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Pathways and Challenges for Biomimetic

Desalination Membranes with Sub-Nanometer
Channels
Cassandra J. Porter, Jay R. Werber,* Mingjiang Zhong, Corey J. Wilson, and Menachem Elimelech*
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sı Supporting Information

ABSTRACT: Transmembrane protein channels, including ion

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channels and aquaporins that are responsible for fast and

selective transport of water, have inspired membrane scientists
to exploit and mimic their performance in membrane
technologies. These biomimetic membranes comprise discrete
nanochannels aligned within amphiphilic matrices on a robust
support. While biological components have been used directly,
extensive work has also been conducted to produce stable
synthetic mimics of protein channels and lipid bilayers.
However, the experimental performance of biomimetic
membranes remains far below that of biological membranes.
In this review, we critically assess the status and potential of
biomimetic desalination membranes. We first review channel
chemistries and their transport behavior, identifying key characteristics to optimize water permeability and salt rejection. We
compare various channel types within an industrial context, considering transport performance, processability, and stability.
Through a re-examination of previous vesicular stopped-flow studies, we demonstrate that incorrect permeability equations
result in an overestimation of the water permeability of nanochannels. We find in particular that the most optimized
aquaporin-bearing bilayer had a pure water permeability of 2.1 L m−2 h−1 bar−1, which is comparable to that of current state-
of-the-art polymeric desalination membranes. Through a quantitative assessment of biomimetic membrane formats, we
analytically show that formats incorporating intact vesicles offer minimal benefit, whereas planar biomimetic selective layers
could allow for dramatically improved salt rejections. We then show that the persistence of nanoscale defects explains
observed subpar performance. We conclude with a discussion on optimal strategies for minimizing these defects, which could
enable breakthrough performance.
KEYWORDS: biomimetic membrane, molecular sieving, reverse osmosis, desalination, aquaporin, carbon nanotube, water purification,
nanochannel, vesicle, stopped-flow

A s stressors like population growth, industrialization, and

climate change threaten to deplete and contaminate our
freshwater resources, larger bodies of saline water could
provide a vast supply of water for drinking, agricultural, and
industrial use.1 However, desalination of these waters requires
energy requirements for the RO stage have drastically reduced
from ∼15 kWh m−3 using the original cellulose acetate
membranes of the 1970s down to only ∼2 kWh m−3, only
∼25% above the practical minimum energy.2
Despite the substantial reduction in energy consumption and
more energy and financial resources than traditional freshwater
overall cost, seawater RO still has room for improvement. While
purification methods.2 Currently, the state-of-the-art technology
for desalination is reverse osmosis (RO) using thin-film- current water permeabilities enable near-optimal performance,
composite (TFC) polyamide membranes.3,4 Fully aromatic
TFC-RO membranes are readily produced at industrial scale Received: July 10, 2020
through interfacial polymerization, whereby a rapid reaction Accepted: September 4, 2020
occurs at the interface of immiscible organic and aqueous phases Published: September 4, 2020
to form a highly cross-linked polyamide selective layer on a
porous support5 (Figure 1). With the advent of the TFC-RO
membrane and energy recovery devices, seawater desalination

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Figure 1. Transition in desalination research from focusing on dense polymers that reject salt by a solution-diffusion mechanism to considering
sub-nanometer channels capable of molecularly sieving ions. In the solution-diffusion panel (left), common reactants for TFC-RO membranes
are represented, which rapidly react at an organic−aqueous interface during interfacial polymerization to form a cross-linked, fully aromatic
polyamide selective layer with characteristic ridge-valley morphology. Salt rejection determined by a solution-diffusion mechanism results from
the higher partitioning and/or diffusion rates of water over ions. In the ion sieving panel (right), common molecular sieves that have been
considered for desalination are shown, with ideal water pathways illustrated. In pores similar in size to water, single-file water transport is
induced. Nanotubes and nanochannels can be synthetic (e.g., carbon nanotubes) or biological (e.g., aquaporins). To produce nanoporous
sheets, sub-nanometer pores where only a few atoms are vacant have been etched in single-layer graphene using chemical oxidation, electron
beam irradiation, doping, and ion bombardment.12 For 2D laminates, the water pathway is through interlayer spaces between sheets. Studies so
far have primarily considered graphene oxide nanosheets for 2D laminates.13,14 The molecular sieving mechanism for ion rejection is by size
exclusion, where highly uniform pores exclude larger solutes and ideally transport only molecules similar in size to water.

increased water-solute selectivity would allow for reduced
operational costs, improved reliability and efficiency, and
enhanced product water quality.4 For example, TFC-RO
membranes inadequately retain chloride and some small neutral
solutes, such as boron in seawater desalination and trace organic
contaminants in wastewater reuse, necessitating extra purifica-
tion steps which increase the cost of desalination.4
Transport through the polyamide layer is well described by
the solution-diffusion model, in which permeants (i.e., water and
solutes) partition into the dense polyamide layer and diffuse
through it (Figure 1).6 The resultant permselectivity of the
membrane is attributed to differences in abilities and rates of
species to dissolve into and diffuse through the polyamide
membrane material.7 Although intrinsic water permeability can
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far exceed salt permeability during solution diffusion, as it does
for polyamide, historical data suggest that it will be difficult to
significantly advance performance with polymeric systems.
Commercial desalination and water purification membranes
typically exhibit a permeability−selectivity trade-off, similar to
the Robeson plot for polymeric gas separations.8−11 Further-
more, despite many decades of extensive research, no polymeric
material has yet surpassed the desalination performance (i.e.,
water permeability, water-salt selectivity, and cost-effectiveness)
of fully aromatic polyamide.
To overcome the limitations of the solution-diffusion-based
polyamide membranes, research focus has shifted toward the
development of desalination membranes that remove solutes via
molecular sieving. In this mechanism of ion rejection, highly
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uniform, rigid pores that are smaller than the diameter of
hydrated salt ions transport water and nearly completely reject
ions by size exclusion (Figure 1). Recent formats of molecular
sieves considered for desalination include nanotubes and
nanochannels, two-dimensional (2D) laminates, and nano-
porous sheets (Figure 1).14 However, these top-down efforts
have failed so far to achieve adequate salt rejection due to the
persistence of defects coupled with the daunting challenges of
tuning interlayer spacing or pore size.12,13,15 Biomimetic
membranes, or composites comprising an amphiphilic matrix
with discrete, aligned nanochannels on a robust support, may
provide a platform for industrial-scale molecular sieves that
overcome the limitations of solution-diffusion-based polyamide
membranes.
After over 3.5 billion years of evolution,16,17 the cell
membranes of modern organisms can perform an array of
highly complicated functions, which rely on a system of complex
transmembrane proteins aligned within the amphiphilic lipid
bilayer. In this system, water and only select ions pass through
channel pores and pumps, depending on the energy and nutrient
needs of the cell.18,19 In pioneering work, Preston et al.
determined that an integral membrane protein formed a
biological channel that selectively transports water in and out
of many types of cells. This protein was the CHannel-forming
Integral Protein of 28 kDa (CHIP28),20,21 later called the
aquaporin. For these discoveries, Peter Agre and Roderick
MacKinnon shared the 2003 Nobel Prize in Chemistry.
Through additional biophysical studies, ion channels also
showed impressive selectivity, inspiring the design of synthetic
ion channels.22−25 Eventually, researchers realized the potential
implications of these channels for industrial-scale water
purification, especially aquaporin in the use of desalination,
and attempted to produce biomimetic membranes, or materials
that mimic the structure and performance of biological
membranes.26−35 While much of the work has focused on
water-solute separations, the biomimetic membrane format also
presents opportunities to develop membranes with tunable
selectivity based on a chosen channel type.
However, translating biological mechanisms into industrial-
scale technology necessitates scale-up by orders of magnitude
from the microscopic size of a cell membrane to tens of square
meters.36 For industrial relevance, the synthesis of a biomimetic
membrane would need to be cost-effective and simple.
Simultaneously, such a membrane would need to be
mechanically stable under RO pressures exceeding 70 bar and
chemically stable during repeated membrane cleaning and
usage.37 Notably, even at the lab scale, sufficiently high-salt
rejection has not yet been achieved for biomimetic desalination
membranes after over a decade of research.37,38 Therefore,
channels, selective layer formats, synthesis strategies, and
support layer types must be carefully considered to attain the
capabilities of this technology. While certain aspects of
biomimetic desalination membranes have been reviewed
recently,37−42 a critical analysis of their performance and their
potential application in water-treatment processes remains
necessary.
In this critical review, we examine efforts toward biomimetic
desalination membranes for water purification in order to
identify the best strategies to realize their full potential for both
desalination and solute−solute selectivity. We first examine
molecular transport, contrasting solution-diffusion with molec-
ular sieving and assessing transport through the mixed matrix of
biomimetic selective layers. We next identify the key character-
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istics of the aquaporin that explain its ultraselectivity and fast
water transport, comparing this biological channel to several
synthetic channels and placing each in industrial context. Using
corrected analysis of reported permeability measurements, we
then show that the water permeabilities of many channels have
been overestimated. Subsequently, we predict best-case-
scenario outcomes for common biomimetic formats, including
membranes with intact vesicles and membranes with planar
biomimetic layers. For the more promising planar format, the
biggest challenge is the presence of nanoscale defects. Through
mathematical models, we estimate the defect density for several
reported biomimetic membranes. We then discuss synthesis
pathways that could limit both the presence of defects and the
effect of defects on transport performance. We conclude with a
discussion on the practicality of biomimetic desalination
membranes and how to best exploit the strengths of discrete
nanochannels as molecular sieves in other applications beyond
desalination.
TRANSPORT BEHAVIOR OF BIOMIMETIC
DESALINATION MEMBRANES
Transport through the Amphiphilic Matrix. The key
components for transport in biological membranes are the
selective transmembrane proteins and the amphiphilic matrix
(i.e., the lipid bilayer) in which these proteins assemble. For
industrial biomimetic desalination membranes, these compo-
nents need to cooperate to reject salt and transport only water.
Molecular transport should predominantly occur through the
incorporated channels that reject solutes by molecular sieving.
Meanwhile, salt flux should be minimized through the
surrounding amphiphilic matrix. The most promising matrices
in biomimetic membranes comprise amphiphilic block copoly-
mers rather than lipids, with the resulting matrix enabling the
alignment of the nanochannels while providing mechanical and
chemical robustness.
Molecular transport through both amphiphilic matrices and
dense polymer membranes (e.g., polyamide selective layers) is
described by the solution-diffusion mechanism.43 In this
mechanism, species dissolve or partition into the membrane
matrix, diffuse in the direction of decreasing chemical potential,
and desorb into the permeate stream. Water flux, Jw, for dense
polymeric membranes is described by44
Jw = A(ΔP − Δπm) (1)
where A is the water permeability coefficient, ΔP is the applied
hydraulic pressure, and Δπm is the osmotic pressure difference
across the membrane.
Rates of both dissolution and diffusion affect overall water and
salt permeabilities, Pw and Ps. For materials with low-water
content, permeability P (for both water and solute) can be
approximated as7,43,45
K pD
P=
d (2)
where Kp is the partition coefficient, D is the diffusivity, and d is
the membrane thickness. The thickness-dependent permeability
as defined here and in the biophysical literature43 is often called
permeance in industrial membrane literature.7,45 Polyamide and
other glassy polymers predominantly have diffusion selectivity
for aqueous separations, whereby differences in D result in
selectivity (e.g., between water and ions). Bilayers predominately
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have solubility selectivity, whereby differences in Kp result in
selectivity.43
The water permeability coefficient A is related to the water
permeability Pw by45
PwVw
A=
̂
RT (3)
where Vw is the molar volume of water, R̂ is the ideal gas
constant, and T is the absolute temperature. Solute flux Js is
related to diffusive salt permeability by the Fickian diffusion
equation:45
Js = BΔCm (4)
where B is the salt permeability coefficient (equivalent to Ps) and
ΔCm is the solute concentration difference across the
membrane. Together, water and solute fluxes under pertinent
operating conditions determine the observed solute concen-
tration in the permeate, Cp, which in turn is related to the
observed solute rejection, Ro, by
Js /Jw Cp
Ro = 1 − =1−
Cb Cb (5)
where Cb is the bulk feedwater concentration of solute.
Transport through Channels. A nanochannel at approx-
imately the size of a water molecule could completely reject
essentially all ions by molecular sieving. If these selective pores
also have sufficient water permeability, typically involving single-
file water transport, they could drastically increase the overall Jw
of a biomimetic layer without affecting Js, thereby reaching salt
rejections above those attainable by current desalination
membranes (see eq 5). To these aims, it is imperative that
desalinating nanochannels: (i) highly reject ions, (ii) permeate
water at a sufficiently fast rate, and (iii) load within the
amphiphilic matrix at acceptable densities.
For convective flow through relatively large channels (i.e., >1
nm in diameter), the macroscopic rules of hydrodynamics apply,
whereby the key determinants to flow are channel length and
diameter, described by Hagen−Poisueille’s law.46,47 For
narrower nanochannels with single-file water transport, bulk
water properties and continuum hydrodynamics cannot apply.
Many models envision nanotubes and nanopores as highly
slippery cylinders with no internal resistances. In these
stochastic models, single-file water transport is described by
either the collective diffusion of a water column based on
random walks or by the movement of a vacancy in the opposite
direction of water flow.46,48,49 Both these treatments ignore the
entrance effects as well as energy barriers often present in
biological pores, like hydrogen-bonding sites and increased
resistance through constriction zones within noncylindrical (e.g.,
hourglass) pores.
To overcome these limitations, a collective diffusion model
was proposed in order to find water permeability through each
channel, Pw,c.50 The most recent of these collective diffusion
models was derived on the basis of the Finkelstein model that
predicts the work required to move a column of water molecules
(represented as hard spheres) through a pipe of a given length by
osmotic force.51 In this newest collective diffusion derivation,
the water diffusivity, Dw, is related to the water permeability:46
Dw vw
Pw,c =
z2 (6)
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where vw is the volume of one water molecule and z is the
distance between each water molecule. Internal and entrance
resistances are incorporated into the effective diffusivity, as
resistances from pore-lining chemical functionalities and
constriction zones cannot be decoupled for a water column.
Additionally, these resistances are currently unpredictable a
priori, requiring molecular dynamics (MD) simulations to
resolve the overall Dw. The single-channel water permeability
can be converted to the channel water permeability coefficient,
Ac, by dividing by the overall channel projected area (i.e., the area
on the basis of the outer and not inner pore diameter) and
subsequently using eq 3.
Overall Transport Through the Biomimetic Mem-
brane. For a high-rejection membrane with relatively large
water flux, the concentration and osmotic pressure changes
across the membrane are not accurately described by the
difference in concentration between permeate and bulk feed due
to the development of an unstirred, more-concentrated layer on
the feed side near the membrane surface. This concentration
polarization effectively increases the osmotic pressure and
depends on membrane permeability as well as operational
factors like applied driving force (typically hydraulic pressure),
crossflow rate, and feed concentration. Film theory is commonly
used to account for concentration polarization for isotropic
membranes when using eqs 1 and 4.44 In the case of discrete,
highly permeable, and selective nanochannels that are spaced
relatively far apart within an amphiphilic matrix, an unstirred
hemisphere surrounding the entrance of each nanochannel on
the feed side forms.52 However, this unstirred hemisphere is
predicted to negligibly exacerbate concentration polarization
due to a sufficiently fast rate of water diffusion from surrounding
regions of the bulk feed, resulting in similar concentration
polarization to conventional polymeric membranes. For densely
packed nanochannels, these unstirred hemispheres could
converge into a much thicker unstirred layer and require special
consideration.52 However, high-density packing is currently
atypical for biomimetic layers. In other words, concentration
polarization for biomimetic membranes is expected to be similar
to that of conventional polyamide membranes.
For both isotropic membranes and molecular sieves, the
energy needed for transport across the membrane, or activation
energy, can be determined using the Arrhenius equation by
varying the temperature during transport. While the activation
energy is necessary to estimate permeability at varied temper-
atures, it is also an important metric for understanding the
mechanism of flow. The activation energy for water flow through
nanochannels (∼4 kcal/mol) is typically much less than for
water flow through lipid bilayers (∼15 kcal/mol), as most
nanochannels are relatively static, whereas thermally driven lipid
motion is required for molecular diffusion.53,54
To calculate the overall water permeability coefficient of the
biomimetic layer, Abm, the channels and amphiphilic matrix are
considered resistors in parallel through the summation of their
respective water permeability coefficients (Aam and Ac):
Abm = ϕcAc + (1 − ϕc)A am (7)
where ϕc is the channel areal coverage. To achieve an overall
acceptable water permeability of a biomimetic layer for a given
application, the target single-channel permeability and pore-
loading density are contingent on one another, as evident from
eq 7. In other words, a pore with lower water permeability may
be sufficient if channel density is relatively high, and vice versa.
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Figure 2. Channel types for biomimetic membranes, including biological as well as bioinspired and bioderived. (a) Single channel water
permeability versus pore interior diameter. The pore interior diameter here is defined as the inner diameter of the most constricted region. For
PAH[4], the pore diameter shown refers to the average width of dynamic voids that formed in channel clusters. Channel permeabilities from
stopped-flow data and simulations were adjusted to 25 °C and corrected for any previous errors in permeability calculation (see SI, Section S1
for details). (b) (left) Overhead view of AqpZ tetramer and (right) side view of single AqpZ channel with characteristic hourglass shape. (c)
GramA dimer as it exists in biological and vesicular environments. In organic solvents, the monomers can intertwine to form a parallel or
antiparallel helix. AqpZ and GramA diagrams were drawn using PyMOL69 with protein sequences from the Research Collaboratory for
Structural Bioinformatics Protein Data Bank (RCSB-PDB, https://www.rcsb.org/), PDB-ID codes 1RC270 and 1NRM.71 (d) Dependence of

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Figure 2. continued

water diffusivity through pores on the number of interior hydrogen-bond points. Data extracted from ref 56. (e) Cyclic peptide nanotubes. (left)
Hydrogen-bonding pattern of pore-forming, stacked cyclic peptides. A polyglycine structure is shown for simplicity; side residues would
typically be present. (right) Modified cyclic peptide with interior peptide-mimicking functional groups. The analogous unmodified cyclic
peptide is radially symmetrical with a fourth primary amine side chain (cyclo[(D-Ala-Lys)4]).72 (f) Single-walled carbon nanotube porins (wide
and narrow) with armchair pattern. Number of carbons approximate those of wCNTP and nCNTP. (g) (left) PAP[5] and (right) PAH[4]
nanochannels with peptide appendages that form interarm hydrogen bonds. (h) (left) Aquafoldamer subunits with “sticky” ends. Differences in
end groups that comprise Aqf1 and Aqf2 subunits are illustrated. (right) Weak hydrogen-bonding pattern of pore-forming, stacked
aquafoldamers. Six subunits are needed to cross a DOPC membrane. (i) Pure water permeability versus total channel areal coverage in a DOPC
bilayer. Single channel permeabilities from (a) were divided by channel cross-sectional area and converted into channel water permeability (A)
coefficients using eq 3. Overall biomimetic layer A coefficients were calculated for various densities of channels within a DOPC bilayer using eq
7. DOPC bilayer hydraulic permeability was taken as 0.15 L m−2 h−1 bar−1.73 The shaded region indicates the water permeability of current
commercial TFC-RO membranes, an adequate range for desalination performance. Permeabilities are listed in Table S1.

KEY MATERIAL COMPONENTS OF BIOMIMETIC
DESALINATION MEMBRANES
Biological Channels. Biological membranes contain an
array of passive transport channels as well as active
cotransporters and pumps with varying functions.42,55 The
broad range of biological activity occurring across the same
amphiphilic matrix suggests that a successful biomimetic
membrane could be tuned for specific applications, from
containing self-aligned channels that reject nearly all solutes
for desalination to more niche applications requiring solute-
selective transport. The proper channel type must be selected for
stability, processability, and performance.
For biomimetic desalination membranes, aquaporins such as
human Aqp1 and bacterial AqpZ that transport only water have
received particular interest. Single-channel water permeabilities
of 2.7 × 10−13 cm3 s−1 for AqpZ and 4.9 × 10−13 cm3 s−1 for Aqp1
have been measured (Figure 2a).56 Aquaporins typically
assemble into tetramers, with each individual protein subunit
containing a water pore that is formed by several membrane-
spanning alpha helices (Figure 2b).42 Aquaporins have been
extensively explored for direct use in biomimetic membranes
and have been functionally reconstituted in lipid and block
copolymer systems. Single channel permeability has been shown
to be similar in different matrices, although reconstitution
efficiency can vary substantially.57 In addition to serving as a
potential membrane selective agent, aquaporins provide a model
for synthetic channels in terms of both the attainable transport
performance and structure−function relationships. Accordingly,
we will briefly review some of the main distinguishing features of
aquaporins.
Aquaporin’s water-solute selectivity with efficient water
permeability is the result of four main attributes: (i) a
predominantly hydrophobic interior, (ii) an hourglass shape
tapering to only ∼0.28 nm in diameter at the constriction zone,
(iii) a series of amino acid residues, asparagine-proline-alanine
(NPA), located before the constriction zone,58−62 and (iv) an
abundance of inward-facing carbonyl groups.63 A channel with
extreme internal hydrophobicity induces a slip boundary
condition where only viscosity is the key limiting factor in
transport rate.64 Moreover, for aquaporin, water is funneled into
a single file, gradually reducing the hydrogen-bond number from
four to two and dissipating bulk-water viscosity.65,66 At a size
around that of water, the constriction zone rejects ions via size
exclusion.58,67 Meanwhile, several features hinder ion transport
via Donnan and dielectric exclusion. Positive poles at the
channel center induced by the two NPA-bearing helices and a
positively charged arginine within the constriction zone prevent
cation transport, while several pore-accessible carbonyl oxygens
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inhibit anion transport.63 The aquaporin completely rejects
protons as well, either by a strong electrostatic repulsion
provided by the NPA motif and the arginine in the constriction
zone or by dipole inversion (i.e., the rotation of water molecules)
that breaks the water wire;60−63 the exact proton-exclusion
mechanisms are currently under debate. Although exclusion of
protons is critical for biological function, it is likely less
important for industrial separations.
Simulations and mathematical analyses have further eluci-
dated some special features of the aquaporin, especially
regarding the influence of channel shape and hydrogen bonding
during water permeation. A continuum hydrodynamics analysis
using finite calculations and subsequent MD simulations
demonstrated that hourglass or cone-shaped channels with
short constriction regions mitigate the large energy barrier
present at the entrance of a water-selective cylindrical pore,
while small cone angles of around 10−30° reduce resistance
when transitioning from funnel to constriction zones.65,66,68 The
smaller the channel length, the larger the optimal funnel angle,
with the greatest observed angle in nature being ∼27° for
Aqp5.66 For a cylindrical pore that induces single-file water
transport, the large entrance effects hinder rapid transport since
each water molecule must simultaneously shed two hydrogen
bonds.66,68 There are a multitude of water-selective protein
channels with varying numbers of hydrogen-bonding sites at
their pore mouths and interiors, including aquaporins that
convey only water (e.g., Aqp1 and AqpZ) and aquaporins for
water and glycerol (GlpF) as well as peptide-based gramicidin
dimers for transporting water and small monovalent cations
(Figure 2c). Notably, the diffusivity of water through each
biological channel is negatively correlated with the number of
hydrogen-bonding sites within the channel itself (Figure 2d).56
In terms of pore diameter of aquaporins, the constriction size
correlates with greater permeability, with GlpF having markedly
higher water permeability. However, when comparing channels
of varying chemistries, there is no correlation between
constriction size and permeability, as other factors predominate
(Figure 2a).
Synthetic Nanochannels. Although protein nanochannels
such as aquaporins can be synthesized using well-established
bioprocessing techniques, these transmembrane proteins can
easily denature and lose functionality, making production much
costlier than for water-soluble proteins. Additional stability
challenges will arise during membrane synthesis and use.
Furthermore, biological channels are not necessarily the most
optimal in terms of pore-loading efficiency and functionality for
industrial processes. Synthetic, bioinspired channels could
overcome the instability and poor processability of their
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biological exemplars and provide a greater opportunity to tune
performance for target applications.
The first generation of self-aligning, synthetic bioinspired
channels were cyclic peptide nanotubes (CPNs), or rings of
amino acids designed in flat, stackable units that can crystallize
into nanotubes hundreds of nanometers long with an internal
pore diameter of ∼1 nm (Figure 2e).72,74 CPN units can in situ
self-assemble into a channel within an amphiphilic matrix.75
Bottom-up, in situ assembly helps avoid issues with physical
mismatch that occurs when lengths of the overall channel and
hydrophobic/hydrophilic regions do not align with the
corresponding surrounding matrix lengths.41 While typical
CPNs transport ions, the pore size can be reduced using a
non-natural aromatic amino acid, thereby inhibiting ion passage.
Simulations have shown that manipulation of the interior-facing
functional group can tune the single-channel water permeability,
reaching a maximum of 1.3 × 10−13 cm3 s−1 (Figure 2a,e).72,76
Recently, it was demonstrated that carbon nanotubes can be
cleaved by ultrasonication to lengths of ∼10 nm, after which
these “carbon nanotube porins” (CNTP) self-align within
amphiphilic matrices due to a predominantly hydrophobic
exterior with hydrophilic oxygen functionalities lining the ends.
Narrow carbon nanotube porins (nCNTP) (Figure 2f) of ∼0.7
nm internal diameter exhibit a water permeability of 3.7 × 10−13
cm3 s−1, while wider carbon nanotubes (wCNTP) of a similar
length but with a larger pore diameter of ∼1 nm had lower
single-channel water permeabilities of 1.9 × 10−14 cm3 s−1
(Figure 2a). These permeability values and several others in
this section were adjusted to account for a fitting error
commonly described in biomimetic membrane literature,
which will be discussed later in detail. The significant increase
in permeability for a narrower nanotube porin indicates the
reduction of water−water hydrogen bonding even within pores
well above the diameter of water. On the basis of measured water
and sodium-chloride permeabilities,54,77 nCNTPs are predicted
to have a water-salt selectivity (Pw/PNaCl) on the order of 105,
resulting in salt rejections >99%. Although CNTPs are more
stable and processable than biological pores, it remains a major
challenge to produce large quantities of narrow carbon
nanotubes with uniform pore diameter.
Other artificial channels of recent interest are imidazole-
derived quartet channels (Imdzl) that form planar supra-
molecular matrices with regularly spaced pores.78,79 These
channels are highly selective for water, forming connected
single-file water wires. Within polymerization solutions and
casting dopants, they may highly cluster to form a planar matrix
with pores without needing the assistance of surrounding
amphiphiles for alignment. However, with a reported pure water
single-channel permeability of at best only around 1.8 × 10−16
cm3 s−1 for Imdzl with left-hand chirality ((S)-Imdzl)
(Figure2a),78 a selective layer solely comprising these channels
would exhibit a permeability of only ∼0.16 L m−2 h−1 bar−1. This
transport rate is only slightly higher than that observed for water
passing through a 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine
(DOPC) lipid bilayer (0.15 L m−2 h−1 bar−1) and roughly an
order of magnitude below necessary to approach minimal
desalination energy.4,73 Highly polar imidazole subunits with
secondary amines provide a large number of hydrogen-bonding
sites, which again is inversely correlated with water permeability
(Figure 2d).
In contrast, pillararene-based channels provide a hydrophobic
interior to allow for relatively fast water transport. The central
constriction in these channels is the pillararene ring, formed by
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linking a defined number of benzenediol units with methylene
bridges. In the first example of pillararene water channels, central
rings with five hydroquinone units were used with hydrazide or
peptide-appended arms, which were stabilized by interarm
hydrogen bonds to form a tubular structure.80,81 The peptide-
appended pillar[5]arene (PAP[5]; Figure 2g) exhibited a sharp
molecular weight cutoff (MWCO) of ∼500 Da and a relatively
low single-channel water permeability of 4.6 × 10−15 cm3 s−1
(Figure 2a). PAP[5]s assembled into dense clusters of parallel
channels, increasing the channel pore packing density. However,
ions readily permeated PAP[5] owing to its inner pore diameter
of 0.5 nm.
In the aim of achieving salt rejection, peptide-appended
hybrid[4]arenes (PAH[4]s) were developed to yield a smaller
central constriction (Figure 2g). The stackable subunit
comprises an arene of alternating recorcinol and catechol
subunits (2 each, 4 subunits total), linked alternately at the meta
and ortho positions, with 8 peptide appendages.82 Similar to
PAP[5]s, PAH[4]s self-assemble to form dense clusters within
amphiphilic matrices. The central constriction of PAH[4] is
relatively impermeable; however, once self-assembled into
clusters, lateral windows provide dynamic voids of 0.2−0.4 nm
width (the 0.5−0.7 nm widths reported in the study referred to
distances between atomic centers), allowing for interconnected
water-wire pathways to hop between channels. The resulting
equivalent “per channel” permeability of PAH[4] is 2.7 × 10−14
cm3 s−1.82 Sodium permeation through the channels was
undetectable, while chloride permeability was measurable but
low. Based on reported chloride and water permeabilities,
pristine biomimetic membranes incorporating PAH[4] could
theoretically achieve water-salt selectivity of at least ∼108, far
exceeding the current selectivity of desalination membranes of
104−105.83
Recently developed aquafoldamer-based synthetic water
channels also have promising performance (Figure 2h).84 In
these aquapores, the helical, crescent-shaped oligomer subunits
have chain-end functionalities which intermolecularly hydrogen
bond, assembling into stacked, one-dimensional nanotubes.
Two versions of these aquafoldamer-based water channels were
created (Aqf1 and Aqf2), both comprising pyridine-based
aromatic polyamide chains beginning with an ester and
terminating with a benzene ring.81 This pyridine chain is similar
in composition to fully aromatic polyamide, but with the
replacement of one methine group in each aromatic ring with a
nitrogen. The nitrogen atom forms hydrogen bonds with
surrounding amide hydrogens to promote a helical curl (Figure
2h). Aqf1 differs from Aqf2 in that it has an additional oxygen
and methylene unit before its terminus, giving Aqf1 a rounded
pore with a diameter of 0.28 nm. Meanwhile, Aqf2 is more
cuboidal at 0.21 × 0.27 nm internally. With these small pore
constrictions, Aqf1 and Aqf2 both transport water in single file
and completely reject ions. Under osmotic gradient, Aqf1
exhibited a higher single-channel permeability of 3.0 × 10−14
cm3 s−1 compared to 2.2 × 10−15 cm3 s−1 for Aqf2 (Figure 2a).
The more hydrophobic, slippery interior of Aqf1 likely explains
this phenomenon, where the hydrogen-bonding energy of water
with the pore walls was found to be 15.1 kcal mol−1 for Aqf1 and
16.1 kcal mol−1 for Aqf2. These channels have only been
incorporated in lipid bilayers; hence, self-assembly and stability
in block copolymer matrices are to be determined.
When maximizing pore loading, the permeability of channels
normalized by their cross-sectional areas should be considered.
That is, a channel with a relatively high flow rate through its pore
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Figure 3. Design considerations for common biomimetic membrane formation strategies. (a) Biomimetic compositions for the amphiphilic
channel insertion matrix. (b) Common nanochannels for desalination, from biological aquaporin and gramicidin to bioinspired channels. All
channels self-align within the channel insertion matrix due to matching amphiphilic pattern. (c) Vesicle-encapsulated mixed-matrix formats,
whereby permeate passes through two layers of a channel-laden matrix in intact vesicles. Lipid or polymer vesicles are either imprinted or fully
encased by nonporous polymer, typically a polyamide formed through interfacial polymerization. (d) Pore-spanning planar layer format,
whereby the solution passes through one layer of channel-laden lipid or polymer matrix. Planar layers are classically formed by rupturing
channel-laden vesicles. Note that vesicles are precursors for all biomimetic membrane formats in (c,d).

may have such a large outer diameter that obtaining a reasonable
overall permeability would require a channel areal coverage that
would destabilize the amphiphilic matrix. For instance, through
simulations, minigramicidin A (MiniGram), a gramicidin
channel with the same diameter as biological gramicidin A
(GramA) but with 8 fewer amino acids in length, has a
permeability slightly less than that of AqpZ (Figure 2a).85
However, MiniGram has an outer diameter of only 0.96 nm per
channel, whereas each AqpZ subunit is approximately a 3 nm × 3
nm square (∼9 nm2 per channel).86,87 As a result, to reach the
typical TFC water permeability of 2−3 L m−2 h−1 bar−1, levels
that allow for near-optimal operation of desalination processes,
MiniGram would need a channel areal coverage of <0.25%,
whereas AqpZ would need a coverage of 2−4% (Figure 2i). As
another example, PAH[4] has a relatively low single channel
permeability (Figure 2a), but only occupies ∼2 nm2 per channel.
Accordingly, just 5−8% areal coverage with PAH[4] would be
needed to reach the target water permeability levels (Figure2i).
The propensity of PAH[4] to form clusters may facilitate
achieving this relatively high pore density without destabilizing
the biomimetic matrix.
Common Biomimetic Selective Layer Formats. Pro-
gress has been made in the development of selective water
channels, particularly synthetic channels that may offer
substantial advantages in terms of processability and cost.
Currently, biological and synthetic channels are available that
could allow for the development of ultraselective biomimetic
membranes, in principle. However, the performance of a
biomimetic selective layer is dependent not only on the chosen
water channel but also to a large extent on the matrix in which it
is incorporated. Therefore, it is imperative to consider the
10901
methods and materials used and the overall format of the
biomimetic membrane.
In general, there are two types of molecules that self-assemble
to form channel-incorporating matrices: amphiphilic lipids and
block copolymers (Figure 3a). Typical lipids used include:
natural lipids like Escherichia coli phospholipid,53,88,89 chicken
egg phosphatidylcholine,57,78,79 or porcine brain phosphatidyl-
serine57,79 as well as synthetically produced lipids like
DOPC,28,29,32,35,90 1,2-dioleoyl-3-trimethylammonium-pro-
pane (DOTAP),30,32,35 or 1,2-dimyristoyl-sn-glycero-3-phos-
phocholine (DMPC).91 Polymer matrices may be formed from
diblock or triblock copolymers, most frequently consisting of
poly(2-methyl-2-oxazoline) (PMOXA) as the hydrophilic block
and poly(dimethylsiloxane) (PDMS) as the hydrophobic
block26,27,33,34,92,93 but sometimes comprising poly(1,2-buta-
diene) and poly(ethylene oxide) (PB-b-PEO).57,73 These
particular polymer systems are not necessarily unique but
nonetheless have been extensively explored, although the higher
fluidity of PDMS over other hydrophobic polymers allows for
better channel insertion efficiency without denaturing chan-
nels.94 Amphiphilic polymer matrices are generally more stable
than lipid bilayers; lipid bilayers can be disrupted by
perturbations as mild as air bubbles. As such, while lipids are
useful in model systems for preliminary explorations, they have
no apparent use in industrial applications. For the best channel
insertion efficiency, the matrix must reasonably match the
physical dimensions of the channel, although bilayer thickness
has been shown to conform to channel heights for some
systems.95 Additionally, the matrix must chemically match the
degree of hydrophobicity/hydrophilicity of the channel
exterior.57
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With a channel type and matrix selected, the synthesis strategy
and overall format will critically impact the performance of the
biomimetic membrane. Channel properties are typically
assessed within model bilayers, usually using lipids. These
bilayers are produced as spherical, nanoscale liposomes through
the rehydration and extrusion of lipid layers or as planar formats
spanning across small apertures, formed by painting lipid/
solvent solutions.96−100 For industrial-scale applications, a
biomimetic active layer would need to be produced on a more
stable support. The most common formats of biomimetic
membranes consist of two general strategies: (i) intact vesicles
encapsulated in a polymeric matrix (Figure 3c)26−30 and (ii)
pore-spanning planar biomimetic layers (Figure 3d).31−35 In
both methods, channel-laden vesicles are precursors, as this
nanostructure is relatively straightforward to synthesize, self-
assembling in water based on amphiphile−solvent interaction.
In aqueous solutions, the amphiphilic biomimetic matrix
thermodynamically favors a sphere to minimize interactions of
unlike phases. In the development of these formats, aquaporins
have most frequently been used because they show relatively
high stability in both polymer and lipid matrices, coupled with
promising transport properties for desalination.26−35
For mixed-matrices incorporating intact vesicles, aquaporin-
laden liposomes or polymersomes are first deposited on a porous
support. Frequently, cross-linkable polymers are incorporated to
stabilize these intact vesicles, especially for pressure-assisted
deposition.27,30 End functionalities on the vesicles can often aid
in bonding the vesicle with the support membrane.26,27,30 The
highest deposition of vesicles included strategies for both cross-
linking the vesicular matrix and bonding the vesicle to the
support, resulting in ∼35% surface coverage by vesicles after
incubation.30 Because vesicle coverages are not typically
reported, we have estimated the coverage based on published
microscopy images (Figure S1).
After immobilizing vesicles, they are surrounded with
(imprinted)26,27 or fully covered in (encased)28−30 a nonporous
polymer. In some cases, the polymeric matrix surrounding the
vesicles is a polyamide formed via interfacial polymerization,28,29
but other polymers have been used as final coating layers. For
example, alternately deposited layers of dopamine and histidine
were used to imprint vesicles.26 In another study, vesicle-
immobilized membranes were dip-coated in initiators and then
monomers of methyl methacrylate and ethylene glycol
dimethacrylate to promote redox free-radical polymerization.27
Additional methods have involved the immobilization of DOPC
vesicles with PEO conjugates and cross-linking the vesicles with
glutaraldehyde to each other as well as a dopamine-coated
support.30 In areas with vesicles, water would theoretically pass
through two biomimetic selective layers (i.e., into and out of the
vesicle) before reaching the support (Figure 3c).
The degree to which the addition of vesicles improves the
performance of nonporous polymers, especially when consider-
ing the highest-performing polyamide material, hinges on the
areal coverage of deposited vesicles. As we will later show,
maximized vesicle coverage is requisite for any improvement in
salt rejection over pure polyamide. However, literature suggests
achieving this level of vesicle coverage is not trivial. We will later
discuss in-depth the limitations of this approach.
For planar formats where the biomimetic active layer spans
across a porous support, channel-laden vesicles are typically
ruptured on the support. Transport would then occur through
just one biomimetic selective layer (Figure 3d).31−35 The
performance of a membrane in this configuration depends on
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the areal coverage of the support, namely the minimization of
defects in the planar selective layer. The addition of attractive
functional groups on the support and/or vesicle outer
surface31−35 and the determination of an optimal rupturing
pressure31 have improved planar coverage. However, sodium
chloride rejection achieved in this format for AqpZ-incorporated
membranes is still only in the range of 20−75%, suggesting full
coverage has still not yet been achieved. The highest reported
salt rejection for a planar biomimetic layer was 97% for a
gramicidin-laden selective layer.31 In this case, a small
proportion of cationic lipid 1,2-dimyristoyl-3-trimethylammo-
nium propane was incorporated into the liposome to aid in
rupturing on a sulfonated poly(ether sulfone) NF membrane,
and an optimized liposome rupturing pressure was determined.
The nonporous nature and relatively high salt rejection of the
NF support, at 60%, likely also helped minimize the impact of
the defects. In subsequent sections, we will further discuss the
typical selective layer defect coverage and its impacts.
Other Biomimetic Formats. Some alternative biomimetic
membrane formats have been explored, aiming to overcome
issues with channel-insertion efficiency and incomplete selective
layer coverage (Figure 4). In general, many of these ideas are
based on lamination of multiple layers with the notion that if one
layer does not fully cover the support, then multiple should.
At a particular channel-to-polymer ratio, block copolymers
and channels can self-assemble into crystalline planar sheets
during slow dilution of detergent or solvent, after which the
sheets can be stacked to form 2D laminates. Carboxylic-
functionalized PAP[5] channels were incorporated into flat
crystalline sheets of PB-b-PEO diblock copolymers as
aggregated microphases (Figure 3a). Because the PAP[5]s
included anionic carboxy groups, the micrometer-sized sheets
could be laminated onto a poly(ether sulfone) microfiltration
support by a layer-by-layer (LbL) technique involving the
cationic polyelectrolyte polyethylenimine. In general, LbL
consists of depositing layers of alternately charged polyelec-
trolytes that adhere via electrostatic attraction. Only four
lamination cycles were needed for full coverage, with a sharp
MWCO of ∼450 Da and water permeability of around 65 L m−2
h−1 bar−1, which is 3-fold greater than industry-leading
composite NF membranes.
The production of these laminated crystalline sheets can take
up to 6 days by way of a slow dilution of detergents. In a
subsequent study, a solvent method reduced the synthesis time
of nanochannel-packed sheets to only 2 h. In this study, other β-
barrel membrane protein nanochannels were incorporated, each
showing sharp and channel-dependent MWCOs with signifi-
cantly higher water permeability than some commercial NF
membranes.101 While these results are exciting, processability
with this approach may be an issue. Additionally, applicability of
this biomimetic format to RO-type separations is unclear, as the
channels used thus far do not inherently reject salt. As with
graphene oxide and MoS2 2D laminates, we can expect that the
rejection performance of any laminated membraneeven when
incorporating salt-excluding channelswill be limited by
interconnected framework defects.13,102 Through these defects,
water and solutes can bypass the biomimetic layer, thereby
hindering overall water-salt selectivity. Such defects would likely
be particularly problematic for small solutes such as sodium and
chloride ions that readily permeate through interlayer materials
(e.g., swollen cationic polyelectrolytes).
Another method to promote multilayering for full coverage of
the active layer relied on a revision of the matrix composition. A
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Figure 4. Alternative biomimetic membrane formats. (a) Laminated
crystalline sheets, where sheets of up to ∼1 μm in side-length are
layered for full coverage. (b) Co-assembly of multilayer lamellar
block copolymer. Biological membranes follow an ABA (hydro-
philic−hydrophobic−hydrophilic) pattern, whereas, here, a BAB
(hydrophobic−hydrophilic−hydrophobic) block copolymer is used
to promote multilayered self-assembly. (c) Blended mixed matrix,
where channels are blended into a polymer solution before casting
or polymerization of a nonporous polymer, typically polyamide
formed through interfacial polymerization. Random alignment of
nanochannels occurs.
cross-linkable triblock copolymer of polyisoprene-b-poly-
(ethylene oxide)-b-polyisoprene was spin-coated onto a silicon
wafer for self-assembly into well-aligned, multilayered lamellae
laying parallel to the surface, confirmed through grazing-
incidence small-angle X-ray scattering.103 These lamellae
could then be transferred to a porous support, in this case
anodized aluminum oxide (Figure 4b). In this system, the BAB
pattern (i.e., hydrophobic−hydrophilic−hydrophobic) was
utilized to promote and stabilize multilayer formation by
fostering cross-linking (polymer bridging) between adjacent
hydrophobic domains. Lithium bis(trifluoromethanesulfonyl)-
imide content in the casting solution was optimized to eliminate
detectable defects by reducing PEO crystallinity. Co-assemblies
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with GramA and PAP[5] increased the water permeability.
Membranes with PAP[5] again exhibited a MWCO of ∼450 Da.
However, for membranes with GramA, the permselectivity of
sodium over chloride was only 0.34, whereas in nature, GramA is
highly selective for monovalent cations over anions,104
suggesting the presence of nanoscale defects. Multilamellar
morphologies such as those used in these studies could
stochastically minimize the overall defect content, but at the
cost of decreased permeability due to the increased selective-
layer thickness.
Biological and synthetic water channels have also been used in
mixed-matrix membranes, whereby channels were directly
incorporated in polyamide films (Figure 4c).105−107 Although
this format lacks the biomimetic channel-aligning matrix, it
resembles biomimetic membranes in that discrete biomimetic
channels are incorporated within a matrix of dissimilar chemical
composition. AqpZ has been protected by complexation with a
β-barrel-forming amphiphilic peptide before incorporation into
polyamide, enhancing permeability.106 The incorporation of
zwitterion-functionalized carbon nanotubes with an average
diameter of 1.5 nm somewhat enhanced salt rejection and
greatly increased water permeability of a TFC-RO mem-
brane.105 While improved performance over controls has been
reported in experimental studies,105−107 no such mixed-matrix
membrane has achieved better performance than the best-
performing commercial membranes. Additionally, this approach
predominantly relies on the intrinsic transport performance of
the polyamide matrix and is generally limited by the defects that
form at the interface between the channel and the glassy
polyamide material.
PERMEABILITY MEASUREMENTS OF CHANNELS AND
BILAYERS
Biophysical Methods to Measure Membrane Perme-
ability. In classic biophysical studies, methods of quantifying
transport through reconstituted membranes were developed to
understand transport through bilayers and protein channels. For
industrial biomimetic membranes, these techniques are essential
for optimizing membrane design. By studying the bilayer alone,
channel loading efficiency can be optimized, where degree of
hydrophobicity and length of each type of block should be
matched between channel exteriors and amphiphilic channel-
insertion matrix.57 For testing the performance of the channel-
laden matrix, two general methods have been utilized: (i)
aperture-spanning patch clamp and (ii) vesicle stopped-flow
studies.
The state-of-the-art system for measuring ion conductance
across a membrane is the patch clamp. To produce a patch
clamp, a pipet tip is touched to the surface of a vesicle or cell
membrane, and a light suction is applied to form a gigaohm-
strength seal. Beyond this point, by adjusting the strength of
pulses of suction or voltage and contact with air, the system can
be setup to attach a full cell/vesicle or a small patch. Extensive
relationships between current and voltage changes and single
channel transport rates have been developed for apertures
containing one channel108−114 and those containing multiple
channels.115,116 However, in the case of developing a
desalination membrane, typical channels should reject all
solutes, prohibiting the measurement of ion conduction across
their orifices. Furthermore, during transport through aquapor-
ins, water molecules are flipped so that electron conductivity
through a fully intact water wire cannot be used as a
confirmation of pore loading. In these cases, methods to
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Figure 5. Concepts for stopped-flow measurements to predict biomimetic membrane permeability and determine efficiency of channel
insertion into amphiphilic matrix. (a) Measuring signal in a stopped-flow apparatus to determine permeability of biomimetic membrane.
Vesicles are loaded with a buffer consisting of a solute that is impermeable through the vesicle selective layer at small time scales. Once exposed
to hyperosmotic solution, vesicles rapidly shrink via water exiting through nanochannels to equilibrate interior and exterior osmolar
concentrations. Either light side-scattering or self-quenching fluorescent dyes are utilized to determine real-time vesicle volume. (b)
Comparison of water permeability results for matrices with AqpZ using the directly fitted flux eq 8, the commonly misused exponential eq 14,
and the good approximation from the exponential eq 12. Permeabilities in stopped-flow were typically taken at lower temperatures and were
temperature-corrected to 25 °C using an activation energy of 3.3 kcal mol−1, an average value from several studies.53,88,89,92 Permeability data
from references a89, b,28 c,35 d,120 e,90 f,123 g,122 and h.57 (c) Comparison of resultant pure water permeability from stopped-flow studies
conducted at various changes in osmolarity across the vesicles, for DOPC and PMOXA-PDMS-PMOXA (MDM) vesicles. Direct fitting of the
correct flux equations as well as the incorrect exponential approximation and two good exponential approximations are provided. Data taken
from literature.73 (d) An example of how AqpZ-loaded vesicles shrink over time when exposed to hyperosmotic solution. Models based on the
commonly used erroneous single exponential fit as well as the use of a differential equation are presented alongside measured data points. Data
was extracted from ref 89. (e) Example of stopped-flow curves to measure transport rates of relatively permeable solutes through MDM using
self-quenching encapsulated fluorophore. Data taken from ref 73.

quantify the transport of water through small ionic concen-
tration changes close to recontituted membranes under osmotic
stress have proven advantageous. A recent example of these
methods uses ion-selective microelectrodes.117,118 Additionally,
the patch clamp can be used to study the slower diffusive
permeabilities of water and neutral solutes through amphiphilic
pure matrices without channels.51,119 However, patch clamping
is not ideal for measuring water permeability since the
membrane area is small.
Stopped-flow methods for vesicular solutions are commonly
used to assess water and neutral-solute permeability. In these
methods, the average volume change of a population of vesicles,
10904
each loaded with multiple channels, can be tracked over a
discrete window of time. In general, a stopped-flow apparatus
allows for the rapid mixing of small quantities of two different
solutions and subsequent measurement of optical changes. By
mixing a solution of vesicles with either a hyperosmotic or
hypoosmotic solution and tracking the average osmotically
driven volume change with time, the permeability can then be
calculated.96
After successfully characterizing the function of the aquaporin
in biophysical work,89 stopped-flow methods with vesicles were
heavily used to determine the aquaporin loading efficiency and
function in efforts toward biomimetic mem-
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branes.26−28,30,32−35,53,57,88−93,120−123 More recent efforts have
paired stopped-flow permeability measurements with techni-
ques such as fluorescence correlation spectroscopy that enable
direct measurement of the number of channels per vesicle.56,81
Signals from either light side-scattering (scattering at 90°) or
fluorescence from encapsulated, concentration-sensitive fluo-
rescent dyes are correlated to vesicle volume. With decreased
vesicular volume, light side-scattering increases, while the
fluorescence of loaded self-quenching fluorescent dyes decreases
(Figure 5a). Fluorescence methods avoid complications with
light scattering, such as optical artifacts from particle motion
immediately after mixing and changes in refractive index.124
Vesicles can also be used to determine solute permeability,
which is especially important for biomimetic membranes
targeting solute−solute selectivity and in processes like
wastewater reuse that aim to eliminate organic, neutral
molecules. In one method used for solutes that are less
permeable than water, the permeable solute of interest is loaded
within the vesicles, which are then mixed with an isotonic
solution containing a relatively impermeable solute. As the
permeable solute exits, vesicles shrink due to water efflux, which
can be used to determine the solute permeability.125,126 A
second method for permeable solutes is to mix vesicles with a
hyperosmotic solution of the pertinent solute.73 After a relatively
fast exit of water, a more gradual increase in vesicle volume
occurs as the solute and water flow back into the vesicle. In cases
where the permeation of a basic or acidic solute is higher than
water permeability, that is, vesicle volume changes are too slow
to reflect species permeability, a pH-sensitive dye can be loaded
into vesicles before mixing with solutions of interest. This
technique has been used for permeation of protons and
ammonia as well as model carboxylic acids (e.g., acetic,
propionic, and butyric acids).73,127
In cases involving previously unexplored matrix materials or
synthetic channels, initial vesicle stopped-flow studies can be
used to optimize these materials and channel incorporation
methods. Once the maximum permeability is determined for
these systems, best-case-scenario desalination performance for
composite, industrial-scale formats of these same materials can
be predicted. However, to accurately predict performance, it is
necessary to correctly translate the measured stopped-flow
signal into permeability, which deserves careful attention.
Common Use of Vesicle Stopped-Flow Equation
Overestimates Permeability. Based on stopped-flow experi-
ments with osmotic gradients, the osmotic water permeability,
Pw, is related to the rate of change in the normalized vesicle
volume, Ṽ (t):128,129
A s,0 ji Cin(t ) y
jj
j − Cout zzzz
dṼ (t )
V0 k V (t ) {
= PwVw
dt ̃
(8)
where Vw is the molar volume of water, As,0 is the initial average
outer surface area of the vesicles, V0 is the initial average vesicle
volume, Cin(t) is the inner vesicular osmolarity with time, and
Cout is the outer osmolarity after mixing, typically taken as a
constant due to the relatively low vesicle concentration.
Similarly, the following equation can be used to determine
solute permeability, Ps:73
A s,0 ij Cin,S(t ) yz
jjC zz
jj out,S − z
Ṽ (t ) z{
dCin,S(t )
V0 k
= Ps
dt (9)
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Review
where Cin,S(t) and Cout,S are the intra- and extra-vesicular
concentrations of the permeable solute. In eqs 8 and 9, the
intravesicular concentrations, Cin(t) and Cin,S(t), are relative to
the initial vesicle volume, while the resulting ratios with
normalized vesicle volumeCin(t)/Ṽ (t) and Cin,S(t)/Ṽ (t)
give the absolute osmolarity and permeable solute concen-
tration, respectively.
Critically, eqs 8 and 9 are algebraic reformulations of eqs 1 and
4, respectivelythe fundamental flux equations for membrane
transport from the solution-diffusion modelwhich have been
translated for vesicles with permeability driven solely by
concentration gradients (i.e., no hydraulic pressure). Accord-
ingly, these permeabilities measured from studies on vesicles
provide the expected permeability for a planar, defect-free
membrane with the same composition as the vesicular walls (i.e.,
the same amphiphilic material and similar channel density). In
other words, Pw and Ps can be used to estimate water flux and
solute rejections for a defect-free membrane, using eqs 1−5.
With modern computer software, it is relatively straightfor-
ward to directly solve for Pw and Ps using eqs 8 and 9 by
numerical iterative fitting. We emphasize that eqs 8 and 9 are the
fundamental flux equations from the solution-diffusion model,
and the direct use of these equations is the most preferred and
most accurate fitting method. However, many early studies used
single exponentials as approximations of eqs 8 and 9.88,130−135
The observed first-order signal rate constant, ks, has frequently
been determined through stopped-flow signal data fitting
using27,30,32,34,91,93
S(t ) = A e(−kst ) + B (10)
where S(t) is the signal intensity with time, A is the amplitude,
and B is the final signal value. Using exponential fits, an early
approximate solution was136
r0ks(1 − b/V0)
Pw ≈
3VwCout (11)
where r0 is the initial average vesicular radius, b is the per vesicle
average volume of the bilayer, and Cout is the initial extravesicular
osmolarity. More recently, it was shown that the permeability
can be approximated within 5% error of the numerical solution
using137
r0ks(Cin + Cout)
Pw ≈
6VwCout 2 (12)
where Cin is the initial intravesicular osmolarity.
Stopped-flow experiments are frequently conducted below
ambient temperature to improve resolution. To predict
performance under normal desalination conditions at ambient
temperature, the osmotic water permeability can be adjusted
ÅÄÅ ÑÉ
1 yzzÑÑÑÑ
using the Arrhenius expression:
ÅÅ −Ea ij 1
Pw(T2) = Pw(T1)expÅÅ Å j
j − zzzÑÑ
ÅÅ R̂ jj T2 T1 {ÑÑÑÖ
ÅÇ k
(13)
where T1 is the absolute stopped-flow experimental temper-
ature, T2 is absolute ambient temperature, and Ea is the
activation energy of water transport through the channel of
interest. The water permeability coefficient of the biomimetic
layer is then calculated from Pw using eq 3.
Although the biophysical community predominantly directly
fits eq 8 to stopped-flow data for determining water
permeability, a longstanding confusion within the biomimetic
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membrane community has led to the incorrect use of an
exponential approximation. In the 1990s, a couple of biophysical
studies presented the osmotic permeability calculation:88,138
roks
Pw =
3Vw(Cout − Cin) (14)
This approximation is nearly identical to eq 11, except that the
volume of the bilayer is neglected andmuch more critically
the permeability is related to the initial concentration difference,
Cout − Cin, rather than just the external concentration, Cout. The
approximation in eq 14 has been extensively used in biomimetic
membrane literature to estimate Pw.26−28,30,32−35,57,90−93,120−123
Unfortunately, this fitting procedure systematically leads to
dramatically higher permeabilities than when using the
fundamental water flux equation, eq 8.73,137
To stress the magnitude of error stemming from the use of eq
14, we have recalculated the vesicular permeabilities for stopped-
flow signals provided by numerous studies involving AqpZ-
laden matrices (Figure 5b; Figure S2). We compared results
from the correct use of iterative numerical methods in Python
coding software (see SI for code) to solve for best fit with eq 8
with the incorrect use of eq 14. Use of the incorrect equation led
to water permeabilities 2−12-fold greater than from a correct
fitting procedure. We also present the use of eq 12 as a good
approximation using single-exponential fitting of stopped-flow
signals, which was generally (but not always) close to the results
from directly fitting eq 8. Typically, we saw that the magnitude of
the error from using eq 14 was mostly related to the solute
concentrations used. This can be more directly illustrated when
the same stopped-flow data are used to solve for water
permeability over a varied range of Cout − Cin using directly
fitted eq 8 as well as exponential approximations, eqs 11, 12, and
14 (Figure 5c).73 Using direct fitting and the approximations in
eqs 11 and 12 tend to result in similar, concentration-
independent permeability, whereas permeability found through
eq 14 shows marked concentration-dependence, which is
aphysical, and especially overestimates permeability at a smaller
difference between intra- and extra-vesicular concentrations
(Figure 5c). We stress that eq 14 with its concentration-
dependent fitting not only yields an inaccurate permeability but
also cannot be used for a valid comparison between channels
from different studies due to dissimilar testing conditions.
These recalculations suggest that all amphiphilic matrices
with AqpZ have exhibited, at best, permeabilities in the range of
1−2 L m−2 h−1 bar−1, resulting from the limited maximum
possible channel loading density. The recalculated permeabil-
ities for unoptimized protein insertion efficiency averaged to
around 1 L m−2 h−1 bar−1,28,35,89,90,120,123 while studies that
optimized insertion reached bilayer permeabilities of around 2 L
m−2 h−1 bar−1 at 25 °C.57,122 The highest water permeability
achieved was 2.1 L m−2 h−1 bar−1 (Figure 5b),123 equivalent to
an AqpZ areal coverage of 2.5% in a DOPC bilayer (Figure 2i).
This water permeability is comparable to that of commercial,
state-of-the-art TFC-RO desalination membranes.
In contrast to water permeability, solute permeability has not
been as well-studied in biomimetic membrane literature.
However, stopped-flow can nonetheless be used to determine
the transport behavior of more permeable solutes and predict
resultant biomimetic membrane performance. Examples of
permeable solute stopped-flow curves with self-quenching
encapsulated fluorophore carboxyfluorescein are presented in
Figure 5e. Rapid self-quenching occurs as water first exits the
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vesicles faster than the permeable solute can enter. A second,
more gradual curve appears as the permeable solute slowly
enters the vesicle and water flows back in to reach osmotic
equilibrium. To find solute permeability, eq 9 is fitted to the
second gradual curve.73 Notably, water permeability can be
found from the same stopped-flow data by fitting eq 8 to the
initial sharp-decline curve.
EVALUATING THE PERFORMANCE OF BIOMIMETIC
MEMBRANES
Modeling Transport Properties Through Composite
Layers. Modeling the expected performance of membranes can
provide a benchmark for membrane fabrication efforts as well as
illustrate the upper performance limits of a particular material.
We modeled transport behavior to predict desalination
performance for biomimetic membrane configurations with
both intact vesicles and pore-spanning planar layers. We focus
on biomimetic desalination membranes with perfectly water-
selective AqpZ. However, similar modeling approaches could
serve for predicting performance of other biomimetic formats.
With knowledge of the transport behavior for each individual
layer in the biomimetic desalinating composite, we modeled the
overall permeability and salt rejection using the resistance
model,139 similar to models used elsewhere for biomimetic
membrane performance.140 The general premise of the model is
to treat molecular flow through a membrane as analogous to
electron flow through a circuit, where the goal is to project the
overall resistance to flow for a given species. This overall
resistance is then simply the inverse of the overall permeability.
As with circuits, membrane components can be considered as
resistors-in-series for vertically stacked components or as
resistors-in-parallel for components that are laterally arranged
(e.g., channels in an amphiphilic matrix, in eq 7). As an example,
an encased vesicle format comprises three types of membrane
composite areas: (i) areas with two bilayers (i.e., entering and
exiting a given vesicle) covered with encasing polymer over the
support, (ii) areas with only encasing polymer over the support
where vesicles are absent, and (iii) defective regions with just the
support layer exposed.
More specifically, the permeability of each layer, Pj, in a total
of n layers for a given type of composite x is incorporated in the
summation:
n
1 1
= ∑
Px j=1
Pj (15)
This resistance model is only valid for composites comprising
solution-diffusion-based membranes. For porous support layers,
we assumed rejection was negligible in cases where the top layer
rejects significantly more than the support. In cases where a
support layer is expected to provide significant resistance to
solute permeation, a real rejection of the support or solute
permeability coefficients should be used.140
From here, the salt and water flux contributions for each
composite type are determined using eqs 1−4. To determine
total water and salt fluxes for the entire membrane, Jt, transport
through the different composite types are considered as
resistors-in-parallel:
m
Jt = ∑ ψxJx
x=1 (16)
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Figure 6. Predicted performance of planar biomimetic membrane formats. (a) Water/salt (sodium chloride) selectivity (left) and water/boron
selectivity (right) with varied permeability of biomimetic layer with nanochannels. For water/salt selectivity, the trends shown are for various
densities of AqpZ, PAH[4], and nCNTP in DOPC. For boron selectivity of AqpZ-loaded biomimetic membranes, matrix types represented
include DOPC, ABA triblock copolymer MDM, and AB diblock copolymer PB-PEO, as boric acid permeates these matrices at different rates.
Amphiphilic matrix water and salt permeability coefficients were retrieved from ref 73 with sodium as the limiting ion. For performance
comparison, data from polyamide RO membranes is also presented, calculated from manufacturer standards.142 Data for optimized channel
insertion areal coverage of 2.5% are pinpointed for AqpZ, Aqf1, PAH[4], and nCNTP as well as the most permeable biological water- and
glycerol-transporting channel GlpF. Single-channel AqpZ and GlpF water permeabilities were taken from stopped-flow studies.56 Water and
salt permeability coefficients of PAH[4] were taken from ref 82 with chloride as the limiting ion. (b) Predicted rejection of sodium chloride
(left) and pure water permeability (right) versus defect coverage for biomimetic membranes in seawater RO. The model includes a biomimetic
active layer on a support. The active layer was modeled with the best-achieved real AqpZ density, resulting in a biomimetic layer permeability of
2.1 L m−2 h−1 bar−1. The range of supports include ultrafiltration and nanofiltration membranes with pores 20 and 1 nm in diameter,
respectively, as well as looser, high-permeability TFC-RO polyamide and tighter, high-rejecting TFC-RO. Reference lines/regions for best-
observed TFC-RO salt rejection (99.85%) as well as the range of water permeability for current commercial TFC-RO polyamide membranes are
included for comparison.140 Models were conducted at standard seawater desalination test conditions (55 bar, 0.55 M NaCl). (c) Permeability
(P in m/s) trends through amphiphilic matrix materials (i.e., no channels) for neutral solutes of varied octanol/water partition coefficients Kow.
These solutes more readily permeate through the channel-incorporating matrix than ions. Data taken from ref 73.

where ψx is the fractional coverage of each composite type x, fluxes, we can calculate the overall observed rejection using eq 4
with m total types of composites. With total water and solute (see SI for more details).
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We used our permeability and rejection models to predict
best-case-scenario performance of defect-free desalination
biomimetic membranes. We also used the models to illustrate
the detrimental effect of defects and the importance of support
layer choice in alleviating defects.140
The Potential of Biomimetic Membranes. A perfectly
formed biomimetic membrane with aquaporins could promise
unparalleled salt rejection. Based on matrix and AqpZ water and
salt permeabilities from vesicular stopped-flow studies, sodium
chloride rejection is predicted to be >99.9999999% for typical
AqpZ loading quantity (Figure 6a, left).73 Meanwhile, boron
rejection can be as high as >99.9% when the matrix comprises an
AB diblock copolymer of PB46-b-PEO20, (Figure 6a, right).
Considering this potential solute rejection, it is clear that no
studies have produced defect-free biomimetic layers that fully
cover the support. Thus far, salt rejection for AqpZ-loaded pore-
spanning planar layers has been in the range of only 20−
75%.32−35 Although optimal vesicle rupturing pressures have
improved rejection for gramicidin-containing membranes,31 the
reported rejection of 97% sodium chloride (or 3% salt passage)
in this single study still cannot compete with current TFC-RO
membranes that exhibit as low as ∼0.15% salt passage.
The impact of defects on performance is markedly influenced
by the transport properties of the underlying support layer(s).140
In particular, a relatively low-permeability, salt-rejecting support
layer mitigates the negative impact of defects on salt rejection.
To illustrate this point, we predicted the performance of planar-
layer biomimetic membranes with increasing defect area, formed
on top of support layers varying from relatively selective RO-
type membranes to highly permeable porous membranes,
modifying the results of our previous study by considering the
true best-case AqpZ-matrix permeability of 2.1 L m−2 h−1 bar−1
(Figure 6b).140 As discussed earlier, increased selectivity and salt
rejection is a critical research goal. Therefore, we consider the
defect areas that would allow for increased salt rejection above
that of currently employed RO membranes. For all support
types, except RO-type layers, just 1% defect area would
undermine the performance of the biomimetic membrane. As
a result, RO-type membranes (i.e., polyamide thin-film
composite) may be ideal supports for biomimetic membranes
to minimize the impact of defects. Additionally, the synthesis
methods of these polymeric membranes are often modified to
adjust their transport properties, with some having relatively
high water permeability and low salt rejection and others having
relatively low water permeability and high salt rejection. In our
model, we consider a high permeability membrane with a water
permeability of 6.1 L m−2 h−1 bar−1 and sodium chloride
rejection of ∼99% as well as a high rejection membrane with a
permeability of 2.3 L m−2 h−1 bar−1 and a salt rejection of
∼99.8%, which are characteristic of the commercial brackish
water and seawater RO membranes XLE and SW30-XLE
(DuPont Water), respectively.141
When using a support layer composed of either polyamide
material, highly selective performance may be achievable with
meaningful defect levels. For example, for a defect density of 1%,
a biomimetic membrane with a support of high-permeability
polyamide would reject ∼99.98% of sodium chloride, while
using a support of high-rejection polyamide would result in
99.996% rejection, corresponding to roughly two orders-of-
magnitude lower salt passage than for current commercial
desalination membranes. Meanwhile, the former would have a
permeability of 1.6 L m−2 h−1 bar−1, while the latter would
permeate at only 1.1 L m−2 h−1 bar−1, which would necessitate
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slightly higher energy usage during seawater desalination.4
While either polyamide support layer could enable promising
biomimetic membranes, there remains a minor permeability/
selectivity trade-off.
Adequate neutral solute rejection is critically important in
most RO applications. Although discrete channels with water-
sized pores can molecularly sieve out essentially all solutes,
lipophilic solutes can permeate through the amphiphilic matrix
by solution diffusion. The affinity for a neutral solute to partition
into and diffuse through the amphiphilic matrix is well-
correlated with the solute’s octanol/water partition coefficient
Kow or the ratio of the chemical’s equilibrium concentration in
the octanol phase versus the water phase (Figure 6c).
Hydrophilic solutes with log Kow less than −0.2 are predicted
to be well rejected (>90%) by a bilayer with 2.5% AqpZ, but for
solutes with Kow higher than this value, rejection would
significantly suffer. Typical neutral organic contaminants
found in wastewater are hydrophobic and have log Kow much
greater than −0.2. These molecules would therefore be poorly
rejected by a biomimetic active layer.73 Likewise, although
boron rejection in a PB−PEO/AqpZ system is predicted to be
>99.9%, it is predicted to be 95−98% when DOPC lipid or an
ABA triblock copolymer of PMOXA-PDMS-PMOXA (MDM)
are used (Figure 6a). These differences in solute transport or
rejection behavior demonstrate the importance of matrix
material in certain applications.140
A salt-selective support layer can minimize the impact of
defects on salt rejection while maintaining sufficient perme-
ability for seawater RO.73,140 The combination of a biomimetic
layer with a high-rejection RO membrane can marry the benefits
of both layers while avoiding their downsides. In seawater RO
and wastewater reuse, a high-rejection polyamide RO
membrane has a relatively high rejection of salt and superb
rejection of larger neutral pollutants due to size-based removal
mechanism (i.e., slower diffusion of larger solutes). The
molecular weights of most neutral solutes present in wastewater
are larger than 80 Da, exceeding typical MWCO for polyamide
RO membranes.73 However, TFC-RO membranes are still not
completely selective for salt, ammonia, boron, or low molecular
weight wastewater organic contaminants, thereby requiring
postprocessing to meet regulatory restrictions for irrigation or
wastewater reuse as well as ultrapure levels for the semi-
conductor and power industries. The combination of both a
high-rejection polyamide RO and biomimetic active layer in a
defect-free membrane is projected to reject >90% of the most
common neutral organic pollutants and >99.9% for the majority.
Even with a 1% defect area, ∼99.7% of boron would still be
rejected.73
Defects Explain Subpar Performance. So far, biomimetic
membranes have not approached the theoretical desalination
performance (Figure 6a, b), suggesting the presence of defects
based on observed transport behavior (Figure 6b). Previously,
defects were not quantifiedeven when vesicle and planar
membrane permeabilities were explicitly measured within the
same studybecause stopped-flow measurements were (i)
decoupled from planar measurements, meaning the osmotic
water permeability was not related to the final biomimetic
membrane permeability, and (ii) the fitting equations used to
predict permeability were wrong, systematically overestimating
possible permeability for a defect-free membrane. We have
estimated the defect coverage to better understand observed
performance. For formats with intact vesicles, the influence of
defects was modeled as areas lacking polymer matrix material,
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Figure 7. Defects for typical biomimetic membrane formats. (a) Defects defined for vesicle-encapsulated mixed-matrix formats, which occur
within the nonporous polymer. (b) Defects defined for pore-spanning planar layer format, which occur within the amphiphilic channel insertion
matrix. Note that in all cases, a defect is essentially an area where the porous support layer directly contacts bulk feed solution. (c) Defect-free
theoretical effective water permeability compared to observed performance. (d) Defect-free theoretical sodium chloride rejection compared to
observed performance. Selection of pure water permeability versus water permeability in the presence of saltwater feed depended on which was
reported in literature. Lower observed rejection and higher water permeability compared to theoretical values suggest presence of defects. (e)
Model-projected coverage of defects based on rejection and permeability models. Defect coverage was deduced by simultaneously minimizing
the sum of square errors between model-predicted water permeability and salt rejection values compared to observed performance reported in
the literature. *Defects projected were 0% for ref 28. **Li et al.35 results did not match model; simple defects could not explain both observed
lower rejection and permeability than the starting NF substrate. For (c−e), blue italic letters refer to the following references: a,26 b,27 c,28 d,29
e,30 f31 g,32 h,33 i,34 and j.35 Note that this study f31 incorporated GramA instead of AqpZ.

while for pore-spanning planar layers, defects were defined as
areas lacking any channel-laden matrix (i.e., exposed support
layer in both cases) (Figure 7a,b). The coverage of vesicles and
whether or not they were imprinted or encased was accounted
for in the models. We first predicted performance of defect-free
membranes for five previous studies incorporating intact vesicles
and five with pore-spanning planar layers, showing that in nearly
every case, defect-free membranes are predicted to have lower
permeability and higher rejection than achieved (Figure 7c,d).
To estimate the defect density, we used the previously defined
transport equations to conduct a least-squares analysis on the
reported permeate flux during salt rejection studies or pure water
permeability (depending on what was reported) and salt
rejection (Figure S3). The predicted defect density of
biomimetic membranes reported in several studies is presented
in Figure 7e. In general, we see that lower defect densities are
estimated for formats incorporating intact vesicles than pore-
spanning planar layers, typically with 0−1% defect coverage for
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the former and between 1 and 10% defect coverage for the latter.
However, as we will further show, formats with intact vesicles
have little chance of surpassing current RO salt rejection unless
vesicle packing density is increased to unrealistic values.
Vesicle-Encapsulated Mixed-Matrices Negligibly Im-
prove Membrane Performance. Just as the performance of
biomimetic membranes hinges on the negative impact of
defects, the enhancement of salt rejection by adding vesicles
within a polymer layer hinges on the areal coverage of such
vesicles. The question then arises whether biomimetic
membranes with intact vesicles have the potential to surpass
the performance of current state-of-the-art desalination
membranes. Thus, we modeled the influence of vesicle coverage
across a range of 0% to 90%, with 90% corresponding to the
theoretical maximum packing density of monodisperse circles
on a 2D plane, which is currently unrealistic to achieve. Typical
coverages are in the range of only 7−20%,26−28,30,92 although
one study achieved a vesicular coverage of ∼35% by using
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Figure 8. Best-case-scenario performance of biomimetic membranes with vesicle-encapsulated mixed matrices. (a) Pure water permeability
(top) and maximum possible salt rejection with no defects (bottom) for varied 2D packing densities of aquaporin-laden vesicles in vesicle-
encapsulated mixed matrices. Two different common commercial TFC-RO membranes, XLE and SW30-XLE (DuPont Water) or high-
permeability and high-rejection RO, were used as model nonporous polymers surrounding the vesicles. Vesicles both encased and imprinted in
nonporous polymer are represented. The biomimetic membranes were modeled on top of an ultrafiltration membrane with pores of 3 nm in
diameter (UF 3). The blue region represents the typical vesicle surface coverage found in literature,26−28,30,92 while the yellow region is the
permeability of current commercial TFC-RO. Note that by using cross-linked vesicles of polyethylene-glycol-conjugated DOPC on a dopamine-
coated support, one study achieved a vesicular coverage of ∼35%.30 The theoretical maximum possible surface coverage of vesicles is assumed
to be the maximum packing density of spheres on a plane, or 0.9. The permeability of the bilayer was assumed to be 2.1 L m−2 h−1 bar−1. (b) A
typical surface coverage of 15% projected area, with calculated flux contributions for water passing through vesicles versus just polyamide over
support layer. (c) Theoretical maximum surface coverage of vesicles at 90% projected area, with calculated flux contributions for each type of
area.

polyethylene-glycol-conjugated DOPC to self-cross-link the
vesicles for stability and later cross-link them to a dopamine-
coated support.30 We used high-rejecting polyamide and high-
permeability polyamide as the model polymers that either
imprint (i.e., surround) or fully encase the vesicles, assuming no
defects are present.
For both imprinted and encased vesicles, which show
insignificant differences in performance, pure water permeability
linearly decreases with increasing vesicle coverage, while salt
rejection improves most rapidly within higher ranges of vesicular
coverage (Figure 8a). We see minimal benefits of added vesicles
for higher permeability polymer matrices. With maximum
imprinted vesicular coverage, high-permeability polyamide
exhibits up to a ∼0.5% increase in rejection, not quite reaching
the initial rejection of high-rejection polyamide, even with the
unrealistic 90% vesicular coverage. At this vesicular coverage, the
permeability of the membrane drops to below that of the high-
rejection polyamide system, rendering the technology overly
complicated with no added benefit in desalination.
For a high-rejection, tight polyamide matrix, maximum vesicle
coverage (i.e., 90%) is projected to yield a 0.12% increase in salt
rejection to ∼99.97%. Although pure water permeability would
drop to 1.2 L m−2 h−1 bar−2 or 0.86 L m−2 h−1 bar−2 for formats
with imprinted and encased vesicles, respectively, 99.97% salt
rejection is an unparalleled level. However, to achieve this target,
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vesicle coverage would have to dramatically increase. Consid-
ering that vesicle surface coverage is typically 5−20%, achieving
nearly 90% ordered packing will certainly be nontrivial (Figure
S1).26−28,30,92
We emphasize that the model we used for vesicle-laden
membranes assumes unidirectional transport and treats vesicles
with channels as an isotropic material. Realistically, multidirec-
tional diffusion may cause species to avoid the well spaced-out
channels in the vesicle bilayers and frequently bypass the vesicles
completely. The only way to avoid this possibility would be with
close to 100% vesicular surface coverage, which is not possible
for spheres on a 2D plane. Finally, this model assumes that
defects are avoided, which may be unattainable considering the
large interfacial areas between vesicle walls and the surrounding
polymer at high vesicle areal coverage. Collectively, our
modeling results and discussion indicate that vesicle-encapsu-
lated mixed matrices negligibly improve membrane performance
and, compounded with the involved complex synthesis, would
likely not be worth pursuing further.
Optimal Biomimetic Desalination Membrane Design.
Although methodological changes can decrease defect density,
to date, even the most well-formed planar bilayers have suffered
from a sufficient defect density to completely undermine the
capabilities of the aquaporin channel. Currently, an absolutely
defect-free bilayer would be difficult to achieve, especially at an
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Figure 9. Concepts for high-performance biomimetic membranes. (a) Synthesis of an ion-rejecting or ion-selective biomimetic membrane. The
presence of functional groups assists in rupturing nanochannel-laden vesicles. Nanoscale defects are filled with a hydrogel reaction solution,
which is subsequently polymerized. The choice of channel would determine whether ions are fully rejected or selectively rejected. (b) Synthesis
of an ultrahigh rejection membrane. Synthesis begins with the formation of a smooth polyamide layer with a dense layer of functional groups.
Functional groups assist in the bottom-up polymerization of the channel insertion matrix, with subsequent insertion of nanochannels, or
rupturing of nanochannel-laden vesicles.

industrial scale. Thus, we propose two approaches that may
allow for defects while still achieving ultrahigh salt rejection
(Figure 9). In the first case, a biomimetic selective layer is
formed on a surface-functionalized porous membrane by
traditional vesicle rupture or by growing a polymer brush
layer,143 followed by channel insertion within the solid-
supported brush layer.144 These processes must be optimized
to minimize defects. A hydrogel coating layer would then fill
defects after biomimetic active layer formation,145 analogous to
silicone defect-sealing layers used in gas separation mem-
branes.139 This hydrogel would decrease the water permeability
of defects, driving more flow through the selective channels.
Depending on the channel type chosen, the membrane would
either highly or selectively reject ions. However, lipophilic
solutes would easily pass through the amphiphilic matrix in this
design (Figure 9a). In the second vision, a smooth polyamide
layer is functionalized with grafting sites to either improve the
top-down deposition and subsequent rupturing of vesicles or to
grow the channel-incorporating matrix from a bottom-up
approach. We envision that such a membrane would highly
reject ions with the biomimetic active layer and also neutral
solutes with the polyamide layer (Figure 9b). This latter design
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could be particularly beneficial in applications such as ultrapure
water production, seawater desalination, and wastewater reuse.
The proposed designs build on previous analyses on ideal
permeability and salt-water selectivity to increase desalination
efficiency4 and the influence of support layer on biomimetic
membrane performance.140 Although the synthesis details of
how to effectively produce these envisioned membranes would
need to be experimentally resolved and may prove challenging,
the suggested schemes piece together methodologies that have
proven effective in other applications. The chemical stability in
various environments as well as mechanical strength to
withstand operating pressures and shear stresses would need
to be investigated and optimized, as is the case for all biomimetic
membranes. The polymeric matrix material will need to be
carefully chosen to enable both functional channel reconstitu-
tion and scalable selective layer synthesis. Furthermore, with the
addition of a polyamide layer, the biomimetic selective layer
would ideally have increased permeability to overcome the
additional resistance. Synthetic channels should be developed
with low outer-to-inner pore diameter ratios so more pores per
channel areal density are present. Perhaps strategically designed
external channel features or matrix compositions could also
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increase channel incorporation. The biomimetic matrix and
channel pairing should be optimized first by use of stopped-flow
experiments with correctly applied permeability equations.
Before attempting new biomimetic formats, best-case-scenario
performance as well as outcomes with typical defect density can
be predicted using similar transport modeling methods as we
demonstrated here.
CONCLUSION AND OUTLOOK
Breakthrough desalination membranes will likely rely on
molecular-sieve-based transport, as is achieved in biological
and biomimetic membranes. In this review, we have shown that
an optimized biomimetic desalination membrane could achieve
salt rejections above those attainable by current RO membranes,
while still maintaining sufficient water permeability. However,
the major challenge for common biomimetic membrane formats
resides in fully covering the substrate with a defect-free, planar
channel-laden bilayer. The performance of a biomimetic
desalination membrane essentially hinges on maximizing
transport through highly selective nanochannels while minimiz-
ing defects.
A significant portion of biomimetic membrane research is
devoted to the development of synthetic channels for higher
stability and smaller per-channel physical footprints than
transmembrane integral proteins. Although the development
of nanochannels is important, we argue that equal attention
should be paid to the general biomimetic platform, including
polymeric matrices that enable functional channel reconstitu-
tion and robust membrane performance. Such work must start
with the optimization of channel reconstitution in vesicles using
stopped-flow studies with correct permeability equations and
then transition to lab-scale planar coupons that include
strategies for overcoming defects. Once successful, only then
can scientists turn to industrial-scale biomimetic formats. Unless
synthesis strategies are optimized, the persistence of defects in
the matrix can completely undermine the capabilities of the most
extraordinary molecular sieves.
Most biomimetic membrane studies have focused on
desalination rather than targeting other applications, likely
because the transport mechanisms of the inspiring aquaporin
initially suggested desalination. However, it is important to
assess if the possibility of biomimetic desalination is worth the
probable convoluted synthesis schemes. Fully aromatic
polyamide TFC-RO membranes already allow for highly energy
efficient seawater desalination and reject most solutes
sufficiently to reach less stringent standards for potable use,
including sodium chloride rejection up to 99.85%. Furthermore,
polyamide membranes are relatively easy and economical to
produce at a large scale and are familiar to the desalination
industry. The most significant room for improvement in
desalination membranes lies with increasing rejection of boron
in seawater desalination, increasing salt rejection in the
production of ultrapure water, withstanding higher pressures
in high-pressure RO toward zero liquid discharge, and
developing highly chlorine-resistant and antifouling materials,
especially for wastewater treatment and reuse. If biomimetic
membranes can achieve one or more of these goals, the relatively
complex synthesis strategies may be worthwhile. Scientists
developing synthesis strategies must consider these particular
goals as well as technoeconomic analyses in comparison with
current commercial TFC-RO standards.
New and emerging applications away from desalination may
best exploit the strengths biomimetic membranes have to offer.
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More niche applications that could benefit from molecular
sieving include recovery of valuable chemicals from waste
streams, analytical separations of chemically analogous and size-
similar species, and high-precision industrial separations. Even
in these situations, strategies to ensure that all transport takes
place through the molecular sieves and not through the
surrounding matrix would be necessary. For instance, as we
have shown, fluid-like biomimetic matrices can readily transport
lipophilic, neutral solutes. For a more impermeable matrix,
perhaps synthetic channels could be incorporated and self-
assemble in polymers above the glass transition temperature and
become immobilized within a glassy polymer upon cooling. In
cases of more random alignment of nanochannels, the addition
of channel exterior functionalities could promote stronger
interactions between channel and surrounding matrix, decreas-
ing the presence of interfacial defects.
The promise of biomimetic membranes lies in the perform-
ance of a well-engineered, highly stable discrete nanochannel,
while its biggest threat resides in the surrounding matrix
material. In general, defects persist in all current efforts toward
molecular sieves that incorporate next-generation nanomaterials
during top-down synthesis. For 2D laminates, interconnected
framework defects override the intended molecular sieving of
interlayer spaces or nanosheet pores, while mixed matrices
incorporating nanotubes or biomimetic nanochannels suffer
from defects like those described in this review. There has been
over a decade of research toward biomimetic membranes using
the top-down approaches discussed in this review. Based on
vesicular formats assessed via stopped-flow, several nano-
channels theoretically promise water-salt selectivity dramatically
exceeding that of current desalination membranes, yet planar
lab-scale biomimetic membranes have not achieved similar
performance. This struggle to transition from a vesicular format
to a small-area planar membrane coupon suggests that scaling up
to the industrial level will be a daunting challenge. However,
through strategic reformatting, biomimetic membranes could
enable a breakthrough performance in RO and find applications
beyond desalination.
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsnano.0c05753.
Descriptions of data extraction for channel permeabilities
and stopped-flow recalculations, Python codes for direct
fitting of the fundamental water flux equation for vesicular
stopped-flow studies (eq 8) and erroneous exponential
approximation (eq 14), further details on transport
modeling; tables with modeling parameters, brief
descriptions of studies that were considered for
calculation of defect density, and single-channel perme-
ability values; figures with example vesicle coverage
estimation using ImageJ, stopped-flow graphs with
overlaid models for eqs 8 and 14, and model sodium
chloride rejection and water permeability (based on
predicted defect density) compared to reported perform-
ance (PDF)
AUTHOR INFORMATION
Corresponding Authors
Jay R. Werber − Department of Chemical and Environmental
Engineering, Yale University, New Haven, Connecticut 06520,
https://dx.doi.org/10.1021/acsnano.0c05753
ACS Nano 2020, 14, 10894−10916
ACS Nano www.acsnano.org
United States; Department of Chemistry, University of Minnesota,
Minneapolis, Minnesota 55455, United States; Email: jwerber@
umn.edu
Menachem Elimelech − Department of Chemical and
Environmental Engineering, Yale University, New Haven,
Connecticut 06520, United States; orcid.org/0000-0003-
4186-1563; Email: menachem.elimelech@yale.edu
Authors
Cassandra J. Porter − Department of Chemical and
Environmental Engineering, Yale University, New Haven,
Connecticut 06520, United States
Mingjiang Zhong − Department of Chemical and Environmental
Engineering, Yale University, New Haven, Connecticut 06520,
United States; orcid.org/0000-0001-7533-4708
Corey J. Wilson − School of Chemical and Biomolecular
Engineering, Georgia Institute of Technology, Atlanta, Georgia
30332, United States; orcid.org/0000-0003-3040-1676
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsnano.0c05753
Author Contributions
C.J.P. primarily wrote the manuscript and conducted transport
modeling. J.R.W. and M.E. gave significant input for overall
article content and organization as primary advisors for this
work. M.Z. and C.J.W. provided technical feedback and helped
revise the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors acknowledge the support received from the
National Science Foundation through the Engineering Research
Center for Nanotechnology-Enabled Water Treatment
(EEC1449500) and via Grant CBET 1437630. The authors
also acknowledge funding from the National Science Founda-
tion Graduate Research Fellowship Program under grant no.
DGE-1752134, awarded to C.J.P. Any opinions, findings, and
conclusions or recommendations expressed in this material are
those of the authors and do not necessarily reflect the views of
the National Science Foundation.
VOCABULARY
Biomimetic membrane, a composite membrane composed of a
biomimetic active layer on a robust support; the active layer
mimics biological membranes by comprising an amphiphilic
matrix that incorporates and aligns biological or bioinspired
nanochannels; aquaporin, the class of biological transmem-
brane proteins responsible for relatively fast and selective
transport of water and, in some variants, glycerol; reverse
osmosis, process by which hydraulic pressure is applied to drive
a solution through a semipermeable barrier against the osmotic
pressure gradient, which is used in desalination; solution-
diffusion model, fundamental model for molecular transport
through isotropic membranes, whereby permeation of a
particular species is dictated by rates of partitioning into the
membrane material and diffusing across it; molecular sieving,
size-based molecular exclusion and transport by a semi-
permeable barrier with rigid, uniform pores
10913
Review
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