Biomimetic membrane for desalination and water reuse 1

BIOMIMETIC MEMBRANES FOR DESALINATION AND WATER REUSE SUN GUOFEI NATIONAL UNIVERSITY OF SINGAPORE 2013 BIOMIMETIC MEMBRANES FOR DESALINATION AND WATER REUSE SUN GUOFEI (B.Eng (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 i DECLARATION r' B:,:, is I hereby declare that the thesis is my original t:.:: 4l "i: tl work and it has been written by me in its entirefy : I l' ii have duly acknowledged *-r " -e- all the sources of :,il *: F' Fj information which have bsen used in the thesis i i L H r^l tJl I * [ degree in any university previously B^-" :, i.: ' ii: 9t@.> &ii 8i , ::) Sun Guofei i:, a;a E:i =' ! li ':j: a- ,:t */-rE i ACKNOWLEDGEMENTS This dissertation would not have been possible without the help of many people First and foremost, I would like to express my deepest appreciation to my supervisor Prof Chung Tai-Shung, in the Department of Chemical and Biomolecular Engineering at National University of Singapore (NUS), for his invaluable support and enthusiastic encouragement throughout my PhD study I would like to thank him for offering me the opportunity to carry out my research in this special topic, and enlightening me in exploring the academic area I would like to thank my mentor Dr Y Li who guided me through the first year of my PhD He was always approachable whenever I have any doubts in research, and was always patient to help A special thank you to Dr A Armugam (Department of Biochemistry, NUS) for her efforts in producing high quality aquaporins and also her valuable suggestions in protein handling I would also like to thank all the collaborators in this biomimetic membrane project, Prof W Meier (University of Basel), Prof S Hua (The State University of New YorkBuffalo), Prof K Jeyaseelan (Department of Biochemistry, NUS), Prof Q Lin (Department of Biological Sciences, NUS), and Prof Y W Tong (Department of Chemical and Biomolecular engineering, NUS), for their generous supports in this project This project was financially supported by the Environment and Water Industry Programme Office (EWI) (NUS grant number: R-279-000-293-272) under the Singapore National Research Foundation (NRF) ii I wish to take this opportunity to thank Dr H Wang, Dr Z Zhou, Dr S Zhang, Dr W Xie, Dr J.C Su, Dr H Zhou, Ms P.S Zhong, Mr J Yong and all my group members who have helped me in one way or another through the years of my PhD study Finally, I am most grateful to my parents and husband for their unconditional love and support iii TABLE OF CONTENT ACKNOWLEDGEMENTS i TABLE OF CONTENT iii SUMMARY vii LIST OF TABLES x LIST OF FIGURES xi Chapter Introduction 1.1 Membranes for desalination and water treatment 1.1.1 RO, NF and FO 1.1.2 Traditional membranes 1.1.3 New generation membranes 1.2 Aquaporin 1.2.1 The aquaporin family 1.2.2 The aquaporin structure 10 1.2.3 Production of AqpZ 14 1.2.4 Functional characterization 15 1.3 Biomimetic membrane 16 1.3.1 Langmuir-Blodgett technique 18 1.3.2 Vesicle adsorption technique 22 1.3.3 Block copolymer membrane 24 1.4 Biomimetic membrane for water treatment 25 1.4.1 Designs and strategies 25 1.5 Motivation, challenges and objectives 28 Chapter AquaporinZ Incorporation via Binary-Lipid Langmuir Monolayers 30 2.1 Introduction 30 2.2 Materials and methods 31 2.2.1 Materials 31 2.2.2 Surface pressure measurement 31 iv 2.2.3 Surface tension measurement 32 2.2.4 LB film deposition and AFM scanning 33 2.3 Results and discussion 34 2.3.1 DDM effects on DPPC monolayer 34 2.3.2 AqpZ incorporation via DPPC-DOGSNTA monolayers 38 2.4 Conclusion 43 Chapter Stabilization and Immobilization of Aquaporin Reconstituted Lipid Vesicles for Water Purification 45 3.1 Introduction 45 3.2 Materials and methods 47 3.2.1 Materials 47 3.2.2 Preparation of Vesicles 48 3.2.3 Characterization of polymerized vesicles 48 3.2.4 Vesicle permeability measurements 49 3.2.5 Preparation of vesicle immobilized membranes 49 3.2.6 Field-emission scanning electron microscopy (FESEM) 50 3.2.7 Nanofiltration studies 50 3.3 Results and discussion 51 3.3.1 Characterization of polymerized vesicles 51 3.3.2 Vesicle immobilization on PDA coated silicon surface 53 3.3.3 Water permeability of polymerized vesicles 54 3.3.4 Vesicle immobilized nanofiltration membranes 57 3.4 Conclusion 60 Chapter Aquaporin-embedded Mixed Matrix Membrane: A Layer-by-Layer Self-assembly Approach 62 4.1 Introduction 62 4.2 Materials and methods 65 4.2.1 Materials 65 4.2.2 Vesicle preparation and characterization 65 4.2.3 Vesicle permeability measurement using stopped-flow 66 4.2.4 Liposome-embedded LbL on a mica surface 67 4.2.5 Liposome-embedded LbL membranes for nanofiltration 67 v 4.2.6 Nanofiltration studies 68 4.3 Results and discussion 69 4.3.1 PLL adsorption on liposomes 69 4.3.2 Water permeability measurement by stopped-flow 71 4.3.3 Embedding PLL-covered liposome in LbL films 72 4.3.4 Liposome embedded membrane for nanofiltration 75 4.4 Conclusion 80 Chapter Highly Permeable Aquaporin-embedded Biomimetic Membrane Featured with a Magnetic-aided Approach 81 5.1 Introduction 81 5.2 Experimental 83 5.2.1 Materials 83 5.2.2 Magnetic nanoparticle synthesis 84 5.2.3 Magnetic liposome preparation 84 5.2.4 Field emission transmission electron microscopy (FETEM) 85 5.2.5 Liposome-embedded LbL membranes formation 86 5.2.6 Vesicle adsorption study by confocal Laser scanning microscope (CLSM) 87 5.2.7 Forward osmosis measurement 87 5.3 Results and discussion 89 5.3.1 Characterization of magnetic liposomes 89 5.3.2 Characterization of the LbL membrane 94 5.3.3 Study the forward osmosis performance 98 5.4 Conclusion 100 Chapter Conclusions and recommendations 102 APPENDICES 105 REFERENCES 106 vi vii SUMMARY Water is transported rapidly through most biological cell membranes Peter Agre and his co-workers revealed the origin of this high water permeability in 1992 with the discovery of the first aquaporin (AQP) protein Inspired from biological membranes where AQPs provide extraordinary water permeability and selectivity, the use of AQPs to fabricate membranes for water purification has recently drawn worldwide attention The discovery of AQPs that facilitate water transport through biological membranes gives us strong support to mimic biological membranes when designing novel membranes for desalination with the aid of these proteins The conventionally used reverse osmosis process for producing high quality drinkable water is an energy-intensive process Developing innovative highperformance membranes for desalination and water reuse that consume less energy has become an urgent issue in recent years Driven by the extraordinary permeability and selectivity of AQP towards water molecules, novel biomimetic membranes consisting of AquaporinZ (AqpZ) for desalination and water reuses are targeted in this study In the first part of the work, a new approach of incorporating the transmembrane protein AqpZ into the lipid bilayer has been developed with the aid of the LangmuirBlodgett (LB) technique Protein incorporation in this study was achieved by combining a pure binary-lipid monolayer with an AqpZ-associated binary-lipid monolayer and a subsequent refolding of AqpZ in the bilayer The binary-lipid monolayer is composed of (1) gel-phase lipids that prevent detergent dissolution and 15 and cloned into the pCR-4 vector using the TOPO cloning kit The positive clones were sequenced and further subcloned into an expression vector, pQE-30 Xa expression vector with ampicillin selection and an amino-terminal containing an affinity tag of histidine residues The E coli strain TOP10F was transformed, grown to 0.6–1 optical density at 600 nm in a broth containing 100 mg/l ampicillin and induced with mM isopropyl-D-thiogalactoside Cells were harvested and lysed by sonication in phosphate buffered saline (PBS, pH 7.4) containing 1% dodecyl maltoside and 0.5 mM phenylmethylsulforyl fluoride The cellular debris was pelleted at 10,000 rpm for 45 and discarded The biological membranes were recovered from the supernatant by centrifugation at 100,000 rpm for 60 AqpZ was solubilized from membranes by agitation in 1% dodecyl maltoside and PBS for 12–16 hours The solubilized protein was bound in batches to Nickel–NTA resin, washed, and then eluted with PBS, 1% dodecyl maltoside and 250 mM imidazole Imidazole was removed using a Bio-Rad desalting column The resulting recombinant AqpZ protein contained a 6-histidine tag at the N-terminal of protein followed by a protease (Factor Xa) digestion site The protein concentration varies from 0.2 to mg ml-1 Further PCR and subcloning steps were conducted to remove the protease site and increase the N-terminal histidine tag from to 10 residues The resulting cloned AqpZ gene with a 10-histidine residue tag was used to further express the AqpZ for our study 1.2.4 Functional characterization The most common approach to study the osmotic transport across AQPs is the light scattering stopped-flow method, by which the water permeability of proteoliposomes can be estimated In the stopped-flow apparatus, AQP inserted 16 proteoliposome solution is rapidly mixed with hypertonic solution, and the osmotic gradient across the membrane will cause the vesicle shrinkage and water efflux The rapid reduction of the vesicular volume was recorded as an increase in the light scattering intensity at an emission wavelength of 577 nm The light scattering signal versus time can be fitted with an exponential decay shown in Equation 3, where Y is the intensity of light scattering signal, A is a negative constant, t is the time of recording, and k is the initial rate constant of the signal increase (s-1) The final osmotic permeability (Pf) of vesicles was calculated with Equation 4, where V0 is the vesicle initial volume, S is the vesicle surface area, Vw is the partial molar volume of water (18 cm3), and osm is the osmolarity difference that drives the size change of vesicles The vesicle diameter was measured by dynamic light scattering (DLS) ! = !!!"#(−!!!) !! = ! ! ! ! !! ! !!"# (3) !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(4)! 1.3 Biomimetic membrane Biological membranes possess superior separation and transportation properties over artificial membranes These properties of biological membranes originate from various kinds of proteins that are incorporated in the lipid barriers Therefore, there is growing interest in the mimicry of biological membranes by reconstituting functional proteins, especially channels, in amphiphilic membranes that are either made up of synthetic lipids or block copolymers Such membranes are termed “biomimetic” membranes Biomimetic membranes are helpful in improving our understanding on basic cellular functions and are also useful in developing biotechnological tools, biomedical devices, or membranes for water treatment 17 Liposome Supported lipid bilayer Black lipid membrane Figure 1.8 Three membrane models that are commonly adopted to mimic biological membranes: liposomes (lipid vesicle), supported lipid membranes and black lipid membranes [52] Among different technologies in the study of biomimetic membranes, three membrane structures are commonly adopted, which are the liposomes, the supported lipid membranes and the black lipid membranes (Figure 1.8) [53] A liposome is a hollow sphere that is enveloped by a lipid bilayer The formation of liposomes is a self-assembly process that is driven by the amphipathic nature of phospholipid molecules Over the last few decades, liposomes were a model system for cell membranes that helped in understanding the function of membrane proteins They are not only popular in catalysis (when used as nanoreactors) and drug delivery studies, but they are also available to form supported lipid membranes via a liposome rupturing process The supported lipid bilayer (SLB) is the most common structure for biotechnology applications [53, 54] The lipid membrane serves as a passive matrix for the embedded proteins, which are typically receptors in biosensors Generally, there have been two approaches taken to synthesize SLBs: the LangmuirBlodgett (LB) technique and the liposome adsorption technique The LB technique allows for the building up of lipid layers by transferring a monomolecular film formed at an air/water interface, which is also termed as the Langmuir monolayer or the Langmuir film, onto a solid support [55] It is also a powerful tool that is used for 18 understanding the physical properties of self-assembled lipid monolayers at an air/water interface and their interactions with bio-active molecules Liposome spreading technique is another method that is used to synthesize the SLBs The bilayer formation in this process depends on the nature of the substrate, the liposomes, as well as the aqueous environment The LB technique and the liposome adsorption technique for the synthesis of supported biomimetic membranes will be extensively discussed in this section The black lipid membrane (BLM) was first described by Mueller [56] and is a freestanding lipid bilayer formed across an aperture (Figure 1.8) in the middle of two aqueous phases As the thickness of the suspended lipid bilayer is only a few nanometers, it appears black or gray when it is imaged optically BLM is a promising design for biomimetic membranes that are used in separation applications by incorporating channel proteins into the bilayer [44] BLMs are typically formed by painting a solvent containing a lipid solution across a hydrophobic partition material (typically Teflon) with an aperture of up to mm in diameter, or by the folding technique where solvent-free lipid monolayers of each side of a partitioning aperture are spread and raised across the aperture (diameters of 50–100 µm) 1.3.1 Langmuir-Blodgett technique LB technology is based on the amphiphilic properties of certain organic molecules to assemble themselves at the air-liquid interface with a minimized free energy to form a Langmuir monolayer This technology was introduced back in the 1920s [57] Amphiphiles comprise two distinct regions: a hydrophilic polar group and a hydrophobic carbon tail Lipids are structural components of cell membranes and are 19 typical natural amphiphiles By spreading a dilute solution of amphiphiles in volatile solvents such as chloroform on the water surface, a Langmuir monolayer can be formed after the volatile solvent is completely evaporated The LB trough is the instrument that is used to study the Langmuir monolayer and transfer the monolayer onto a solid substrate (Figure 1.9) When the amphiphiles spontaneously form a monolayer on the aqueous surface, the surface tension will be changed The monomolecular film on the liquid surface reduces the free energy of water because of the interactions between the hydrophilic group and the water molecules at the surface [58] The reduction of the surface tension of a pure liquid surface in the presence of a Langmuir monolayer is known as the surface pressure, π: π = γ0 – γ where γ0 is the surface tension of the pure liquid and γ is the surface tension with the monolayer coverage For a pure water surface, γ0 is 72.8 mN m-1 The surface pressure can be monitored by a pressure sensor known as the Wilhelmy Plate The surface pressure increases as the surface monolayer is compressed with two barriers and decreases when the barriers are expanded The area per molecule, which is the geometric area of the monolayer divided by the number of monomers on the surface, is recorded together with the surface pressure, and the pressure-area (π-A) isotherm diagram can be plotted A typical π-A isotherm diagram is shown in Figure 1.10 When a π-A isotherm is measured, the quantity of amphiphilic monomers that is initially spread is very low, such that monomers are far apart and maintained in a two-dimensional gaseous phase The surface pressure is usually less than 0.5 mN m-1 in this phase When the monolayer is compressed by two 20 barriers, the intermolecular distance decreases and the monolayer will come into contact with the liquid phase, followed by the solid phase In the liquid phase, the monomers start to experience compaction but still have a certain degree of freedom Upon further compression, the monomers are closely packed and the molecular area (A) is almost equal to the cross-sectional area of the amphiphilic molecules Thus, the monolayer is in a solid phase where the hydrocarbon chains are crystallized If the compression continues, the monolayer will become unstable and finally “collapse”, which is reflected by a sudden reduction in the surface pressure In general, the π-A isotherms offer information about the surface monolayer, including the phase transition, stability, and the re-orientation of the monomers, among other parameters Pressure sensor Hydrophilic substrate Dipping arm Moving barrier Hydrophobic substrate Figure 1.9 The schematic diagram of an LB trough and the vertical monolayer transfer technique onto a hydrophilic substrate or a hydrophobic substrate [59] 21 Figure 1.10 A pressure-area (π-A) isotherm plot of a phospholipid, dipalmitoylphosphatidylcholine (DPPC) with an indication of the twodimensional phases of the monolayer at different regions LB films can be formed by the vertical passage of a solid substrate through the monolayer surface, which is also known as LB deposition (Figure 1.9) When the surface monolayer is compressed to a condensed liquid phase or a solid phase, a monolayer can be transferred onto a hydrophilic substrate by raising the substrate out of the interface, or onto a hydrophobic substrate by dipping the substrate below the surface Ridge monolayers can also be deposited onto a hydrophobic substrate by touching the monolayer horizontally from the top, which is also known as the Langmuir-Scharfer (LS) technique To deposit lipid bilayers onto a hydrophilic surface such as mica, LB deposition is carried out to transfer the first monolayer, followed with a horizontal LS deposition to form a complete bilayer [60] The LB technique offers the possibility to control and optimize every step of the LB film formation, such as the monolayer formation, the molecular area, and the 22 deposition procedures It is also a powerful method for studying protein-lipid interactions [61] by injecting protein solutions into the subphase of the monolayer However, the operation of LB trough requires stringent experimental conditions to obtain reproducible results, because the system is very sensitive to the environmental changes Therefore, every detail of the LB trough experiment has to be precisely controlled, such as the purity of the amphiphiles, the trough cleaning, the water quality, the temperature control, the substrate preparation, the speed of barrier movement, and many other factors 1.3.2 Vesicle adsorption technique McConnell et al [62] was the first to recognize that the spreading of small unilaminar vesicles (SUV) on a hydrophilic support occurred readily and was a versatile approach in forming SLBs Although the exact mechanism of this selforganization process is still not clear, it is known that this process generally involves three steps, namely vesicle adsorption, vesicle rupture, and the eventual formation of planar lipid bilayers [53] Depending on the vesicle size, vesicles may fuse together before rupturing Many factors could enhance the efficiency of the vesicle rupture process, such as electrostatic interactions between the vesicle and the substrate [6365], osmotic stress [66], presence of calcium ions in the solution [67], increased temperature [68], and hydrophilic substrates [69] Concerning the unsatisfactory stability of the lipid bilayer that is deposited directly on the solid surface, several improved SLB designs have been introduced Liposomes can be spontaneously disrupted on self-assembled monolayers (SAMs such as thiols on gold or silanes on glass or silica) to form a uniform and highly stable lipid bilayer 23 (Figure 1.11B) [70] However, there is a loss of membrane fluidity in the bottom layer, hence transmembrane proteins cannot be incorporated into this type of membrane The other strategy to form stable SLBs is spreading liposomes onto a polymer cushion covered support (Figure 1.11C) In comparison with the simple SLBs and the SLBs formed on a SAM, the presence of the polymer layer prevents any unfavorable contact of proteins with the substrate, while the fluidity of the lipid bilayer is well preserved Many types of polymer cushions have been developed, including cellulose [71], polysaccharide [72], dextran [73], polyelectrolytes [74, 75] as well as lipo-polymer tethers [76] The lipo-polymer is a synthetic polymer that consists of a hydrophilic polymer chain such as polyethylene glycol and a lipid-like molecule Figure 1.12 shows a few examples of the lipo-polymer molecules Typically, the lipid-like molecules are inserted into lipid bilayers and tethered to the polymer cushion that can be covalently bonded to the substrate The lipo-polymer tethers provide various methods where the stability of SLBs can be enhanced A B C Figure 1.11 Liposome adsorption techniques: (A) Liposome spreading directly onto a solid surface to form an SLB; (B) liposome spreading on to a self-assembled momolayer to form an SLB; (C) liposome spreading on a polymer cushions to form an SLB [52] Electrochemical methods require electrically conductive surfaces Ottova et al [15] recently reviewed formation, characterization, patterning and application of so called supported planar lipid bilayers (s-BLMs) on various metal surfaces or silver In particular, 24 beensuch as platinum, gold,suited to allow for an gold has proven to be ideally easy formation of highly oriented self-assembly layers, on phospholipid derivative [18, 19], or a hydrophobic hairyrod polymer [20] Alkanethiols adsorb spontaneously from solution on gold forming densely packed almost defect-free monolayers A second monolayer can be attached on the first hydrophobic one by various techniques In case of the Langmuir-Schäfer dip a lipid monolayer is transferred horizontally from the air-water interface to the hydrophobic O O OH O O H O 45 N O O Si O O O O O P O O OH H N N O O O O O O O O n = 10, 11, 12 OH O P S O O O O HN S H N O O O O O P O O O O SH O OH O O 93 O O O NH2 O P O O O O HN n O O P O OH S n O HO S O O O O O O O O O S O HS N H H N O OH O N H OH H N O O N H H N O O O N H OH H N HO P O O O Fig Different synthetic lipid molecules used to build up polymer supported and tethered lipid bilayers Figure 1.12 Examples of synthetic lipo-polymers that are used to build-up polymer-cushined/tethered SLBs [76] 1.3.3 Block copolymer membrane Amphiphilic block copolymers can undergo self-assembly into structures that mimic lipid bilayers Such block copolymers can be synthesized as either di-block (AB) or tri-block (ABA) structures, where A is a hydrophilic block and B is a hydrophobic block The block copolymers could be assembled into a variety of structures in aqueous solution, and their structures are determined by the ratio of the hydrophilic to hydrophobic blocks as well as their individual conformations [77, 78] The most wellknown block copolymer for biomimetic membranes is polymethyloxazolinepolydimethylsiloxane-olymethyloxazoline, which has been investigated extensively for membrane protein incorporation [38, 79-81] S 25 1.4 Biomimetic membrane for water treatment 1.4.1 Designs and strategies In 2007, Kumar and his co-workers reconstituted AqpZ into block-copolymer vesicles, which demonstrated the great potential of AQP-based biomimetic membranes [38] Since then, different designs have been proposed to apply AQPs in water treatment applications such as forward osmosis [82-86], nanofiltration [87], and reverse osmosis [88] Three typical designs have been summarized in Table 1.2 Recently, Kaufmann et al have proposed a design to fabricate an RO membrane by covering a commercial NF membrane with lipid bilayers using the vesicle adsorption method In this design, lipid vesicles are positively charged so that they can be ruptured onto the NF membrane, which carries strong negative charges They have demonstrated that a continuous lipid bilayer could be formed on the NF membrane surface, similar to a mica surface, and the membrane permeability dropped dramatically after the lipid bilayer coverage However, a functional reconstitution of AQP in this membrane and the permeability of the AQP-embedded membrane have not been demonstrated The membrane stability against hydraulic pressure is also unknown In another design, several efforts have been made to fabricate the large-scale black lipid/polymer membranes (BLMs/BPMs) for water purification [82-85] The hydrophobic membrane scaffold to form the free-standing membrane was scaled up to a 24ì27 aperture array with a diameter of 300 àm for each aperture [82], while a smart automation technique was developed to fabricate membranes on this aperture array to ensure reproducible results A complete design of this biomimetic membrane 26 was conceptually introduced by Nielson [89], where the biomimetic membrane matrix was sandwiched between cushions and porous supports (Table 1.2) However, the long-term stability of the free-standing film in the membrane matrix is a major concern for water purification applications, and further studies on the membrane performance have not been published yet A vesicle-threaded membrane was proposed in Montemagno’s patents [90, 91] In this conceptual design, polymerized proteoliposomes are linked into a porous membrane matrix after reconstitution with AQP and a layer of densely packed proteoliposomes function as a selective barrier for water purification A cross-linking of both vesicles and the entire membrane could enhance the overall stability, because the pristine lipid bilayer is too weak against the harsh operating conditions such as elevated temperatures, high hydraulic pressures, and osmotic stress However, no experimental results have been found in the literature using this method In the second part of our work, we developed a biomimetic membrane similar to this design and demonstrated the NF performance of the membrane 27 Table 1.2 State-of-the-art designs of biomimetic membranes for water treatment Designs Supported lipid bilayer on NF membrane Supported BLM/BPM matrix Vesicle-thread conjugate membrane Possible Applications Advantages and Disadvantages Experimental Results Publications or Patents RO Easy to fabricate; but maybe not stable due to lack of covalent bonding AQP incorporation is not demonstrated Kaufman et al [88] US 20110084026 FO High flux; but the stability of the free-standing film is a concern Membrane formation was demonstrated, but no FO results Nielsen et al [41, 44, 82-85, 89] EP 885 477 B1 EP 937 395 A1 RO/NF/ FO Good stability due to conjugation; flux may be compromised Not found from the literature Montemagno et al US 2011/0259815 A1 US 2012/0043275 A1 28 1.5 Motivation, challenges and objectives Although the RO process meets the requirements for producing high quality drinkable water, it is still an energy-intensive process Developing innovative high-performance membranes for desalination and water reuse has become an urgent issue in recent years Inspired from biological membranes where AQPs provide an extraordinary water permeability and selectivity, the use of AQP proteins to fabricate membranes for water purification has now become a cutting-edge research topic The discovery of AQP water transport proteins provides solid evidence for fast transmembrane water transport and gives us a strong motivation for mimicking biological membranes to design novel desalination membranes with the aid of these proteins To our best knowledge, the science and engineering of how to create an AQP-embedded biomimetic membrane and how to manipulate the water transport proteins within the membrane have not been disclosed in detail yet However, there are numerous challenges involved when transiting from conceptual demonstrations to real applications In all the designs mentioned above, there are several common issues in the development of biomimetic membranes Firstly, the essential problem is in minimizing membrane defects The lipid film or block copolymer film is thin (