3.1. Characterization of AAO substrates
The surface morphology of a substrate plays an important role in the fabrication of AQP biomimetic membranes; therefore, we analyzed the surface morphology of the AAO substrate through FESEM and AFM. Generally, an AAO substrate is fabricated through a 2-step anodic aluminum oxidation process to achieve a uniform honeycomb array of nanoporous structures25. This 2-step anodic oxidation process increases the surface roughness of the AAO substrate mainly due to the self-alignment of the porous layer during the first step of anodization as a base for nanopores formed in the second anodization process, which results in the formation of a nanodimple pattern, increasing the surface roughness. Figure 2(a) and 2(b) shows FESEM images of inclined AAO substrate after one (Fig. 2b) and two (Fig. 2a) steps of anodic oxidation. The presence of nanodimples can easily be seen in Fig. 2(a), while they are absent in Fig. 2(b), which consequently reduces the surface roughness of the AAO substrate. The surface roughness (Rq) of the 2-step sample is 35.9 nm, whereas that of the 1-step sample is 19.6 nm, as shown in Fig. 2(c) and 2(d). Channel-type pore formation using the 1-step anodic aluminum oxidation was verified by taking a cross-sectional image of AAO substrates. The pore size was measured using a previously published computational image processing method 26 and 100 FESEM images each of the AAO samples. Furthermore, the effectiveness of 1-step anodic aluminum oxidation in producing channel-like through pores was verified by analyzing the cross-sectional FESEM images of AAO substrates. Figure 2(e)-(h) shows the cylindrical pore structure of different AAO substrates with diameters ranging from 50nm to 125nm. This demonstrates that through 1-step anodic aluminum oxidation, a uniform cylindrical channel-like porous substrate with a wide range of controlled pore sizes can easily be synthesized with minimum surface roughness.
3.2. Characterization of AqpZ protein and AqpZ liposomes
SDS-PAGE analysis was performed on each fraction from the purification process. It was confirmed that GFP-AqpZ was expressed and purified by the major band (red box, size ~ 70kDa) of the elution fraction (Fig. 3a). In addition, the green fluorescence of the purified protein was confirmed by microscopy (Fig. 3b), indicating the successful fusion expression and purification of GFP-AqpZ. The presence of AqpZ in liposomes affects their structural characteristics and alters their properties, including size and surface charge. The size analysis of liposomes before and after AqpZ incorporation was analyzed by DLS intensity analysis in Fig. 3(c). The liposomes show an average size (Zavg) of 164 ± 44 nm with a polydispersity (PDI) of 0.236 ± 0.151, while the AQP liposomes show a decrease in size to 134 ± 21 nm with a PDI of 0.015 ± 0.002, the low PDI shows a uniform size distribution of AQP liposomes in solution, which is a highly required attribute for water permeability performance as well as uniform coating on the substrate surface. The decrease in liposome size is due to the bending and compression of the lipid membrane near the extracellular region of the AQP protein27. Similarly, the AQP surface contains a strong negative charge, which changes the liposome charge from slightly positive (1.41 ± 0.1.2 mV) to strongly negative (-17.44 ± 2.3 mV) in Fig. 3(d). These observations validate the high incorporation efficiency of AQPs into liposomes with uniform size distribution and surface charge, a prerequisite for the fabrication of high performance AQP biomimetic membranes. Furthermore, the activity of AQP liposomes was analyzed using a stopped-flow light scattering apparatus. The analysis was performed on liposomes with and without AQPs. The sample solution was mixed in a fixed ratio with a hyperosmolar sucrose solution using a stopped-flow mixing system. The osmotic gradient across the liposome-lipid bilayer leads to water efflux, which reduces the size of the liposomes and increases their light scattering cross-section. The variation in scattered light intensity was analyzed using a fluorescence spectrometer (Fig. 3e). The liposomes show a low kinetic constant (≈ 15.5 ± 5.7 s-1), while the AQP liposomes show the much higher rate constant (≈ 172 ± 20.15 s-1) and reach a stable position in a short time interval of < 0.05 s. Similarly, the liposome sample shows a lower net permeability (Pf = 35.12 ± 11.36 µm/s) compared to the AQP liposome (Pf = 350.61 ± 7.89 µm/s) calculated by Eq. 2. The increase in permeability of AQP-liposomes is due to the presence of AqpZ proteins, which provide an additional pathway for water molecules to move across the liposome bilayer in a short interval of time as compared to the slow diffusion of water molecules in the control liposome sample.
3.3. AQP AAO membrane fabrication
The coating processes prior to AQP immobilization on the AAO substrate are essential to fabricating a membrane with both high water permeability and salt rejection. Successful coating in each step was confirmed via XPS, AFM, and FESEM analyses of the membranes. A surface quantitative elemental analysis following each coating process (APTES, PDA, and AQP liposomes) was performed using XPS. Figure 4 shows the analysis results for substrates after each coating process. The silane functional group in APTES results in an increase in the concentration of silicon (Si) on the substrate surface after coating, which can be identified by analyzing the Si2p peak area (Fig. 4(b); compare the uncoated substrate in Fig. 4(a)). The Si2p peak area decreases upon coating of PDA and AQP liposomes on the substrate (Fig. 4(c) and (d)). Moreover, the membrane without AQP liposomes has a low phosphorus (P2p) signal (Fig. 4(e)), whereas after AQP liposome coating, the concentration of phosphorus atoms (P2p) increases, indicating the accumulation of lipid phosphate groups and confirming the presence of AQP liposomes on the substrate.
The bare substrate without any coating has uncovered pores, as shown in Fig. 5(a). After silane functionalization and PDA coating, the pore size decreases, and a thin layer of PDA is clearly visible on the substrate (Fig. 5(b)). Liposome immobilization was performed by using previously reported electrokinetic phenomena7, as this method ensures uniform coating of liposomes on the substrate pores without aggregation (Fig. 5(c)). After AQP liposome fixation, a protective layer was introduced by alternate coating of PDA and histidine in three cycles. The self-polymerizing molecule PDA binds with amine groups on the silane-functionalized AAO and the lipids. This coating layer protects the AQP liposomes and fills the voids if any between the AQP liposomes and substrate, to restrict the diffusion of solutes and minimize defects. Histidine can also bind dopamine and helps increase the salt rejection property because of its zwitterionic property.
The surface roughness was also analyzed using AFM after each step. The decrease in the surface roughness of the AQP membrane indicates the coating quality or uniform coating layer of proteoliposomes on the substrate surface, which ensures the functionality of the aquaporin protein after coating7,28,29. The formation of liposome/proteoliposome aggregates (which may affect the structure and performance of the aquaporin protein) on the surface during coating indicates the formation of valleys, which consequently increases the surface roughness and thus decreases the performance of the AQP membrane. Therefore, roughness is an important parameter to analyze the quality of the coating on the substrate. The bare AAO substrate has the highest surface roughness (Rq) of 19.6 ± 3.2 nm Fig. 5(d), while the surface roughness decreases to 12.4 ± 2.53 nm after surface functionalization with APTES and PDA in Fig. 5(e), this decrease in roughness indicates that the surface functionalization covers the valleys of the pristine substrate and forms a cushioning support layer for uniform AQP liposome coating on the substrate. The surface roughness further decreases to 9.16 ± 1.09 nm after AQP liposome coating, as shown in Fig. 5(f). This further decrease in surface roughness shows that AQP liposomes have been uniformly coated on the substrate and smoothes the valley on the pristine AAO substrate. The AQP AAO membrane surface morphology after water purification was also analyzed using FESEM and, as shown in Fig. S2, there are no notable changes.
3.4. Water purification ability
To evaluate the performance of AQP-coated membranes, a laboratory-built custom FO chamber was used with an eddy-promoting spacer. The addition of the spacer creates turbulence that facilitates mass transfer. The water permeability of the AQP AAO membrane was tested under the FO system using a 2000 ppm NaCl FS and a 1 M sucrose DS. Unlike the uncoated AAO substrate, which did not reject salt due to its large pore size and high porosity, the liposome coated AAO membrane without AQP proteins showed a decrease in water flux to 8.2 ± 1.59 LMH (L/m2h) and a reverse solute flux of 2.4 ± 0.53 GMH (g/m2h) in the FO test (Fig. 6). The low reverse solute flux was likely due to the complete coating of the liposomes on the AAO substrate surface, which has a low permeability to ions, since the lipid bilayer membrane is intrinsically non-permeable to ions.
On the other hand, the AQP-containing AAO membrane showed high water permeability: the water flux was as high as 27.6 ± 3.6 LMH, with a reverse solute flux as low as 1.6 ± 0.26 GMH, demonstrating the activity of AQP in the liposomes. The AQP water channels provided additional pathways for water flow, increasing water flux while rejecting solute or salt compared to the liposome-coated membrane without AQP. To demonstrate the power of these aquaporin pathways to allow the transport of solute-free water, we also calculated the specific reverse solute flux (SRSF), a key parameter indicating the selectivity of the FO membrane. The SRSF is the ratio of the movement of solute particles to the movement of water. The lower the SRSF value is, the higher the selectivity of the membrane. The AAO membrane with AQP shows a lower SRSF value (higher membrane selectivity) compared to the control AAO membrane (without AQP) mainly because of the increased water permeability and low solute diffusion due to the presence of highly surface-charged aquaporin proteins, which increase both the water permeability and the solute rejection. In addition, the ion rejection is mainly based on the lipid membrane (liposomes). This barrier is similar for membranes with and without AQP, but the water permeability increases significantly due to the presence of the aquaporin water channel, which in turn decreases the SRSF value. Furthermore, a high reverse solute flux value in the control membrane (liposomes) could indicate some defects in the membrane coating. Since the membrane coating was performed by electrokinetic interaction, where the modified particles move in the direction of the electric field, which is directly influenced by the charges of the particles (in this case liposomes)30–32, the low charge of the liposomes may leave some defects, which in turn shows high salt movement across the membrane. Table 1 shows a comparison of the membrane reported on here with previous literature reports. Our membrane shows superior water purification performance mainly due to the optimized fabrication of a substrate with a low surface roughness and a channel-type porous structure, which supports the water permeability performance of AQP. As previously reported membranes mostly utilized polymeric substrates which has low porosity as well as non-channel like pores which are unable to support the water permeability performance of pristine aquaporin liposomes as shown by several biophysical and stopped flow analysis7,14,33. Moreover, these membranes were fabricated mostly using interfacial polymerization (IP), in which the proteoliposomes were covered with a selective layer, most usually polyamide, so that most of the water permeability as well as the salt rejection was depended only on the characteristics of this selective layer or interfacial polymerization rather than on the proteoliposomes or AQP-liposomes13,33,34. Although we reported an electrokinetic coating method in our previous studies to uniformly coat a commercially available membrane for high water permeability, this membrane still does not fully support the permeability capabilities of aquaporin protein mainly due to the low porosity7. Finally, Doung et al. utilized the commercially available alumina substrate to fabricate aquaporin biomimetic membrane for water purification and it shows salt rejection performance of only 45% due to the coating defects17. It was observed that the defects in the reported study occur due to the non-optimization of the surface morphology of the substrate as well as the coating methodology. The uncontrolled chemical coating of aquaporin planar bilayer on the surface leads to defects or uncoated pores, because the vesicle rupture leads to several shortcomings including irregular planar bilayer thickness, limited control over planar bilayer orientation as well as the uniformity or thickness control. Therefore, we overcome these problems by fabricating a substrate with a channel like structure with controlled surface morphology as well as employing a uniform proteoliposomes layer on the substrate surface, which offers high salt rejection with high water permeability capabilities.
Table 1
Comparison of different FO membranes
Membrane | Water Flux [LMH] | SRSF | Operating Conditions | Ref |
Aquaporin inside a thin film composite membrane | 8.8 | 0.45 | DS: 1.5 M NaCl; FS: DIW | 35 |
Graphene oxide aquaporin thin film nanocomposite (GO-AQP-TFC) | 24.1 | 0.37 | DS: 0.5 M NaCl; FS: DIW | 36 |
Aquaporin A/S hollow fiber membrane (AQP-HF) | 13.2 | 0.13 | DS: 1 M NaCl; FS: DIW | 37 |
Aquaporin biomimetic membrane | 22.31 | 0.13 | DS: 1 M NaCl; FS: DIW | 38 |
TFC-layered silica-polysulfone (PSf) | 31 | 0.24 | DS: 1 M NaCl; FS: DIW | 39 |
Silane-functionalized aquaporin-coated AAO membrane | 27.6 | 0.11 | DS: 1M sucrose; FS: 2000 ppm NaCl | This study |
3.5. Transportability and storability of the AQP-coated membranes
Previously reported AQP membranes have strict environmental requirements for storage, including a limited range of temperatures and humidity compatible with continuous hydration of the membrane before testing or after fabrication33,40–42. These stringent requirements limit the transport, storage, and practicality of AQP membranes, thus precluding their industrial adoption and commercialization. A recently reported study shows that proteoliposomes, even in solution form at room temperature, lose their functionality within a week33, this also in good agreement with the previously reported studies mentioning the degradation or rupture of liposome structure in dry environment. To verify this, we stored the membrane under ambient conditions (25°C, 35% humidity) for 7 days without DI water, and analyzed the membrane surface morphology by FESEM analysis. The membrane shows the ruptured liposome structure (Fig. S2), indicating that the dry environment makes the liposome structure brittle and prone to rupture, due to the evaporation of the suspension or water layer 43. To overcome these hurdles, we devised a cryodesiccation method in which AQP AAO membranes were freeze-dried for storage and then rehydrated immediately before use, as shown in Fig. 7(a). Briefly, we froze the fabricated AQP AAO membranes at -80°C and then sublimated the ice under low pressure in a vacuum chamber. After complete removal of the ice, we stored the sample under the same environmental conditions (25°C and 35% humidity) for at least 7 days. The surface morphology of rehydrated membrane was analyzed by performing by FESEM analysis (Fig. 7(b)) and found that the liposomes remained intact on the substrate without any noticeable defects. Furthermore, to verify the activity of AqpZ activity after cryodesiccation water purification analysis was performed, the stored desiccated membrane was rehydrated with DIW at 4°C for at least 1 hr44. After rehydration, water purification was carried out using the FO system described above. The rehydrated membrane showed minimal degradation of water purification performance in terms of water flux (26.9 ± 3.3 LMH) and reverse salt flux (2.8 ± 0.3 GMH), as shown in Fig. 7(c), which corresponds to ~ 98% of the performance shown by the AAO membrane before cryodesiccation. The statistical analysis shows no significant difference in the results of both membranes before and after cryodesiccation. This shows that the AQP liposomes remained intact following freeze drying. We also verified the surface structure of the rehydrated membranes. In short, the freeze-drying process preserves the AQP liposome structure as well as its function; thus, these membranes can be stored in a desiccated state semi permanently and transported anywhere without specific environmental or storage conditions such as temperature and humidity.