 (DE3), Rosetta (DE3), C43 (DE3), and C41 (DE3). LB liquid medium consisted of 5 g yeast extract powder, 10 g tryptone, and 10 g NaCl per 1 L. LB solid medium consisted of 5 g yeast extract powder, 10 g tryptone, 10 g NaCl, and 15 g agar per 1 L. (b) Clone picking: Pick the clones and place them in 20 mL of 50 μg mL -1 Amp in LB liquid medium and grown in a shaker at 37 °C for 12 h; (c) Inoculation: The above bacterial solution was added to a solution containing 50 μg mL -1 Dilute the Amp 100-fold in 2 L TB liquid medium and continue growing in a shaker at 37°C for 2-3 h until the cell density OD 600 nm =0.6-1.0; the composition of TB liquid culture medium is 47 g TB culture medium and 4 mL glycerol in 1 L; (d) Induction culture: Add 0.1-1.0 mM IPTG to the above solution and continue culturing on a shaker to obtain E. coli cells rich in aquaporins. The culture conditions are 37°C for 4 h or 16°C for 12 h.

4. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 1, wherein: The mussel biomimetic co-precipitation method in step (2) is to add 0.5-2.0 mg mL -1 Aqueous solution of tea polyphenol derivatives and 0.5-2.0 mg / mL -1 The tea polyphenol derivatives and polyethyleneimine aqueous solution were mixed in a 1:1 volume ratio and co-deposited on the surface of the microfiltration base membrane at room temperature for 0.5-2 hours; wherein, the tea polyphenol derivatives and polyethyleneimine formed a complex through Michael addition and Schiff base reaction, and migrated and deposited to the gas / liquid or liquid / solid interface; then the membrane surface was thoroughly washed with deionized water to remove unbound reagents, thereby obtaining a positively charged porous microfiltration base membrane.

5. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 4, characterized in that: The polyethyleneimine has a molecular weight of 600-10,000 Da; the tea polyphenol derivatives are organic molecules containing a dihydroxyphenyl moiety and its derivatives, including anthocyanidins, dopamine, flavanols, tannic acid, gallic acid, flavonols, flavonoids, flavanones, isoflavones, chalcones, resveratrol, gallic acid, lignans, ferulic acid, and xanthones.

6. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 4, characterized in that: The microfiltration base membrane in step (2) includes cellulose acetate membrane, cellulose nitrate membrane, mixed ester membrane, regenerated cellulose membrane, polyethersulfone membrane, polytetrafluoroethylene membrane, polyvinylidene fluoride membrane, polyvinylidene fluoride membrane, nylon membrane, and ceramic membrane.

7. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 1, characterized in that: In the step (3), 0.32-1.60 mg of the cell membrane solution rich in water channel protein is deposited onto the surface of the porous basement membrane prepared in step (2) at a pressure of 0.1-1.0 bar by vacuum filtration, and then the cell membrane solution rich in water channel protein is cross-linked for 25 minutes using 0.5-2.0 wt.% of a cross-linking agent to prepare a water channel protein biomimetic nanofiltration membrane.

8. The method for preparing the aquaporin biomimetic nanofiltration membrane according to claim 1, characterized in that: The cross-linking agent used in step (3) is a molecule containing not less than two functional groups, wherein the two or more functional groups are the same or different functional groups, and the functional groups are amino, carboxyl, hydroxyl, acyl chloride, aldehyde, N-hydroxysuccinimide ester, maleic anhydride, epoxy and siloxane; commonly used cross-linking agents include glutaraldehyde, paraformaldehyde, N-[α-maleimidoacetate] succinimide ester, bis-[2-(succinimidyloxycarbonyloxy)ethyl] sulfone, 5-(bis-N-succinimidyl [pentanediol] ester), disuccinimidyl glutaric acid, disuccinimidyl suberate and γ-glycidyloxypropyltrimethoxysilane.

9. A water channel protein biomimetic nanofiltration membrane prepared according to any one of the preparation methods of claims 1-8.

10. Use of the aquaporin biomimetic nanofiltration membrane according to claim 9, characterized in that: The aquaporin biomimetic nanofiltration membrane is widely used in the separation and concentration fields of sewage treatment, biology, medicine, electronics and food, and can efficiently pass water molecules and efficiently intercept salt ions and dye molecules.

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