Article Cite This: Langmuir 2019, 35, 7285−7293
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Toward a Better Understanding of the Nature-Inspired Aquaporin Biomimetic Membrane Hui Xian Gan,†,‡ Hu Zhou,†,§ Hui Juan Lee,‡ Qingsong Lin,†,§ and Yen Wah Tong*,†,‡ †
NUS Environmental Research Institute (NERI), National University of Singapore (NUS), Singapore 117411, Singapore Chemical and Biomolecular Engineering, National University of Singapore (NUS), Singapore 117576, Singapore § Department of Biological Sciences, National University of Singapore (NUS), Singapore 117543, Singapore ‡
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S Supporting Information *
ABSTRACT: The biomimetic membrane technology may unlock unprecedented membrane separation capabilities to solve the increasing need for clean water. Despite the efforts in exploring numerous membrane preparation methods, the membrane performance achieved to date is still far from the theoretical predictions. To overcome this bottleneck, a deeper understanding of the role of the channels or vesicles immobilized on the membrane would be required. In this work, we seek to quantify the amount of vesicles immobilized per unit area of membrane and correlate it with the membrane performance. The results show that, although the vesicles successfully immobilized onto the membrane increase with an increasing vesicle concentration, less than 4% of the vesicles loaded onto the membrane successfully remains on the membrane after interfacial polymerization. Furthermore, an increase in the amount of vesicles remaining on the membrane may not always result in improvement in membrane performance. To the best of our knowledge, this is the first time that a study has been performed to determine an accurate relationship between the vesicles immobilized and the biomimetic membrane performance.
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INTRODUCTION Over the past 50 years, conventional membrane technology, such as the interfacial polymerized composite membrane and asymmetric membrane fabricated via the phase inversion method, has matured to become the key solution for waterscarcity issues.1,2 However, greater innovation will be required to cope with the rapid increase in global population and the associated need for clean water.1,3 Inspired by nature, there has been great interest to incorporate selective water channels, such as aquaporin (Aqp) water channel proteins, onto a membrane in attempt to achieve membrane capabilities superior to the performance using the conventional “poreless” membrane filtration.3 Aqp is a transmembrane water channel protein that can allow water to pass through quickly while maintaining rejection toward all other solutes.4 However, the protein needs to be reconstituted in an amphiphilic environment resembling a cell membrane to maintain its structure and functionality.5 Theoretical calculations based on Aqp osmotic permeability have shown that the Aqp biomimetic membrane may attain membrane permeability of 167 μm s−1 bar−1, which is 2 orders of magnitude higher than a typically commercial polymeric membrane.6 Since then, numerous biomimetic membrane fabrication approaches have been investigated and can be broadly classified under the vesicle immobilization membrane or planar layer immobilization membrane.5 Briefly, for the vesicle-immobilized membrane, the water channels are © 2019 American Chemical Society
supported in intact vesicles on the membrane substrate and polymerization is performed to seal the gaps between the vesicles. Conversely, for the planar layer immobilization membrane, water channels are embedded in the planar polymer or lipid layer and supported by the membrane substrate. On the basis of the membrane performance reported to date, the vesicle immobilization method is considered to be more efficient.5 Although the planar layer immobilization membrane is expected to provide higher water flux, with water passing though the channels once instead of twice, the thin planar layer is also highly susceptible to defects and salt leakage.5 Despite the large number of membrane-fabricating strategies explored, the membrane performance achieved to date is still far from ideal.5,7 Some of the common challenges faced are the difficulties in scaling up the size of the biomimetic membrane as a result of the formation of defects5 and the large amount of water channels required for modification with an increase in the membrane size.8 Thus far, only the strategy developed on the basis of the conventional interfacial polymerization method to prepare the biomimetic membrane has successfully progressed to the commercialization and long-term testing stage.5,9 Nonetheless, the exact amount of biomimetic vesicles Received: February 7, 2019 Revised: May 10, 2019 Published: May 13, 2019 7285
DOI: 10.1021/acs.langmuir.9b00380 Langmuir 2019, 35, 7285−7293
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DNA sequence encoding AqpZ with a N-terminal 6× His affinity tag was transformed into E. coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, CA, U.S.A.). Cells from a single colony were inoculated in lysogeny broth (LB) medium with 100 μg/mL ampicillin and grown overnight at 37 °C for 2 h before harvesting. The cells in a 1 L culture was harvest by centrifugation at 6000 rpm for 15 min and resuspended in 10 mL of lysis buffer containing 20 mM Tris−HCl (pH 8.0), 100 mM MgSO4, 1 mM phenylmethanesulfonyl fluoride, and 0.1 mg/mL deoxyribonuclease I. After which, the cell resuspension was subjected to sonication and the lysate was centrifuged at 10000g for 30 min to remove the insoluble material. Finally, the membrane fraction was recovered from the supernatant by centrifugation at 140000g for 1 h. For AqpZ extraction, the membrane fraction was resuspended in a solubilization buffer [1% n-dodecyl-β-maltoside (DDM) in a buffer containing 20 mM Tris−HCl (pH 8.0) and 100 mM NaCl] and incubated overnight at 4 °C. The insoluble material was pelleted via centrifugation at 140000g for 45 min. DDM-solubilized AqpZ was bound to the cobalt resin under gentle shaking at 4 °C for 3 h in the presence of 5 mM imidazole. After which, the protein-bound cobalt resin was washed with 10 column volumes of buffer containing 20 mM Tris−HCl (pH 8.0), 100 mM NaCl, 10 mM imidazole, and 0.2% DDM. Lastly, AqpZ was eluted with the washing buffer supplemented with 150 mM imidazole. E. coli Vesicle Preparation and AqpZ Reconstitution. E. coli lipid vesicles were prepared using the dialysis incorporation method according to a previously reported protocol.2 Briefly, E. coli lipid was dissolved in chloroform and dried under vacuum to form a thin homogeneous film. Next, the thin lipid film was rehydrated in 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS) buffer adjusted to pH 7.4 with sodium hydroxide (NaOH). The lipid was mixed with AqpZ and octyl glucoside (OG) detergent in 0.1 M 3-(Nmorpholino)propanesulfonic acid sodium (MOPS−Na) buffer until homogeneous. The mixture was then transferred to a 10 000 molecular weight cut-off dialysis cassette for dialysis against 0.1 M MOPS−Na buffer over 3 days. During the course, dialysis buffer was replaced with fresh buffer daily. Vesicle Characterization. Vesicle size was measured via dynamic light scattering (Zetasizer, Nano ZSP equipped with a helium−neon laser beam at 633 nm, Malvern Instrument, Ltd., Malvern, U.K.). Three measurements were made to obtain an average value for each reading at 25 °C. Vesicle permeability was studied using a stopped-flow apparatus spectrometer (Applied Photophysics, U.K.) method. A total of 0.5 mg/mL of the vesicle solution was rapidly mixed with sucrose buffer at 0.6 osmol/L concentration at 25 °C. The high osmolarity of sucrose buffer will drive a water efflux from the vesicles, which can be recorded in the form of an increasing light-scattering signal. The initial gradient of the light-scattering curve can be fitted to an exponential equation to represent the rate of vesicle shrinkage, k. The osmotic water permeability was calculated using eq 12
or proteins required to bring about an improvement in membrane performance either remains unknown or varies broadly across different works and membrane performance strategies.9−11 Notably, the amount of vesicles required to prepare a biomimetic membrane may contribute significantly to its production cost. Therefore, an accurate method to determine the amount of biomimetic vesicles or channels on the biomimetic membrane is important. Several quantification methods have been proposed to provide a more accurate measurement of biomimetic vesicles and membrane performance. However, most of the methods only cater to measuring the amount of Aqp reconstituted into vesicles. These methods include freeze-fracture transmission electron microscopy (FF-TEM),5 fluorescence correlation spectroscopy (FCS),5 small-angle X-ray scattering (SAXS),5 gel electrophoresis,2 and inductively coupled plasma mass spectrometry (ICP−MS). A few methods, such as Fourier transform infrared spectroscopy (FTIR),5,9 field emission scanning electron microscopy (FESEM),9−11 and confocal microscopy,11 have been proposed to show the presence of proteins or vesicles on the biomimetic membrane. These methods only provide qualitative information about the presence of biomimetic components on the membrane. However, the results may not be representative of the entire samples because only a small, non-representative sample size can be examined each time. On top of that, it is technically challenging to prove that the spherical structures shown on FESEM are truly vesicles. Conversely, confocal microscopy requires vesicles to be labeled with fluorescence dye prior to visualization. Dependent upon the labeling chemistry, there could be much non-specific binding of the fluorescence dye onto the membrane surface. The unremoved fluorescence dye and membrane substrate may result in false-positive results. In our previous work,2 ICP−MS has been demonstrated as a useful method to accurately determine the incorporation efficiency of aquaporin Z (AqpZ) into vesicles. In this work, ICP−MS is proposed as a method to accurately determine the amount of lipid vesicles immobilized on the biomimetic membrane. To the best of our knowledge, this is the first time that a method has been proposed to accurately quantify the amount of biomimetic vesicles per unit area of membrane and to correlate it with the membrane performance. While the analysis method proposed is the same, the sample preparation and considerations for quantification on the solid membrane samples are different from the vesicle solutions. These would be further discussed in the next section. The proposed quantification method was applied to study (1) the efficiency in loading vesicles at selected steps of interfacial polymerization and (2) the influence of loading different concentrations of vesicles and vesicles of different lipid/protein mass ratio (LPR) on the membrane performance. This method is anticipated to be useful for selection, optimization, and design of the biomimetic membrane fabrication method.
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Pf =
k
( )V Δ SA V0
w
osm
(1)
where Pf is the osmotic water permeability (m/s), SA is the vesicle surface area (m2), V0 is the initial vesicle volume (m3), Vw is the partial molar volume of water (0.018 L/mol), and Δosm is the osmolarity difference that drives the vesicle shrinkage (osmol/L). The vesicle permeability can be further converted into the effective membrane permeability (LMH/bar), Aeffective, using eq 27
MATERIALS AND METHODS
Materials. Escherichia coli lipid was purchased from Avanti Polar Lipids. Dialysis cassettes were purchased from Thermo Fischer Scientific. Polysulfone polymer pellet was kindly provided by Solvay. Trace-metal-grade nitric acid was purchased from Fischer Scientific. All of the other chemicals and solvents were purchased from SigmaAldrich, unless otherwise mentioned. Methods. Expression and Purification of AqpZ. The expression and purification of AqpZ were performed according to those previously reported.2 The modified pET plasmid containing the
Aeffective =
Pf Vw 1.57 1 RT 2 0.278
(2)
where Pf is the osmotic water permeability (μm/s), Vw is the partial molar volume of water (18 cm3/mol), R is the universal gas constant (83.1 cm3 bar mol−1 K−1), and T is the temperature (298.15 K). Assuming that all of the vesicles are spherical in shape and do not overlap, the total effective area provided by the vesicles would be 1.57 7286
DOI: 10.1021/acs.langmuir.9b00380 Langmuir 2019, 35, 7285−7293
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Figure 1. P measurements using ICP−MS. (a) Representative calibration curve for ICP−MS measurement. (b) Samples with different concentrations of lipid vesicles or membrane spiked with different concentrations of lipid vesicles were prepared in three independent experiments. The P concentrations of the samples were determined using ICP−MS (n = 3). ij Cf − C p yz zz × 100% R = jjj j Cf zz k {
μm2 per 1 μm2 of filtration membrane. Because the solution will have to pass through the vesicle twice, the actual membrane permeability will be halved. On the basis of unit conversion, 0.278 μm s−1 bar−1 is equal to 1 LMH/bar. Membrane Preparation. Membrane Casting. Membranes were prepared according to a previously reported method.10 A casting solution with 16% (w/v) polysulfone, 2% lithium chloride, and 5% polyethylene glycol (molecular weight of 600) were completely dissolved in N-methyl-2-pyrrolidone solvent and degassed. The dope solution was then casted onto a glass substrate and transferred immediately into a water bath at room temperature. The substrates were washed thoroughly with excess water. Substrates were kept soaked in deionized water at 4 °C until further use. Interfacial Polymerization. The vesicles and 1.5% m-phenylenediamine (MPD) were incubated on the polysulfone membrane substrate for 10 min. Next, excess solution was removed from the membrane by drying the membrane vertically for 20 min, followed by gentle blowing with nitrogen gas. After which, 0.1% trimesoyl chloride (TMC) dissolved in n-hexane was poured over the surface of the substrate to allow for interfacial polymerization for 1 min. Excess TMC solution was removed, and the membranes were flushed with excess deionized water and kept in ultrapure water at 4 °C until further characterization. The vesicle loading was controlled by varying the concentration of vesicles and keeping the volume of vesicles and MPD solution loaded constant. All membranes were incubated with vesicles and MPD in 0.1 M MOPS−Na (pH 7.4) buffer, except for W0×, which was prepared with MPD in water, without vesicles added. Membrane Characterization. FESEM. The membrane morphology was observed using FESEM (JEOL JSM-6700LV). Membrane samples were frozen in liquid nitrogen before transferring to a freeze dryer for drying overnight. The cross sections of samples were prepared by freeze fracturing the membrane in liquid nitrogen before drying in a freeze dryer. All samples were coated with a sputtering coater (JEOL LFC-1300). Nanofiltration Test. Each membrane was nitrogen-pressurized under 5 bar at a magnetic stirring speed for 600 rpm using a dead end stirred cell (HP4750 stirred cell, Sterlitech) to determine its pure water permeability and salt rejection performance. The active membrane area is 14.6 cm2. Prior to each data collection, membranes were conditioned under 5 bar for at least 3 h. The pure water permeability was calculated by weighing the mass of collected permeate over time. For the salt rejection test, the feed solution was prepared by dissolving sodium chloride in deionized water at a concentration of 500 ppm. The concentration of the feed and permeate solutions were determined using a conductivity meter (SevenCompact conductivity meter S230, Mettler Toledo). The salt rejection (R) was calculated as in eq 3
(3)
whereby Cf and Cp are the respective salt concentrations of the feed and permeate solutions in parts per million. Microwave Digestion. Membrane samples with a surface area of 14−20 cm2 or known volume of lipid samples were weighed and digested in 8 mL of 65% concentrated nitric acid in a Ethos One microwave digester (Milestone, Inc., Shelton, CT, U.S.A.) at 200 kW. The digested sample solution was then diluted to 50 mL with ultrapure water. The solutions were filtered through a syringe filter of 0.22 μm pore size before ICP−MS analysis. ICP−MS. The phosphorus concentration in each microwavedigested sample was measured with an Agilent 7700x ICP−MS system. The operating conditions are summarized in Table S1 of the Supporting Information. For all of the measurements, the sample uptake was directed through the Agilent integrated autosampler with a micromist nebulizer. A series of known dilution of Periodic table mix 1 ICP−MS standard solution was used to obtain a calibration curve for the phosphorus content of the membrane samples. All reported phosphorus readings have been corrected by subtracting the blank reading before normalizing the reading to 20 cm2 of membrane based on mass. The average mass of an unmodified polysulfone membrane is 53.93 ± 0.29 mg/20 cm2, and the average mass of an interfacial polymerized polysulfone membrane is 54.40 ± 2.81 mg/20 cm2.
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RESULTS AND DISCUSSION Theoretical Calculations of Vesicle Coverage on the Membrane. The amount of vesicles loaded onto the membrane can be designed in terms of the number of times (×) the vesicles would be able to fully cover the membrane surface area. Using the calculations presented by Grzelakowski and co-workers7 together with an approximation of molecular weight of the lipid vesicles, the vesicles required for a single layer of non-overlapping vesicles to fully cover the membrane is 2 μg/cm2 = 0.003 nmol/cm2 (please refer to the Supporting Information for the detailed calculations). Accordingly, the vesicles loaded on the membrane can be loaded in excess based on the number of vesicle layers, and this is summarized in Table S2 of the Supporting Information. For the rest of this work, the amount of vesicles added will be presented on the basis of the number of times in excess loaded and with the prefix C for control lipid vesicles without AqpZ and the prefix Z for lipid vesicles with AqpZ incorporated. As shown in Table S2 of the Supporting Information, the amount of lipid vesicles that is required to fully cover the 7287
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Figure 2. Interfacial polymerization method to prepare the biomimetic membrane. The P concentration was measured for samples at different steps of membrane preparation (numbered and boxed).
Figure 3. (a) Percentage (%) of lipid loaded that remains in solution, loss or adsorbed on the membrane based on the P measurement for membranes prepared with different amounts of control vesicles loaded (1×, 10×, 50×, and 250×). (b) P concentration of the membrane at the adsorption step, after polymerization and after the filtration test, for membranes prepared with different amounts of vesicles loaded (1×, 10×, 50×, and 250×).
membrane surface area is low. Therefore, ICP−MS is proposed as a quantification method to leverage upon its ability to detect a broad number of elements with high sensitivity. Using this method, the membrane area analyzed can be accurately recorded on the basis of the sample mass. In this work, phosphorus (P), an element that is present at about 4 wt % in the E. coli lipid, was measured to determine the amount of lipid present on the membrane. Notably, P is also present in other commonly used lipids, such as 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) and 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), at around 4 wt %, making this quantification applicable to most of the biomimetic membranes that are prepared with lipid vesicles. Validation of the ICP−MS Quantification Method. From Figure 1a, the P concentration can be measured linearly up to 1000 ppb using ICP−MS for the P concentration above 10 ppb. Next, to confirm that the lipid concentration can be correlated with the P concentration measured, a series of
known concentrations of lipid vesicle solution were subjected to microwave digestion and their respective P concentrations were measured using ICP−MS. The blank without sample and the control sample with only unmodified polysulfone support substrate show P readings of 30.79 ± 2.50 and 31.67 ± 2.06 ppb, respectively. The P reading of the blank was subtracted from the samples before correcting based on the mass in the rest of this work. As shown in Figure 1b, the P concentration correlates proportionately with the concentration of lipid present in the sample. Unlike lipid vesicle solution, the polymeric membrane samples cannot be digested completely into a clear solution using the microwave digestion method. Therefore, the digested solution has to be filtered prior to ICP−MS analysis. Figure 1b shows the P concentration of unfiltered vesicles, filtered vesicles, and filtered membrane spiked with vesicles at various known lipid vesicle concentrations. A one-way analysis of variance (ANOVA) test was conducted to compare the effect 7288
DOI: 10.1021/acs.langmuir.9b00380 Langmuir 2019, 35, 7285−7293
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Langmuir of filtration on the P concentration of unfiltered vesicle samples, filtered vesicle samples, and filtered membrane spiked with vesicles. The test shows that the filtration step has no statistically significant effect on the P concentration of the three groups of samples at different lipid concentrations [C1×, F(2,6) = 0.56, p = 0.59; C10×, F(2,6) = 0.24, p = 0.79; C50×, F(2,6) = 0.19, p = 0.83; and C250×, F(2,6) = 0.24, p = 0.80]. Because the filtration process does not affect the P reading of samples, all of the samples in the rest of this work are filtered before ICP−MS analysis to ensure a fair comparison. Evaluation of the Interfacial Polymerization Method for Biomimetic Membrane Preparation. The ICP−MS method was applied to quantify the amount of biomimetic vesicles per unit area in the selected step of the biomimetic membrane preparation procedure. The selected steps at which samples were analyzed are numbered and boxed in Figure 2. The percentage of lipid that remains in each step of membrane preparation compared to the total amount of lipid loaded was calculated according to eq 4 and presented in Figure 3a. For these calculations, the P concentrations of unmodified polysulfone membranes with a known amount of vesicles spiked were used for the total P concentration. lipid (%) =
P concentration (ppb) × 100% total P concentration (ppb)
Figure S1 of the Supporting Information). Because the vesicle diameter ranges from 100 to 200 nm (Please refer to Table S3 of the Supporting Information), the polymerized layer would be able to embed no more than two layers of vesicles. Therefore, the polymerized layer will lose its ability to stabilize and maintain the vesicles embedded in the polymerized layer once the vesicles on the membrane exceed two layers. Furthermore, the greater amount of vesicles adsorbed on the substrate may also influence the interfacial polymerization process and in a more defective and loose polymerized polyamide layer. As shown in Table 1, for the membrane with Table 1. Vesicle Coverage on the Membrane Surface Calculated on the Basis of Equation 5 vesicle coverage on the membrane surface (%) vesicles loaded (× in excess) 1 10 50 250
vesicles adsorbed 3.77 23.49 69.18 263.04
± ± ± ±
1.63 11.79 1.96 13.29
polymerized membrane 7.57 23.01 83.24 197.52
± ± ± ±
7.28 8.00 13.84 43.09
tested membrane 6.91 25.24 72.87 175.99
± ± ± ±
9.14 19.10 7.89 3.15
250× vesicles loaded, the surface coverage of vesicles decreases from 263.04 ± 13.29% (greater than two layers of vesicles) for the vesicle adsorption step to 197.52 ± 43.09% (equal to two layers of vesicles) for the polymerized membrane. After the filtration test, the surface coverage further decreases to 175.99 ± 3.15% (less than two layers of vesicles). This is probably due to the loss of vesicles, which are partially embedded on the surface of the polymerized layer, which may be driven by the high pressure applied or the direct contact with a high concentration of salt solution. These findings can help to identify specific improvements for the membrane preparation method. The poor loading efficiency of less than 4% vesicles via the physical adsorption method will lead to wastage of a large proportion of the highvalue biomimetic vesicles. Hence, surface modification would be necessary to improve the efficiency of capturing high-value biomimetic vesicles on the membrane. Alternatively, there should be ways of recovering the vesicles that do not remain on the membrane for further use. On the other hand, the observations from vesicles loaded at 250× in excess also show that it is important for the polymerized layer to cover and stabilize the vesicles to prevent the loss during filtration. Otherwise, Aqp should be incorporated into vesicles that can be further stabilized and immobilized on the membrane to prevent loss of vesicles during filtration. Correlation of Lipid Immobilized with Membrane Performance. In the next part of the work, the quantification method was applied to understand the factors influencing the performance of the biomimetic membrane. The two main factors under consideration are the amount of vesicles loaded and the LPR of the vesicles loaded. Because the preparation steps after vesicle absorption do not have a large influence on the amount of lipid remaining up to vesicles loaded 50× in excess, the rest of the P measurements were performed on membranes after the membrane filtration test. Membranes with Different Concentrations of Control Vesicles. To decouple the effect of the amount of Aqp loaded onto the membrane from the membrane performance, control lipid vesicles without AqpZ would be used for studying the effect of varying the vesicle amount loaded.
(4)
Figure 3a shows that less than 4% of the total amount of lipid vesicles that were loaded onto the membrane still remains on the biomimetic membrane after the vesicle adsorption step and more than 80% of the vesicles loaded remains in the excess solution removed from the membrane. On the other hand, lipids that were neither deposited on the membrane nor remain in the solution account for up to 18.65% of the total lipid loaded. This can be attributed to sample loss during the multiple handling and transferring steps. The deposition of vesicles via physical adsorption is highly inefficient, and a large proportion of the high-value biomimetic molecules does not remain on the membrane. Using the quantification method, the physical absorption of vesicles on the membrane surface was identified as the limiting step in immobilization of vesicles on biomimetic membranes. The P concentration measured for membranes with vesicles adsorbed, after polymerization and after the filtration test, is shown in Figure 3b. A one-way ANOVA test was conducted to compare the effect of further modifications on the amount of lipid that remains on the membrane during adsorption, polymerization, and the filtration test. The test shows that further modification steps after vesicle adsorption have no statistically significant effect on the P concentration of the samples for lipid loaded up to 50× in excess [C1×, F(2,6) = 0.14, p value = 0.87; C10×, F(2,6)] = 0.02, p value = 0.98; and C50×, F(2,6) = 1.31, p value = 0.34]. However, there is a statistically significant difference in the P reading between different steps after adsorption for lipid loaded at 250× in excess. Accordingly, a Turkey post hoc statistics test was performed to determine the specific groups that are different. The results show that the significant difference lies between the P reading at the vesicle adsorption step and after the filtration step. This could be attributed to the loss of lipid from the membrane during polymerization and testing. The current interfacial polymerization method produces a polyamide layer, which is 200−300 nm in thickness, when observed under FESEM (please refer to 7289
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Figure 4. (a) Membrane performance (5 bar and 500 ppm of NaCl) of membranes prepared via interfacial polymerization with different amounts of control vesicles loaded (0×, 1×, 10×, 50×, and 250×). W0× and C0× refer to membranes prepared without any vesicles loaded in water and 0.1 M MOPS−Na buffer, respectively (n = 3). All other membranes were prepared with MPD and vesicles in MOPS buffer. (b) Membrane morphology under FESEM.
During the preparation of the biomimetic membrane using the interfacial polymerization method, the vesicles were added with the MPD solution onto the membrane. However, the biomimetic vesicles are typically prepared in buffer with controlled pH, and this may affect the polymerization. Furthermore, it may cause the results of membrane prepared with different amounts of vesicles to be incomparable. Figure 4a shows that the preparation of the membrane with MPD in buffer instead of water increases the water flux of the membrane from 6.5 ± 0.8 to 8.4 ± 1.6 LMH. On the other hand, the salt rejection decreases slightly from 92.8 ± 3.2 to 90.8 ± 3.1%. To avoid the varying influence of buffer on the membrane prepared at different vesicle loadings, apart from sample W0×, for which MPD was dissolved in water, all of the other samples in this work were prepared with MPD and vesicles in 0.1 M MOPS−Na (pH 7.4) buffer solution. The size and permeability of the control lipid vesicles without AqpZ incorporated are measured using dynamic light scattering (DLS) and stopped-flow light scattering (SFLS), respectively. The diameter of the control vesicles is 131 ± 2 nm, with a polydispersity index of 0.13 ± 0.03, and the kinetic rate constant is 10.11 ± 1.21 s−1. With the measured diameter and kinetic constant, Pf of the control vesicles is 40.8 ± 5.4 μm/s. Using eq 2, the maximum membrane permeability possible with the membrane surface fully covered with a single layer of control vesicles is 0.09 LMH/bar, which is at least an order of magnitude lower than the average flux of a typical interfacial polymerized reverse osmosis membrane, 1 LMH/ bar. With this information, the addition of control lipid vesicles to the interfacial polymerized layer is expected to lower the flux of the membrane. Ideally, the addition of vesicles to the polymerized layer should not introduce defects to the polymerized layer and result in reduced salt rejection. The water flux decreases up to 10× in excess control vesicles loaded, and this is in good agreement with the theoretical predictions. However, a further increase in vesicles loaded would result in a significant deterioration in salt rejection down to 84.8 ± 2.6% (C50×) and 66.8 ± 9.3% (C250×). Figure 4b also shows that the membrane morphology shows increasing distinctive spherical structures on the surface of the
polymerized layer with increasing vesicles loaded. These observations can be correlated with the percentage surface coverage of the vesicles on the membrane, which can be calculated on the basis of eq 5, whereby 100% refers to a membrane fully covered with a single layer of vesicles. coverage (%) = percentage of lipid (%) × vesicles loaded (× in excess)
(5)
The vesicle coverage on the membrane surface at different loadings was calculated on the basis of the P concentration measured and summarized in Table 1. The salt rejection of the membrane decreases when the vesicle coverage occupies more than 23% of the polymerized layer. This could be attributed to the increasing susceptibility to defects with the larger amount of lipid loaded onto the membrane polymerized layer. The membrane performance test was performed on the membrane with lower vesicle loading between 0× and 25× in excess. The results confirm that the salt rejection is indeed maintained when the vesicle loading is maintained below 10× (please refer to Figure S2 of the Supporting Information). Membranes with AqpZ-Reconstituted Vesicles at Different LPRs. Most of the work published on biomimetic membranes places their emphasis on optimization of LPR to prepare vesicles with highest permeability before immobilization onto the membrane. This is based on the hypothesis that vesicles with higher Pf would theoretically be able to result in greater flux improvement. However, the previous discussion has shown that vesicle coverage above 23% on the membrane may result in defects and, therefore, a decrease in salt rejection. This part of the work aims to determine and compare the influence of AqpZ-reconstituted vesicles on the membrane at different LPRs at different vesicle loadings. The size and permeability of the AqpZ-incorporated vesicles at different LPRs and their respective control lipid vesicles prepared by the addition of an equal volume of buffer (20 mM Tris−HCl, 100 mM NaCl, and 0.2% DDM) instead of AqpZ were measured using DLS and SFLS, respectively, and summarized in Table S3 of the Supporting Information. Accordingly, Pf and Aeffective can be calculated using eqs 1 and 2 and summarized in Figure 5. Given that the permeability of the 7290
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Figure 5. (a) Vesicle permeability of lipid vesicles prepared at different LPRs (n = 3). Vesicle permeability was calculated using the SFLS and DLS measurements and eq 1. Control vesicles were prepared with the addition of an equal volume of buffer without AqpZ. (b) Membrane permeability of vesicles (LMH/bar) calculated on the basis of vesicle permeability and eq 2.
Figure 6. (a) Water flux of the biomimetic membrane prepared via interfacial polymerization at different vesicle loadings (1×, 50×, and 250×) and different LPRs (400, 200, 100, 50, and 25). (b) Salt rejection of the biomimetic membrane prepared via interfacial polymerization at different vesicle loadings and different LPRs (n = 3). 7291
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NaCl, 10 bar, and cross-flow mode). This is equivalent to an improvement of 6.96 LMH from 13.69 to 20.65 LMH under 5 bar. Similarly, a high salt rejection of 97.2% was maintained for membranes with Aqp-incorporated vesicles. However, as a result of the lack of information on the volume of vesicles loaded per unit area of membrane and accurate quantification of the amount of vesicles successfully immobilized, the underlying reason for the remarkable membrane performance improvement without the need for an ultrapermeable vesicle is unknown.
interfacial polymerized membrane without the addition of vesicles is 1.7 LMH/bar in this work, only vesicles with Aeffective higher than that may bring about a flux enhancement. On the basis of calculated Aeffective (Figure 5b), only vesicles containing AqpZ with a LPR lower or equal to 50 will be able to bring about a flux improvement. The membrane performance results are shown in Figure 6 (the corresponding P concentration of the membranes is shown in Table S4 of the Supporting Information). At vesicles loaded at 1×, the addition of vesicles can result in a flux improvement of 2.6 LMH (at LPR of 50), with salt rejection maintained above 90%. This is consistent with the theoretical calculations presented above. However, a further increase in vesicle loading beyond 10× loading may introduce significant defects in the polymerized layer that bring about significant deterioration in salt rejection across all LPRs and a large standard deviation. The results show that, while Pf of vesicles plays an important role in flux enhancement, the salt rejection appears to be largely maintained by the concentration of the biomimetic vesicles immobilized on the membrane. Notably, the experimental results deviate from the theoretical predictions to a large extent. This is because the theoretical calculations assume that the vesicles remain intact on the polymerized layer and that there are no defects between the polymerized layer and the vesicles. However, in the actual situation, there could be incompatibility between lipid vesicles and the interfacial polymerized layer. Therefore, the choice of amphiphilic materials for AqpZ incorporation is vital. The material should be able to support the functionality of water channels, while retaining good compatibility with the polymerized layer and maintaining high stability are vital. On the other hand, it should also allow for reconstitution of a large amount of AqpZ, such that a high Pf is significantly highly than the permeability of the polymerized layer that can be attained. Comparison to Literature Values. There are two key works published on the interfacial polymerization for the preparation of an Aqp-incorporated biomimetic membrane.9,10 However, a direct comparison between the results obtained from different publications is highly challenging as a result of the difference in experimental conditions used and resultant performance. Nonetheless, the amount of vesicles or proteins added may contribute substantially to the membrane production cost and should not be neglected when considering the cost effectiveness of the biomimetic technology. Zhao et al. produced DOPC lipid vesicles with Pf of 600 μs/ s at a molar of LPR 200, which is equivalent to a weight LPR of 13. As such, work by Zhao et al. used more than 10× the amount of Aqp compared to this work to achieve similar permeability to Pf of E. coli lipid vesicles at a weight LPR of 200. Zhao and co-workers presented significant improvements in membrane performance from 16 to 20 LMH (10 mM NaCl, 5 bar, and cross-flow mode), this improvement of 4 LMH is comparatively much higher than all conditions reported in this work, even for membranes modified with vesicles of higher Pf. Furthermore, a high salt rejection of 96−97% was maintained, even with the addition of vesicles. Qi et al. published another work on the long-term stability of Aqp biomimetic membranes. Similar to the current work, E. coli lipids were used for incorporation of Aqp to prepare vesicles with Pf of 1000 μm/s. However, the LPR is not reported, and hence, the amount of Aqp incorporated is unknown. This resulted in a good improvement in membrane permeability from 2.68 to 4.13 LMH/bar at 10 bar (10 mM
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CONCLUSION In this work, an ICP−MS quantification method is validated as a viable method to determine the accurate amount of lipid vesicles immobilized per unit area of biomimetic membrane. The method was applied to determine the limiting step, physical absorption of vesicles, in loading biomimetic vesicles onto the membrane. From the quantification results, less than 4% of vesicles added was adsorbed onto the membrane, with the loss of high-value protein and vesicles significant. Accordingly, future work may consider methods to improve the retention efficiency of vesicles on the membrane, such as a stronger interaction between the membrane and vesicles or recycling the vesicle solution. Next, the method was applied to understand the correlation between the membrane performance and concentration of vesicles loaded or LPR of the vesicles loaded. The results show that the concentration of vesicles loaded has a greater influence than the LPR of the vesicles, whereby the amount of vesicles on the membrane surface should be limited, because beyond a certain amount may result in the introduction of substantial defects in the polymerized layer, regardless of the amount of protein added. It is anticipated that the method and results presented in this work will enable a better understanding and, hence, improvements in preparation methods of biomimetic membranes.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.9b00380.
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Calculations for the amount of vesicles required to fully cover the membrane with a single layer of vesicles, membrane (cross section) morphology under FESEM (Figure S1), membrane performance (5 bar and 500 ppm of NaCl) of biomimetic membranes prepared with control vesicles loaded (Figure S2), ICP−MS operating conditions (Table S1), mass of lipid loaded per unit area of membrane to provide different coverages of vesicles on the biomimetic membrane (Table S2), SFLS and DLS results of biomimetic vesicles prepared at different LPRs (Table S3), and P concentration of the interfacial polymerized membrane prepared at different vesicle loadings and different LPRs (Table S4) (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hui Xian Gan: 0000-0001-6486-245X Yen Wah Tong: 0000-0002-6004-6284 7292
DOI: 10.1021/acs.langmuir.9b00380 Langmuir 2019, 35, 7285−7293
Article
Langmuir Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Wai Fen Yong, You Kang Lim, Poh Geok Per, and Chee Kin Chay for their help in this work. REFERENCES
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DOI: 10.1021/acs.langmuir.9b00380 Langmuir 2019, 35, 7285−7293
