Aquaporin–graphene interface: relevance to point-of-care device for renal cell carcinoma and desalination

Abstract

The aquaporin superfamily of hydrophobic integral membrane proteins constitutes water channels essential to the movement of water across the cell membrane, maintaining homeostatic equilibrium. During the passage of water between the extracellular and intracellular sides of the cell, aquaporins act as ultra-sensitive filters. Owing to their hydrophobic nature, aquaporins self-assemble in phospholipids. If a proper choice of lipids is made then the aquaporin biomimetic membrane can be used in the design of an artificial kidney. In combination with graphene, the aquaporin biomimetic membrane finds practical application in desalination and water recycling using mostly Escherichia coli AqpZ. Recently, human aquaporin 1 has emerged as an important biomarker in renal cell carcinoma. At present, the ultra-sensitive sensing of renal cell carcinoma is cumbersome. Hence, we discuss the use of epitopes from monoclonal antibodies as a probe for a point-of-care device for sensing renal cell carcinoma. This device works by immobilizing the antibody on the surface of a single-layer graphene, that is, as a microfluidic device for sensing renal cell carcinoma.

1. Introduction

Permeation of water through the cell membrane is a critical event in maintaining an even hydrostatic pressure for billions of cells present in a living system. Cell membranes contain pores or ‘channels’ which were not understood until Peter Agre discovered the first water channel and was awarded the Nobel Prize in Chemistry in 2003 (https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2003/agre-lecture.html). For billions of cells to be able to function, the coordination of water movement is required; this involves intricate and elaborate communication between the cells. The signals transferred between cells consist of ions or small molecules. These trigger cascades of chemical reactions that cause our muscles to tense, our eyes to water—indeed, they control all our bodily functions (https://www.nobelprize.org/mediaplayer/index.php?id=550).

Water channels consist of aquaporins, membrane proteins found in living cells, which facilitate highly efficient water transport in and out of cells. Aquaporin water channels only allow water molecules to selectively pass through the channel and each aquaporin water channel transports up to 1 billion water molecules per second. The high water permeability characteristic of mammalian red blood cell membranes is now known to be caused by aquaporin, AQP1 (figure 1). This channel freely permits movement of water across the cell membrane, but the cell membrane is not permeable to other small, uncharged molecules or charged solutes. AQP1 is a tetramer with each subunit similar to an hourglass containing an aqueous pore (figure 2a,b). Additionally, some aquaporins, e.g. human AQP5 (mainly localized in cells proximal to air-interacting surfaces) or spinach SoPIP2;1, incorporate a gating mechanism. Channel closure can be a consequence of dephosphorylation of two conserved serine residues under drought, or of the protonation of a conserved histidine residue following a drop in cytoplasmic pH during flooding (figure 2c).

Figure 1.

Functional schematic view of water passage through human AQP1 (Protein Data Bank (PDB) id: 4CSK) [1]. (a) The extracellular vestibule and the intracellular vestibule of the channel contain water in bulk solution. Arginine-195 and histidine-180 provide fixed positive charges to repel proton passage. A single water molecule forms hydrogen bonds with the side chains of highly conserved asparagines-76 and -192. Partial positive charges are provided by the orientation of the two α helices that enter but do not entirely span the bilayer. (b) Top down view of the aquaporin water channel. Residues facing the channel are coloured in yellow. The residues H180 and R195 are labelled to show the size-restriction gate.

Figure 2.

Aquaporin crystal structure. (a) Hourglass shape of human aquaporin AQP1 (PDB id: 4CSK) [1]. The central part of the protein is composed of two half-helices (green and orange). (b) Water pathway (red dashed line) across AQP1. The protein is showed as grey van der Waals spheres. (c) Open (loop in green) (PDB ID: 2B5F) and closed (loop in red) (PDB ID: 1Z98) [2] conformations of spinach aquaporin SoPIP2;1.

The aquaporins reported to date crystallize as homotetramers with each monomer forming an independent transmembrane channel. Such tetramers display extended hydrophobic interactions between monomers [3]. However, the requirement for tetramerization of aquaporins that forms four apparently independent channels remains enigmatic [4]. The crystal structure of the atomic resolution of human AQP1 tetramer [1] imposed into the lipid bilayer in the periodic simulation box is shown in figure 3.

Figure 3.

Membrane protein simulation system. Top (a) and side view (b) of the tetramer simulation system of a human AQP1 (PDB id:4CSK) [1] embedded in a pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) bilayer. In (b), the front monomer is removed for clarity. Water molecules in the water channel of AQP1 are shown as transparent blue van der Waals spheres.

2. Aquaporin superfamily: major intrinsic proteins

Major intrinsic proteins (MIPs) constitute a large superfamily of transmembrane protein channels that are grouped together on the basis of homology. The MIP superfamily includes three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. The aquaporins are water-selective membrane channels expressed in almost every organism and involved in the bidirectional transfer of water and small solutes across cell membranes. The aquaglyceroporins are permeable to water, but also to other small uncharged molecules such as glycerol, urea or ammonia. The third subfamily includes so-called superaquaporins (S-aquaporins) or subcellular aquaporins, a third subfamily present in animals but not in plants, fungi or bacteria with uncertain permeability [5].

Aquaporins are proteins with molecular masses around 30 kDa (monomer size) [6]. Three-dimensional structures of several aquaporins have been determined and the quaternary structures of the proteins reveal that they all form homotetramers where each monomer acts as a functional unit. Sequence homology similarity suggests that the functional units of all members in this superfamily are predicted to have six hydrophobic, membrane-spanning α-helices connected by five loops of variable length that delimit a polar channel with two wide vestibules and a narrow pore. Two of the connecting loops, namely B and E, interact with each other from opposite sides through two highly conserved (Asn-Pro-Ala) motifs conserved throughout the aquaporin family, forming a seventh transmembrane region that contributes to the pore region of aquaporins.

To date, 13 isoforms of aquaporins have been discovered in mammals (AQP0–AQP12), nine of which are localized in different parts of the renal tubular epithelium [7]. Two main groups of aquaporins are distinguished: (i) classical aquaporins, permeable only to water molecules (AQP0, AQP1, AQP2, AQP4, AQP5), and (ii) aquaglyceroporins, permeable to other small molecules, such as glycerol and ammonia (AQP3, AQP7, AQP9, AQP10) [8]. In addition, a third group has been recently isolated, the so-called unorthodox aquaporins (AQP11 and AQP12), which share low homology with other proteins from this family [9]. AQP6 and AQP8 are classified as unorthodox aquaporins; however, due to their ability to transport other small molecules, the present review will discuss them along with the rest of the aquaglyceroporins located in the kidneys [7]. The aquaporin family is implicated in numerous physiological processes as well as the pathophysiology of a wide range of clinical disorders. The critical residues affecting channelling performance of AQP1 are shown in table 1, while the other aquaporins are detailed in the electronic supplementary material, table S1. Some mutations were also introduced artificially to increase our understanding of the mechanism of water permeability and/or to change selectivity.

Table 1.

Mutations in AQP1 affecting its permeability and selectivity.

AQP1	C189S	C189S mutant of AQP1 induces the same CO2 permeability as the wild- type AQP1 but the C189S-dependent increase in CO2 permeability is insensitive to p-chloromercuribenzene sulfonate (pCMBS).	[10,11]
AQP1	ΔT157, ΔT239 (lacking)	Inactivation of any of two protein kinase C (PKC) phosphorylation sites T157 and T239 abolishes the positive regulation of AQP1 water permeability by PKC.	[11,12]
AQP1 (human)	H180A/R195 V	This double mutant changes the electrostatics of the selectivity filter region and facilitates proton entry from the bulk water into the narrow AQP1 channel. The double mutation drastically drops the overall free-energy barrier by approximately 20 kcal/mol via simultaneously relaxing the direct electrostatic interaction (by R195 V) and the dehydration effect (by H180A).	[13,14]
AQP1 (rat)	H180A	Replacing H180 with alanine increases the channel diameter and reduces the dehydration penalty for the proton. The H180A mutant passed ammonia significantly faster than the wild-type. The mutation did not affect water permeability.	[15]
AQP1 (rat)	F56A/H180A	Joint mutations of residues F56 and H180 in the selectivity filter of rat AQP1 have been shown to lower its selectivity. Replacement of both residues enlarged the maximal diameter of the aromatic/arginine (ar/R) constriction threefold and enabled glycerol and urea to pass. F56A/H180A mutant passed ammonia significantly faster than the wild-type.	[15]
AQP1 (rat)	H180A/R195 V	Individual or joint mutations R195 in the selectivity filter of rat AQP1 have been shown to lower its selectivity experimentally. In particular the substitution of R195 was shown to allow the passage of protons. The double mutant did not affect water permeability but passed ammonia significantly faster.	[15]
AQP1 (rat)	R195S	The mutation changes the orientations of water molecules along the channel and, therefore, influences proton permeability. The enlarged channel radius allows for the existence of two water molecules at the constriction region.	[15]
AQP1 (human)	Y186F, Y186A, Y186N	Mutants created to investigate whether a tyrosine residue in loop E of AQP1 was involved in water permeability. Y186F conferred high water permeability comparable to that seen in wild-type AQP1 while Y186A and Y186N mutants did not show significant changes.	[16]
AQP1 (human)	A73M	A73M yeast-expressing cells did not exhibit osmosensitivity in comparison with wild-type AQP1-expressing cells.	[17]

3. AQP1 point-of-care device

Aquaporins have important biological roles and have been implicated in several pathophysiological conditions, suggesting a great translational potential in aquaporin-based diagnostics and therapeutics. Recently, overexpression of AQP1 has been associated with many types of carcinoma as a distinctive clinical prognostic factor. This has prompted researchers to evaluate the link between AQP1 and cancer biology [18]. AQP1 is overexpressed in multiple human cancers, including those of the biliary duct, bladder, brain, breast, cervix, colon, lung, nasopharynx and prostate. In the case of colon cancer it has been suggested that AQP1 is especially involved in the early stages of cancer tumorigenesis [19]. Additionally, its expression was reported to be associated with clinical characteristics known to be prognostic, such as histological grade and the status of lympho-vascular invasion and nodal involvement. These findings suggested a link between AQP1 and cancer biological functions, which act to drive cancer development and progression. Various hypotheses have been advanced to explain the critical role of AQP1 in tumorigenesis and especially resistance to apoptosis has been proposed as a part of the mechanism underlying enhanced cell proliferation of AQP1-expressing cells [20,21].

Testing for AQP1 alongside other urine biomarkers such as perilipin-2 (PLIN2), hypoxia-inducible factor 1α (HIF-1α), carbonic anhydrase IX and vascular endothelial growth factor (VEGF) would allow for a more sensitive and specific diagnosis of renal cell carcinoma (RCC) as well as a more accurate prognosis [22,23]. Urine concentrations of AQP1 or PLIN2 were not increased in patients with common non-cancerous kidney diseases, non-cancerous kidney tumours or bladder or prostate cancer. Thus, common kidney disease and non-renal urological cancers do not confound the ability of AQP1 and PLIN2 to detect clear cell and papillary cancers, suggesting that these biomarkers have potential for population screening and/or differential diagnosis.

At the present time, there exists no point-of-care (POC) device for an accurate and easy way of sensing AQP1 in urine. We have begun a design of an AQP1-detecting device, and other selected biomarker-detecting POC devices, based on a similar approach to sandwich enzyme-linked immunosorbent assay (ELISA; a plate-based assay technique) [24]. The complicated nature of standard western blot procedures precludes its clinical implementation for renal cancer screening. While the ELISA test is a solid-phase method and requires a solid support, usually a polystyrene microtitre plate, such a plate would not be required in our method, since a single graphene sheet would play the role of the solid support. A specific AQP1 antibody [25] would be bound through special linker molecules to the surface of a single-layer graphene embedded in a microfluidic channel [26]. A scheme of a single-layer graphene attached to a peptide epitope of AQP1 and interacting with AQP1 is shown in figure 4. The urine sample would be sucked into these microfluidic channels and delivered to AQP1-specific antibodies immobilized on a graphene sheet attached to a field-effect transistor (FET) [26]. AQP1 molecules (if present in the urine sample) would bind to these antibodies, changing the electrical potential of the graphene layer. The changes in electrical potential would be measured and their magnitude would be proportional to the number of AQP1 molecules bound to the graphene layer, reflecting the concentration of AQP1 in the urine sample.

Figure 4.

Schematic of an AQP1 antibody linked to a graphene interacting with a tetramer of AQP1. So far two antibodies have been developed: linking to the extracellular loop of AQP1 (198-GSAVLTRNFSN-208) and to the intracellular loop (249-GQVEEYDLDDDINSRVEMKPK-269) [25].

The proposed method would not require the secondary antibody linked to an enzyme, which is necessary in standard ELISA tests. The proposed method of AQP1 detection is more advantageous over ELISA and western blot because of its direct measurements. While it is expected to have similar accuracy to ELISA it is much quicker and easier to perform (it does not involve long incubation times like ELISA and western blots do). Additional detection of PLIN2 could increase assay efficiency and enable widespread implementation [27]. Further improvements in the AQP1 detection ELISA tests may potentially reduce the AQP1 background coming from common non-cancerous kidney diseases and bladder and prostate cancer, which can lead to false-positive RCC tests.

The monitoring of patients with RCC using a POC device measuring AQP1, and potentially other biomarker levels, could also be useful to track disease progression because different levels of such biomarkers can be found at various stages of tumorigenesis. Recently, it was also found that the serum levels of many biomarkers, including AQP1 and AQP4, were increased in response to injury in mice [28]. This suggests that new types of enhanced diagnostics in injury-related neurological disorders, multi-system deficits and possibly cancer could be developed.

4. Properties and applications of spinach aquaporin

The water permeability characteristics of spinach aquaporin (SoPIP2;1) have been well described [2,5], and therefore, it is a good candidate for application in this technology. The expression of aquaporins was upregulated in response to drought and salinity, and conferred water stress tolerance in plants. Aquaporins are involved in many functions of plants, including nutrient acquisition, carbon fixation, cell signalling and stress responses. The high selectivity and water permeability of SoPIP2;1 make it particularly interesting for biomimetic water desalination and water filtration technology.

The tertiary structure of SoPIP2;1 containing six tryptophan (Trp) residues in the primary sequence provides intrinsic fluorophores for analysing structural fluctuations by fluorescence spectroscopy using the Trp fluorescence emission spectrum, which is sensitive to both the polarity and the dynamics of the environment surrounding the aromatic side chain. Plasencia et al. [5] used circular dichroism spectroscopic studies to probe the structural stability of spinach aquaporins, SoPIP2;1, and concluded that SoPIP2;1 can exist as a stable folded protein in non-ionic n-octyl-β-D-glucoside detergent micellar solutions and that the protein can be transferred from detergent micellar solutions and reconstituted into selected phospholipid membranes, preserving its structural characteristics. It is likely that more suitable reconstitution systems exist for SoPIP2;1 than those studied in the present work. In order to efficiently test a range of systems, new methods are called for. Presently, we are working on developing a new microscopic method that will allow us to test at the same time the incorporation and distribution of the protein in different membrane systems and evaluate the yield of the incorporation and characterize the protein functionality.

Plant aquaporin channel gating is triggered by dephosphorylation of two conserved serine residues, or by the protonation of a conserved histidine residue. The X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 Å resolution and in its open conformation at 3.9 Å resolution has been validated by molecular dynamics simulations [2]. In the closed conformation, loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation, loop D is displaced up to 16 Å and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm (figure 5).

Figure 5.

Proposed gating mechanism for aquaporin (spinach aquaporin SoPIP2;1) (PDB id: 1Z98) [2]. (a) Overview of aquaporin in the closed conformation at pH 8. Interactions between loop D (in yellow) and a Cd²⁺-binding site at the N-terminus insert a hydrophobic plug indicated by L197, thereby occluding the water-conducting pore. The Cd²⁺ ion and water molecules in the water-conducting channel are shown as yellow and red spheres, respectively. Residues involved in gating by phosphorylation (S115 and S274) as well as gating by pH (H193) are indicated. (b) Close-up view of H193. In the protonated state, an alternative rotamer of the H193 side chain (in white) may be adopted which is within the hydrogen-bonding distance of D28.

5. Scaling-up expression and purification of recombinant aquaporins

Aquaporin-mediated water transport is a prominent example of how Nature has evolved an effective mechanism for purifying water, and many technologies based on biomimetic membrane transport are now attracting considerable commercial interest. However, successful reconstitution and stabilization of functional proteins in biomimetic membranes depend on suitable choices of both detergent and host lipid membrane components.

Recombinant aquaporins have been expressed only in laboratory-scale quantities for screening, functional, regulatory or structural studies [29,30]. One of the main obstacles in protein production is that membrane protein overexpression in vivo is hampered by their complex structure, hydrophobic transmembrane regions, host toxicity, and the time-consuming and low-efficiency refolding steps required. Recent developments of high-expression systems may, however, provide insights into how large-scale aquaporin production may be realized. These include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and baculovirus/insect cell-based systems; for a recent review, see [31].

6. Status of aquaporin membrane development

Kumar et al. [32] suggested that membranes with very high permeability and salt rejection may be constructed based on aquaporin protein function. Based on the measured water permeability of AqpZ-containing proteoliposomes, these authors postulated that AqpZ-based biomimetic membranes can potentially achieve a membrane permeability as high two orders of magnitude more permeable than existing commercially available seawater reverse osmosis (RO) membranes. However, a major issue remains unresolved: because the membrane is constructed from nanoscale elements (the aquaporins), how can the biomimetic membrane be scaled-up and stabilized to two dimensions suitable for industrial applications? Several design strategies have recently been proposed (figure 6).

Figure 6.

Schemes for the fabrication and water purification mechanism of the E. coli AqpZ-vesicle-imprinted membrane. (a) Schematic of the AqpZ-vesicle-imprinted membrane preparation: (1) AqpZ polymer vesicles, (2) porous cellulose acetate membrane substrate, (3) AqpZ vesicles immobilized on the porous membrane, (4) AqpZ-vesicle-imprinted membrane, and (5) cross-section of the AqpZ-vesicle-imprinted membrane. (b) Water purification mechanism of the AqpZ-vesicle-imprinted membrane under pressure. Water molecules in the feed solution will penetrate the entire membrane in three steps: (1) passing from the feed solution to the vesicles through the AqpZ water channel located at the polymer bilayer facing the feed solution, (2) passing from the vesicles to the substrate membrane through the AqpZ located at the polymer bilayer facing the substrate membrane, and (3) penetrating the porous hydrophilic substrate membrane into the permeate solution. (Reprinted with permission from Xie et al. [33]. Copyright © 2013 Royal Society of Chemistry.)

These include membranes established across multiple micrometre-scale apertures either as free-standing lipids or polymer membranes or as membranes stabilized by polymeric support materials. Other approaches rely on nanoporous support material onto which membranes are deposited. These include charged lipid vesicle depositions onto commercially available nanofiltration membranes where the recipient surface was either cross-linked polyamide or sulfonated polysulfone, both of which are negatively charged at pH 7; rupture of aquaporin-containing polymersomes on methacrylate-functionalized cellulose acetate membranes with detergent-stabilized His-tagged aquaporin added to monolayers with nickel-chelating lipids; and proteopolymersome deposition onto polycarbonate track-etched substrates coated with gold and functionalized with photo-active acrylate groups.

7. Forward osmosis membrane

Forward osmosis (FO) is a membrane separation technology powered by the osmotic pressure gradient. Unlike RO, which needs external pressure to function, FO is driven by the osmotic pressure difference across a semipermeable membrane. It has recently gained wider attention in many applications, such as seawater desalination and power generation [29,30,34–37]. There are several FO plants operating now for seawater desalination using full-scale FO membranes with satisfactory results reported [38]. FO processes operate close to atmospheric pressure and rely on the osmotic pressure gradient across a semipermeable membrane for freshwater extraction from feed saline water. Freshwater crosses the FO membrane from the feed to the draw solution side of the membrane and dilutes the draw solution. Diluted draw solution is, typically, sent to thermal or membrane treatment processes for freshwater extraction and draw solution regeneration and reuse. An ideal draw solute should be characterized by the ability to ensure a high osmotic gradient, substantial water flux and efficient recovery at minimal energy consumption [39]. For osmosis-driven desalination, an ideal draw solute should have zero toxicity and low cost among its characteristics [40]. Many solutes suitable for water desalination have been developed, among which some are classified as (i) inorganic-based draw solutions (solutes: CaCl₂, KHCO₃, MgCl₂, etc.) or (ii) organic-based draw solutions—the solutes are non-electrolytes but can generate high osmotic pressure due to their high solubility (e.g. glucose, fructose, ethanol). In recent years, highly hydrophilic nano-sized magnetic particles, functionalized by polyacrylic acid, have been discovered to be crucial in the application of draw solutes in FO desalination as this engineering can yield high osmotic pressure and high water flux [41]. Magnetic separators can be used to recycle the magnetic particles. In general, the FO process does not require high energy for operation and most of the energy consumption would be incurred in the regeneration process [42,43]. It can be coupled with membrane and thermal processes for seawater treatment; for example, FO has been suggested for the pretreatment of feed water to thermal and membrane processes [44,45] to provide high feed quality, especially in the case of high-fouling feed waters. The FO process can operate at high efficiency without the need for frequent cleaning due to the reversible fouling nature. However, there are several challenges facing the application of FO process. One of these challenges is the FO membrane; initially, the membranes used in the FO process were inefficient because of the severe concentration polarization (CP) phenomenon which takes place on the feed and draw solution side of the membrane and which results in a sharp decrease in water flux across the FO membrane.

There has been a plethora of attempts to develop an efficient FO membrane [45–48]; most of these attempts were laboratory-scale experiments. The results shown in the electronic supplementary material, table S2, reveal that experimental work was successful in the development of a high-permeability FO membrane which achieved a staggering water flux of 32 l/m²h using 0.5 mol NaCl draw solution and deionized water as the feed solution. This was almost double the water flux achieved by the HTI cellulose tri-acetate (CTA) membrane using the same salinity gradient resource (electronic supplementary material, table S2). Using a modified polyvinylidene fluoride membrane enhanced water flux because of the larger pore size but caused higher reverse salt diffusion. One of the strategies to improve the performance of the FO process is by increasing the membrane permeability, which compensates for flux loss due to internal and external CP. Aquaporin membranes exhibit water flux higher than that in the traditional CTA and polyamide membranes. Using such a high water permeability membrane will revolutionize the FO process. The low fouling tendency and operating pressure of the FO process will increase the lifetime of the aquaporin membrane and improve the performance of FO process. Future research work should focus on the development of an aquaporin membrane with properties suitable for the FO process such as high water flux and thin porous structure to reduce the effect of CP.

8. Aquaporin and graphene reverse osmosis membranes

Nanometre-scale pores in a single layer of graphene have been shown to be an effective sieve in separating NaCl from water (figure 7).

Figure 7.

Scheme of separating NaCl from water using nanoporous graphene. (Reprinted with permission from Cohen-Tanugi & Grossman [49]. Copyright © 2012 ACS Publications.)

Cohen-Tanugi & Grossman [49] have reported from classical molecular dynamics the desalination performance of membranes as a function of the membrane's ability to prevent salt passage, which depends critically on pore diameter (pore size varies from 1.5 to 62 Ų) with adequately sized pores allowing for water flow while blocking ions. According to simulation results, the maximum pore diameter that will reject salt ions is 5.5 Š(pore size approx. 24 Ų), while the minimum pore diameter required for water passage is 3.8 Š(pore size 11.3 Ų) [6]. The results of simulations also indicate that the water permeability of nanoporous graphene is several orders of magnitude higher than that for conventional RO membranes, and that this material may have a valuable role to play in water purification.

A lipid bilayer with aquaporin channels imprinted in it should have somewhat smaller permeability than nanoporous graphene, because of the smaller pore size and similar or smaller pore density (depending on how many aquaporin units are immersed in the membrane). But still we expect a permeability improvement, relative to a standard RO membrane, of one to two orders of magnitude. Under these conditions, the energy required for purifying water will drop significantly, potentially making desalination a much more widely applicable technology. Although it is likely that fluxes will still be modest, low operating pressures could make the technology commercially attractive due to low energy cost. The challenges for aquaporin membranes will be robustness and longevity.

9. Conclusion

Our interest in AQP1 stems from its important role in RCC and a critical need to design efficient POC devices. Owing to application of AQP1-specific antibodies immobilized on a graphene layer attached to a FET, the measurement process would be much simpler and faster than in standard biomedical assays. Recombinant aquaporin (from spinach or bacteria E. coli) films can potentially improve water desalination at reduced operational costs and low energy consumption. Despite the high performance of aquaporin membranes, it will still be a long time before these membranes are available for commercial application. Techno-economical issues need to be addressed before aquaporin technology can be applied for large desalination projects.

Data accessibility

Additional data can be found in the electronic supplementary material.

Authors' contributions

J.J. performed all computations required for molecular structures, made all figures of proteins and graphene and participated in writing the paper; A.S. selected literature information and completed table 1. S.F. analysed computed systems and participated in writing the paper. A.A. and A.O.S. contributed to the section on forward and reverse osmosis in desalination. J.R.E. contributed to the development of ideas on renal cell carcinoma detection. P.L. contributed to the expression of aquaporin and site-directed mutations. K.R. and N.B. contributed to the development of cartridges using aquaporin biomimetic membrane, S.R. contributed the idea of fundamental mechanisms in the aquaporin–graphene interface. P.M.A. was involved in the development of single-layer graphene. Initial ideas on aquaporins are credited to V.R. Expression of aquaporins from plasmids in Saccharomyces cerevisiae and a large number of thermostable aquaporins was performed in V.R.'s laboratories. V.R. coordinated the study and drafted the manuscript. All authors gave final approval for publication.

Competing interests

All authors declare no competing interests.

Funding

V.R. acknowledges the Rothschild Foundation, NIH, NSF, USAFOSR and the Wallace H. Coulter Foundation for early support of research at Florida International University.