Research Article

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 26653−26661 www.acsami.org

Tailored Robust Hydrogel Composite Membranes for Continuous

Protein Crystallization with Ultrahigh Morphology Selectivity
Lin Wang,† Gaohong He,†,‡ Xuehua Ruan,‡ Daishuang Zhang,† Wu Xiao,† Xiangcun Li,†
Xuemei Wu,† and Xiaobin Jiang*,†
†
State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Engineering Laboratory for Petrochemical
Energy-efficient Separation Technology of Liaoning Province, Dalian University of Technology, Dalian, Liaoning 116024, China
‡
School of Petroleum and Chemical Engineering, Dalian University of Technology at Panjin, Panjin 124221, China
*
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ABSTRACT: The tailored and robust hydrogel composite membranes (HCMs) with diverse ion adsorption and interfacial
nucleation property are prepared and successfully used in the continuous lysozyme crystallization. Beyond the heterogeneous
supporter, the HCMs functioning as an interface ion concentration controller and nucleation generator are demonstrated. By
constructing accurately controlled nucleation and growth circumstances in the HCM-equipped membrane crystallizer, the target
desired morphology (hexagon cube) and brand-new morphology (multiple flower shape) that differ from the ones created in
the conventional crystallizer are continuously and repetitively generated with ultrahigh morphology selectivity. These tailored
robust HCMs show great potential for improving current approaches to continuous protein crystallization with specific crystal
targets from laboratorial research to actual engineering applications.
KEYWORDS: hydrogel composite membrane, continuous protein crystallization, morphology selectivity,
nucleation and growth control, lysozyme

■ INTRODUCTION
Biomacromolecule and protein crystallization processes play an
important role in analyzing the complex molecular structure
through X-ray diffraction (XRD) analyses, which substantially
promotes the development in biological research and drug
design.1−8 Simultaneously, the manufacture of the protein
crystal requires many target properties (fully grown crystal
surface and proper size are beneficial to accurate molecular
structure analysis; various morphologies are beneficial to
revealing sufficient multiple-dimensional information and so
on). Thus, stable operational materials and platform to achieve
this aim are dependent on the advanced technology in the
actual and continuous applications, which is also of importance
for the fine chemical, pharmacy, biological production and so
on. Both the crystals characterized by XRD or performed as
the pharmacy and biological products require perfect quality
and multiple crystal morphologies.9,10 As for the accelerating
nucleation and modified growth, the continuous and accurate
supersaturation control should be ensured, especially for the
protein crystallization following the two-step nucleation
mechanism.11,12 The devices with the microscale structure
can also improve the crystallization efficiency and process
control.13−16
In principle, robust materials mounted in a suitable
crystallizer for controlled heterogeneous nucleation and highly
selective crystal surface growth will facilitate the continuous
protein crystallization. However, the existing traditional
crystallization methods (batch crystallization, liquid−liquid
diffusion, vapor diffusion, etc.) for bio-macromolecules
crystallization have great limitations in efficiency, reproduci-
bility, and accurate crystal morphology control because of the
sensitivity of the intrinsic characteristics of biomacromolecule
to multiple biological, physical, and chemical conditions.1,17−19
The key approach to solving these problems is developing an
advanced crystallization platform equipped with the specific
robust materials with heterogeneous nucleation interface.
Recently, hydrogel composite membranes (HCMs) have
been applied in membrane crystallization (MCr) operation as a
Received: May 21, 2018
Accepted: July 16, 2018
Published: July 16, 2018

© 2018 American Chemical Society 26653 DOI: 10.1021/acsami.8b08381

ACS Appl. Mater. Interfaces 2018, 10, 26653−26661
ACS Applied Materials & Interfaces Research Article

Figure 1. (A) Preparation process of the detailed HCMs. (B) Infrared spectra of PP, prepolymer, PEGDA HCM, and HCM with different
PEGDA−NIPAM molar ratios (from 1:1 to 5:1). (C) DSC analyses of hydrogels with different PEGDA−NIPAM molar ratios (values in the
image: glass transition temperature, Tg). (D) SEM images of PP membrane, PEGDA HCM, and PEGDA−NIPAM HCM. (a) PP flat sheet
membrane. (b) Fully swollen PEGDA HCM with fingerprint-like surface morphology. (c) PEGDA membrane partially swollen. (d) Cross-section
SEM image of PEGDA HCM with fingerprint-like interface structure. (e) Fully swollen 2:1 HCM. (f−i) SEM images of cross sections of HCMs
with different hydrogel layer thickness. (j) Cross-sectional SEM of HCM with prolonged UV radiation duration. (k) Cross-section SEM image of
HCM immersed in water 14 °C for 4 weeks. (l) Cross-section SEM image of HCM applied in lysozyme MCr process for 7 days.

promising novel crystallization technology to accelerate the
crystallization process.20 HCMs act not only as a mass transfer
interface but also as a heterogeneous nucleation interface to
control the diverse properties of crystals via modifying the
nucleation and growth processes.21−23 Nevertheless, the
modification and optimization of the membrane interface are
still a challenging and limited research, such as the stability,
mechanical strength, and control effectiveness of the multi-
functional composite interface at harsh conditions. Con-
sequently, membrane modification employed in MCr has
attracted extensive attention and interest, and abundant
outstanding works had been reported.20,24,25 Hydrogel is one
kind of soft materials with 3D networks derived from specific
monomers and cross-linkers through different synthetic
methods, which have been exploited for various applica-
tions.26−31 The combination of hydrogel and microporous
membrane enhances the mechanical properties and retains the
intrinsic characteristic of hydrogel.27 In addition, HCMs have a
great influence on promoting the biomacromolecules crystal-
lization because of its superiority regarding reducing random
collisions and restraining convection in the biomacromolecules
MCr process.20,32
Herein, N-isopropylacrylamide (NIPAM) and poly(ethylene
glycol)diacrylate (PEGDA) are introduced as the functional
monomer and cross-linker, respectively, to obtain a series of
HCMs with thin hydrogel layers and tailored function of
regulating the interface ion concentration. The ratio of
monomer to cross-linker and polymerization conditions were
optimized to yield the mechanically stable composite hydrogel
membrane that can endure the prolonged continuous
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crystallization procedure. Additionally, as one of the classic
thermo- and pH-sensitive material, the NIPAM−PEGDA
hydrogel surface can provide diverse heterogeneous nucleation
free energy with high accuracy even under confined solution
compositions and operating temperatures. Fabricated HCMs
can simultaneously perform the functions of continuous ion
concentration control and crystal nucleation modification.
With realizing the above functions, the MCr devices equipped
with the developed HCMs were utilized to grow the lysozyme
crystals (as a classic model protein) continuously and
parallelly, which is aimed to develop a new approach to
continuous protein crystallization with high crystal morphol-
ogy selectivity and stable performance.
■ RESULTS AND DISCUSSION
Here, NIPAM (functional monomer) and PEGDA(cross-
linker) were used to fabricate thin hydrogel layers supported
by porous polypropylene (PP) membranes. To enhance the
chemical affinity between hydrophobic PP membrane and
prepolymer and synchronously maintain a defect-free thin layer
on the PP membrane surface, we had employed a new
fabrication approach of HCMs (Figure 1A): (1) first, the
prepolymer was coated by using a scraper on a piece of clean
and smooth glass plate. The ultimate thickness of the
composite layer is set at a proper value (about 5 μm); (2)
PP membrane then covered the prepolymer and acted as the
support layer to create an oxygen reduction circumstance,
which ensures the efficiency of the subsequent cross-linking
reaction under an oxygen-insufficient environment; and (3)
the “sandwich assembly” (PP membrane−prepolymer−glass
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Figure 2. (A) Schematic of the ion swelling-crystallization test on the fabricated HCMs. (B) Swollen sketch of PEGDA HCM and PEGDA−
NIPAM HCM. (C) SEM images of salt ion adsorption on HCMs with different PEGDA−NIPAM molar ratios (numbers in the images: crystal
distribution density, particle number per μm2). (D) Distribution statistics of NaCl crystals on different hydrogel membranes. (CV, coefficient of
variation).

plate, from top to bottom) was placed under a UV lamp for 30
min to initiate the cross-linking reaction. Then, the fabricated
HCMs were transferred from the glass plate and soaked into
ultrapure water to remove the unreacted discrete prepolymer.
Fourier transform infrared spectroscopy−attenuated total
reflection analysis indicated the successful polymerization
reactions (including PEGDA self-polymerization and
PEGDA−NIPAM copolymerization), shown in Figure 1B.
Compared to the prepolymer, the peaks of PEGDA HCM
disappear at 3070, 1680, and 990 cm−1, which confirms that
CC moieties had been completely consumed after UV
radiation. With different PEGDA−NIPAM molar ratios (from
1:1 to 5:1), the characteristic peaks of N−H appeared at 3310
and 1539 cm−1 (because of the effect of O−H at around 3500
cm−1, the peak at 3310 cm−1 is dependent). The characteristic
peak of amide-derived CO appeared at 1652 cm−1, and the
CC moieties were also completely consumed. In addition,
the formation of hydrogel layers did not affect the molecule
structure of the PP support membrane (shown in Figure S1). A
series of HCMs were completely established using the
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proposed fabrication approach. Additionally, the differential
scanning calorimetry (DSC) analyses demonstrated that the
glass transition temperature (Tg) of hydrogels increases with
mounting NIPAM percentage (Figure 1C), which also
qualitatively verified the increased average molecular weight
of hydrogel polymer. It also means that the size of mesh
structure in hydrogel can be regulated by changing the ratio of
the monomer (NIPAM) to cross-linker (PEGDA), which is
significant for the swelling and mass transfer of the
crystallization solution.
Beyond the structural regulation of HCMs at the molecular
scale, the stable functional surface and composite architecture
are more crucial for the continuous operation. Compared with
the porous surface of PP membrane (Figure 1Da), PEGDA
HCMs can readily possess fingerprint-like and wrinkled
morphology upon swelling after being soaked in aqueous
solution [Figure 1D(b,d)]. However, the hydrogel layer could
recover the smooth surface after drying, a specific HCM image
revealing this sensitive property is shown in Figure 1D(c), and
the swollen part is covered with regular wrinkles, whereas the
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Figure 3. Schematic diagram of the dynamic interfacial MCr assisted by HCMs and the molecular formation of functional cross-linked network.
(G*nucleation, the critical nucleation energy; Tfeed, the temperature of the feed crystallization solution; vflow,b, the velocity of the feed flow in the bulk
solution; vflow,m, the velocity of the feed flow at the membrane surface; Cs,b, the concentration of the crystallization component in the bulk solution;
and Cs,m, the concentration of the crystallization component at the membrane surface.)
dry part displays a smooth surface. In comparison, the scanning
electron microscope (SEM) images of fully swollen PEGDA−
NIPAM HCMs did not present the fingerprint-like morphol-
ogy of PEGDA HCM, conversely, a smooth surface [Figure
1D(e)]. It is likely attributable to the SEM testing conditions, a
vacuum and dehumidifying environment, evaporating a certain
amount of water. Compared to PEGDA HCM, the mesh size
of PEGDA−NIPAM HCMs (from 1:1 to 1:5, the molar ratio
of PEGDA to NIPAM) is too large to trap enough water to
maintain the hydrated state. As a result of the faster water loss
rate and lower equilibrium moisture content in PEGDA−
NIPAM HCMs in the vacuum and dehumidifying environ-
ment, the wrinkled morphology could not be probed by SEM.
For the continuous crystallization, persistent fluid drag force
requires a stable and robust HCM, that is, the hydrogel layer
and the support membrane bond tightly with each other.
Considering the possible deformation of the hydrogel layer
during the polymerisation reaction, a thinner hydrogel layer
will be conducive to the strong bonding between the hydrogel
layer and the support membrane, which is verified by the
experimental results [Figure 1D(f−i) ]. With increasing
thickness, the hydrogel composite layer tends to detach from
the supporting membrane. A proper thickness of the composite
hydrogel layer is around (or less than) 5 μm to ensure the
stable HCM structure throughout repeated tests. When pro-
longing the UV-radiation duration and the contact time
between supporting membrane and prepolymer, the composite
layer deeper infiltration into the porous support membrane
caused greatly tight bonding, which will result in another
structural damage. The hydrogel layer will destroy the integrity
of the support membrane even as the hydrogel layer thickness
is 4.9 μm [Figure 1D(j)]. With the proposed approach, the
fabricated HCMs have stable and robust performance in two
different tests: (1) immersed into water at 14 °C for 4 weeks;
(2) installed in the flat sheet membrane module for lysozyme
crystallization in the MCr process for 7 days. Figure 1D(k,l)
shows the virtually same cross-section structure of HCMs as
the original one after the tests, both of which confirm that the
composite layer had been firmly attached to the PP membrane
by controlling an appropriate hydrogel layer thickness and
polymerization reaction conditions.
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To demonstrate the different mesh structure of the HCMs
and reveal the significant impact of diverse hydrogel mesh
structure on ion adsorption and surface mass transfer, an
innovative swelling-crystallization test was carried out on the
fabricated HCMs via NaCl aqueous solution (the classic
crystallization agent solution for the lysozyme crystallization).
PEGDA HCM, 5:1 HCM, 3:1 HCM, and 1:1 HCM were
immersed into 1 mol L−1 NaCl solution for 7 days at 14 °C
(Figure 2A). After complete swelling, the treated HCMs were
placed under the same conditions (room temperature and the
same humidity and atmospheric pressure). The salt crystals
were formed on the hydrogel surface then observed by SEM
and analyzed using relevant image analysis software. NaCl
crystals with diverse shapes, average sizes, and size
distributions were obtained on the hydrogel surfaces with
different cross-linking structures (Figure 2C,D). The energy
dispersive X-ray (EDX) spectroscopy analysis results validates
that the crystals on the surface of HCMs are all NaCl crystals
(shown in Figure S2).
In the case of the PEGDA-based hydrogel, the salt crystals
formed on the surface are the classic square shapes with a high
distribution density (16.9 crystals per μm2) and wide size
distribution (mean size ∼0.1 μm, coefficient of variation (CV)
equal to 44.5). With the increase of NIPAM proportion in the
fabricated hydrogel layer (where the cross-linked molecule
mesh size is larger than the self-polymerized PEGDA, shown in
Figure 2B), the NaCl crystals varied from a cross-flower shape
(5:1) to a branched shape (3:1) and then to a long rod-like
shape (1:1). According to the classic nucleation and growth
theory,33−35 the polynuclear growth dominates the crystal
growth process when the rate of crystal nucleation growth
exceeds 20% of the diffusion-controlled rate. If the acquired
polynuclear growth rate is high and dominates the crystal
growth process under certain diffusion and operation
conditions, the resulting crystal morphology tends to be
rough, with numerous small dendrites and dispersive structure.
As for the hydrogel layers with different mesh sizes, the
increasing cross-linked molecule mesh size will lead to a quick
and uniform drying process. It induces the polynuclear growth
and creates highly branched crystal structure.
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In addition, the average sizes and size distributions obtained
on the different hydrogel surfaces (shown in Figure 2D) also
validate the diverse nucleation rates and crystal growth rates,
which suggests that the hydrogel interface with different
molecular mesh structures regulates the crystallization process.
PEGDA−NIPAM HCMs demonstrate the potential for
adjusting the crystal morphology and acquiring larger crystals.
Therefore, these results reveal the developed hydrogel
materials with a promising ability to regulate and control the
crystal morphology and size distribution.
Overall, considering the relatively low temperature required
of the lysozyme crystallization (from 10 to 14 °C), the
PEGDA−NIPAM HCMs with lower NIPAM percentage is
adopted to ensure the Tg of the material far from the operative
temperature. Thus, the hydrogel material can maintain the
proper mesh structure for the ion absorption and water transfer
flux. In addition, to achieve the potentially high throughput
production, the 5:1 HCM is chosen for the following
continuous protein crystallization in comparison with the
original PP membrane and PEGDA HCMs, providing
sufficient nucleation spots and nucleation rates.
Further understanding on the developed dynamic MCr
assisted by HCMs and the formation process of functional
cross-linked network was shown in Figure 3. With increasing
the stripping solution concentration on the permeate side of
HCMs, the concentration gradient of the crystallization
component Cs (black line in the figure) increased and reached
the maximum value at the hydrogel surface because of the
concentration polarization effect.36,37 Thus, the protein
reached the supersaturated metastable state in the boundary
layer of the HCMs, whereas the bulk solution was still in the
unsaturated or critical saturation state. This concentration
gradient increased the probability of the protein nucleation on
the hydrogel surface by constructing a stable density phase of
protein on an ideal two dimensional surface.38 In contrast, the
velocity of the feed flow (the component diffusion coefficient
and micromixing efficiency are the same) sharply decreased
from the interface of the boundary layer to the surface of
HCMs under the dynamic flow shearing effect. It will definitely
suppress the self-assembly of protein molecules from cluster to
nucleus in the metastable state.14 The inverse gradient of
concentration and velocity in the boundary layer of HCMs
surface, which are the crucial thermodynamic and kinetics
factors to induce the protein nucleation and provide the
conflicting effect on the critical nucleation energy G*nucleation
distribution (red line in the figure). Rather than on the
interface of the boundary layer-bulk solution or the surface of
HCMs, the initial nucleation prefers to occur at certain
locations away from the surface of HCMs. Subsequently, the
generated nuclei can be transferred from the boundary layer to
the bulk solution and then migrate out of the membrane
module, which achieves the continuous crystallization.
In addition, the thermo- and pH-sensitive HCMs provided
*
the extra function to further modify Gnucleation with high
flexibility under the limited operation conditions (T, pH, and
so on) because of the featured cross-linked network. The
hydrogel network composed of NIPAM and PEGDA is
thermo- and pH-sensitive because of their functional groups.
Hydrophobic amide groups of NIPAM form strong hydrogen
bonds with water. As the temperature rises to a critical point,
the hydrogen bonding of amides and water disappears, and the
interaction between amides and amides is enhanced so that the
mesh is contracted and the hydrophobicity is enhanced (the
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contact angle measured results demonstrated this conclusion in
Tables 1 and 2). While, with the decrease of pH, the gel grid
Table 1. Contact Angles under Different Temperatures of
HCMs
contact angle (deg)
HCMs type (PEGDA−NIPAM) 8 °C 25 °C 60 °C
1:1 45.5 50.7 55.6
2:1 38.1 42.0 53.9
3:1 46.7 47.5 52.4
4:1 42.6 47.3 52.8
5:1 32.9 45.5 58.5
shrinks, which leads to the enhancement of hydrophobicity,
but the ester carbonyl of PEGDA forms hydrogen bonds to
enhance the hydrophilicity of the material in the acid
environment. Under the combined action of the two aspects,
the HCMs finally showed the hydrophilic enhancement under
the condition of pH decreasing in the acid solution. As a
consequence, the nucleation generated under different
concentrations and fluid fields possesses diverse crystal forms
and distinct growth paths, which eventually achieve the
controllable, continuous crystal harvest with a range of
morphologies.
We further assembled the fabricated HCMs into the
membrane-crystallizer devices for the possible continuous
protein crystallization (Figure 4A). Other than in situ protein
crystallization on the membrane surface (or other substrates),
the varying ion concentration of NaCl on the feed side of the
membrane and the tunable flow velocity ensure the stable
crystal nucleation and growth in the boundary layer. The
whole set of the device was placed in a low temperature
chamber to provide the constant crystallization conditions.
Subsequently, the manufactured crystals in the membrane
crystallizer with a variety of morphologies are then transferred
to the storage tank (Figure 4B). To harvest the protein crystal
product with a high-throughput and validate the device
stability, the experiments are carried out with several parallel
MCr systems (Figure 4C, up to five parallel membrane
crystallizers can be running via the multichannel constant-flow
peristaltic pump), which enables scale-up in the laboratory and
investigates factors impacting the protein crystallization.
Applying this continuous MCr device, 11 classic tests were
carried out by introducing the original PP membrane, PEGDA
HCMs, and PEGDA−NIPAM HCMs under various temper-
atures, pH values of lysozyme solution, and stripping solution
concentrations. Five different crystal morphologies obtained in
the tests are defined as follows:
(i) Rod-like shape, aspect ratio (AR) > 4;
(ii) Long hexagon shape, 4 > AR > 2;
(iii) Hexagon plate shape, 2 > AR > 1;
(iv) Hexagon cube, hexagon shape, possesses similar length,
width and height in dimension, perfect particle fluidity, a
better shape than (i−iii) to avoid agglomeration;
(v) New polyhedron, multidirection grown flower-like
shape, only found in the PEGDA−NIPAM HCM-
equipped membrane crystallizer, which manifests a
totally different morphology following the polynuclear
growth mechanism.
PEGDA HCM preferred to form hexagon and hexagon cube
crystals so that the proportion of rod-like crystals decreased at
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Table 2. Contact Angles of HCMs under Different pH Values

contact angle (deg)
buffer pH PEGDA HCM 1:1 HCM 2:1 HCM 3:1 HCM 4:1 HCM 5:1 HCM
3.6 40.30 37.50 33.92 33.95 30.50 41.80
4.0 41.30 41.50 40.50 42.17 40.67 42.08
4.6 44.80 50.67 42.00 47.50 47.25 45.40

Moreover, the rod-like crystals and long hexagon crystals

Figure 4. (A) Schematic of the experimental setup of the continuous
MCr process. (1) Stripping solution (MgCl2) reservoir. (2) Lysozyme
solution (15 mg mL−1 lysozyme, 25 mg mL−1 NaCl, 30 mg mL−1
glycerol in 60 mL of sodium acetate−acetic acid buffer) reservoir. (3)
Flat sheet membrane module manufactured by polymethyl meth-
acrylate. (4) Peristaltic pump. (B) Schematic of membrane crystallizer
for the protein crystals growth. (C) Actual experimental devices of
continuous protein crystallization (from left to right: single membrane
crystallizer, multichannel parallel MCr system, an environmental
chamber with low temperature and low humidity).
pH = 3.6. Compared with the case pH = 3.6, the crystals were
only composed of hexagon and hexagon cube at pH = 4.6.
disappeared through pH regulation.
Under the investigated conditions, PEGDA−NIPAM HCMs
presented an ultrahigh morphology selectivity, predominantly
manufacturing the hexagon cube and a novel polyhedron
(flower) shaped crystals. As mentioned above, the target
morphology formation relies on the modified crystal surface
diffusion-controlled growth and polynuclear growth mecha-
nism. The flexibility and controllability of the relevant material
and devices on modifying the different crystal growth
mechanisms is the determining factor. By changing the
operation temperature, we can specifically obtain the hexagon
cube and the new-polyhedron (or flower) shaped crystals with
both selectivities exceeding 97%, and the formation of the rod-
like, long hexagon and hexagon plate shapes can be completely
inhibited in the PEGDA−NIPAM HCM-based membrane
crystallizer. The reason for the difference in crystal
morphologies is that the flexible hydrogel networks show
different swelling properties at diverse pH and temperature.
The diverse adsorption of salt ions and lysozyme molecules by
the microgrid leads to different supersaturations and
nucleation conditions at the hydrogel interface. As shown in
Figure 3 (the schematic diagram of the cross-linking reaction
of NIPAM and PEGDA), the microgrids of flexible hydrogel
consisting of ester carbonyl and amide groups afford a platform
for Na+, Cl−, and lysozyme molecules, which provide the
temporary stabilization of amorphous lysozyme (AL) nano-

Figure 5. Frequency of the different crystal morphologies manufactured via the original PP membrane and the corresponding HCM-based MCr
process. (AR, aspect ratio; Cstrip, concentration of the stripping solution; and ΔG*HEN/ΔG*HON, the ratio between heterogeneous nucleation energy
and homogeneous nucleation energy).

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Figure 6. Optical microscopy and SEM images of terminal lysozyme crystal products: (A) test #1, rod-like, 7 to 10 days; (B) test #3, long hexagon,
7 to 10 days; (C) test #6, hexagon plate, 7 days; (D) test #9, hexagon cube, 9 days; and (E) test #11, new-polyhedron, 6 days.
particles. Thus, the AL undergoes phase transition and causes
the different morphologies to be stabilized, which met the
reported results.24
Considering the thermo-sensitivity of NIPAM and pH-
sensitivity of PEGDA, PEGDA−NIPAM HCMs presented
different contact angles of the lysozyme solution at different
temperatures (shown in Figure 5). The adjustable surface free
energy can dramatically influence the heterogeneous nuclea-
tion energy, which extends the crystal growth mechanism
controlling scope at the limited operation parameters. The
hydrophilic HCMs can decline the initial heterogeneous
nucleation energy to 3−10% of that of the original hydro-
phobic PP membrane, which enables the protein nucleation at
a relatively low supersaturation degree and shortens the growth
duration. In addition, ΔGHEN* substantially decreased to 1/10
or 1/20 of ΔGHON*, presumably owing to the incorporation of
the NIPAM. Additionally, the surface free energy of PEGDA−
NIPAM HCMs still is readily adjusted via varying the system
pH and temperature, which may be of importance during the
accelerated nucleation and growth process. It is notable that
under the extremely low ΔGHEN * , the thermal−sensitive
interface of PEGDA−NIPAM HCMs can further reduce the
ΔGHEN
* /ΔGHON * from 0.029 to 0.021, when the operation
temperature decreased from 14 to 10 °C. In the case of
PEGDA HCMs, the corresponding ΔG*HEN/ΔG*HON increases
from 0.057 to 0.059, which shows no sensitive response to the
temperature variation. The high thermal sensitivity of
PEGDA−NIPAM HCMs endows the developed MCr devices
with the highly adjustable and accessible property, allowing us
to generate the target hexagon cube or new polyhedron shaped
crystals just via changing the operation temperature.
In principle, increasing the stripping solution concentration
can accelerate the concentrating process of the lysozyme
solution and promote the protein nucleation. On the other
hand, it may also increase the risk of the uncontrolled
nucleation and growth (e.g., tests #3 and #4 in Figure 5, the
crystal morphology distribution gets dispersed as Cstrip
increases from 0.30 to 0.35 mg L−1, the percentage of the
rod-like crystals increased from 36 to 54%). For comparison,
tests #10 and #11 in Figure 5, the crystal morphology shows
the single polyhedron (or flower) shape with a very high
uniformity (97 and 98%, respectively) when Cstrip increases
from 0.30 to 0.35 mg L−1. Thus, this superior selectivity
demonstrates the importance of the fabricated HCMs in terms
of the screening of the crystals with the target morphology and,
more importantly, with respect to developing the crystals with
brand-new morphology. In addition, increasing research
indicated that the nonclassical polymer crystallization occurs
through the mesoscale self-assembly of (bio) macromolecular
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nanocrystals to result in diverse crystal morphologies.39−41 The
presented results in this work that met the new concept for
macromolecular crystallization is of importance for materials
science, chemistry, nanoscience, structural biology, and so on.
Besides the high crystal morphology selective function, the
PEGDA−NIPAM HCM-equipped membrane crystallizer also
acts as an efficient and stable platform for continuously
harvesting the desired protein crystals with target shape
(Figure 6). The classic morphology and size of the final
lysozyme crystal products generated the prominent influence
of the membrane crystallizer and the membrane materials
employed. To quantitatively evaluate the advantage of the
fabricated HCMs, the production capacity (PC, kg products·
m−3·h−1), which is a classic definition in industrial crystal-
lization aspect, is introduced and calculated as follows
m
PC = P
Vτ (1)
where mP is the mass of the crystal products; V is the volume of
the crystallizer; and τ is the crystallization duration. In contrast
to the test performed with the PP-based membrane crystallizer,
the PEGDA−NIPAM HCM-based crystallizer can maintain
the high morphology selectivity even when the Cstrip increased
to 0.35 g mL−1 (the frequency of the new polyhedron shape
approaches 0.98 in test #11). With the intensified stripping
process, the nucleation and protein crystallization process can
also be accelerated, and the crystallization duration is
shortened from 9 (test #10) to 6 days (test #11) when the
average size of the terminal crystals is the same (around 100−
150 μm). For the crystal products obtained in tests #1, #3, and
#6, the rapid and uncontrolled nucleation at the high
supersaturation level inhibits the crystal growth, and most of
the obtained products were around 10−30 μm in size (Figure
6A−C), together with the low morphology selectivity. As a
comparison, under the stable nucleation and growth at the
lower supersaturation degree, the obtained product in
PEGDA−NIPAM HCM-equipped MCr (Figure 6D,E) pre-
sented perfectly smooth crystal surface with larger size, which
could be conducive to the XRD analysis and biological
purpose. Additionally, well-controlled nucleation and crystal
growth in the membrane module reduce the required retention
time and the size of the tank for storing crystal products, that
is, the PC can be substantially enhanced, which shows the
potential of the proposed HCM-equipped membrane crystal-
lizer for harvesting the crystals on a large scale.
■
CONCLUSIONS
In summary, we have developed a series of tailored and robust
HCMs for continuous protein crystallization. The effective
DOI: 10.1021/acsami.8b08381
ACS Appl. Mater. Interfaces 2018, 10, 26653−26661
ACS Applied Materials & Interfaces Research Article

combination of hydrophilic hydrogel prepolymer and hydro-
phobic PP membrane affords the long-term stable perform-
ance, accurate mass transfer, and protein crystallization control.
In addition to the heterogeneous support for the crystallization
process, PEGDA HCM and PEGDA−NIPAM HCM function-
ing as an interface of ions concentration regulator and
nucleation generator have been demonstrated experimentally.
To reveal the superior functions of the developed HCMs
regarding crystal growth modification and morphology
selection, the advanced membrane crystallizers with selected
PEGDA−NIPAM HCMs were established to realize the stable
high-throughput crystal production.
To shift the protein crystallization from an inefficient and
stochastic way to a stable and specific one, the robust
PEGDA−NIPAM HCMs with intrinsically effective pH and
MCr Process for Continuous Lysozyme Crytstallization. The
feed side of the protein membrane crystallizer consisted of lysozyme,
NaCl, and glycerol. The pH values of HCH3COOH−NaCH3COOH
buffer were fixed at 3.6 and 4.6. Lysozyme (900 mg) was dissolved in
30 mL of HCH3COOH−NaCH3COOH buffer (15 mg mL−1). Next,
1.5 g of NaCl was dissolved in 30 mL of HCH3COOH−
NaCH3COOH buffer (25 mg mL−1) to behave as a crystallization
promoter agent, then mixed together, and 1.8 g of glycerol was added
(30 mg mL−1), which was used as an additive to accelerate the
crystallization process. The other side involved 60 mL of MgCl2
solution (250, 300, 350, 400 mg mL−1). At 10 and 14 °C, each side of
the solution was circulated simultaneously in a 30 mL flat-sheet
membrane module. The flow rates of lysozyme-containing and MgCl2
solutions were regulated by using a peristaltic pump. (Lysozyme,
0.103 mL s−1, equivalent to 0.131 m s−1; MgCl2, 0.328 mL s−1, 0.104
m s−1).
* /ΔGhom
Calculation of ΔGhet * . The ratio between the heterogeneous

ÅÄÅ ÑÉ3
thermal sensitivities demonstrate its role in screening the and homogeneous nucleation free energy is expressed as follows34

Å (1 + cos θ)2 ÑÑÑ

= (2 + cos θ)(1 − cos θ)2 ÅÅÅÅ1 − ε ÑÑ
crystals with the target favorable morphology (hexagon cube),

ÅÅÇ (1 − cos θ)2 ÑÑÑÖ

and more interestingly, the crystals with brand-new morphol- *
ΔG het 1
ogy (the multiple flower shape) were discovered. The PC of *
ΔG hom 4
the proposed MCr process can be easily enhanced by (2)
assembling the developed HCMs into the multichannel parallel ΔG*het, heterogeneous nucleation free energy; ΔG*hom, homogeneous
membrane crystallizer systems and can accomplish scalability nucleation free energy; θ, contact angle; and ε, porosity, for the
of continuous protein crystallization, which may provide a new membrane in this work, εPP = 0.1, εHCMs = 0.

■
perspective on the development of flow chemistry and crystal
engineering. In addition, this research provides in-depth insight ASSOCIATED CONTENT
into the production of protein crystals, applied to numerous *
S Supporting Information
fields such as pharmacy and biophysics, and into the The Supporting Information is available free of charge on the
fundamental understanding of crystallization mechanisms. ACS Publications website at DOI: 10.1021/acsami.8b08381.

■ EXPERIMENTAL SECTION
Materials. PP flat sheet membranes of ∼40.0 μm thickness and
XRD, EDX spectroscopy analysis, contact angles
measurement, and mechanical property analysis results
∼0.1 surface porosity were provided by the Institute of Chemistry, (PDF)
Chinese Academy of Sciences, Beijing, China. NIPAM (analytical
pure) was purchased from Sigma-Aldrich. PEGDA (molecular weight
of PEG is 400) was purchased from RYOJI Organic Chemical
Industry Co., Ltd. The photoinitiator (Irgacure2959) was provided by
■ AUTHOR INFORMATION
Corresponding Author
BASF. The lysozyme was provided by Solarbio. Sodium chloride, *E-mail: xbjiang@dlut.edu.cn. Phone: +86-411-84986291. Fax:
magnesium chloride, and sodium acetate (NaCH3COOH) were +86-411-84986291.
provided by Tianjin Damao Chemical Reagent Co., Ltd. Acetic acid ORCID
(HCH3COOH) was provided by Aladdin. The glycerol was provided Gaohong He: 0000-0002-6674-8279
by Sinopharm Chemical Reagent Co., Ltd.
HCMs Fabrication. The prepolymer was prepared by mixing
Wu Xiao: 0000-0003-1810-7562
PEGDA and NIPAM at different molar ratios (1:1 to 5:1), and then, Xiangcun Li: 0000-0003-1647-676X
Irgacure2959 was added as a photoinitiator with a mass fraction of Xuemei Wu: 0000-0002-0930-7602
2%. First, the mixed prepolymer was stirred with a magnetic stirring Xiaobin Jiang: 0000-0003-0262-4354
bar for 24 h until completely dissolved. The prepolymer was coated Notes
by using a scraper on a piece of clean and smooth glass plate. Next,
The authors declare no competing financial interest.

the PP membrane covered the prepolymer as a support layer to create
an oxygen insufficient circumstance. The glass, prepolymer, and PP
support had already comprised a monolithic sandwich construction.
The “sandwich construction” was placed under a UV lamp for 30 min
to induce the polymerization and cross-linking of monomers. Finally,
the fabricated HCMs were removed from the glass plate, soaked into
ultrapure water for 12 h to extract the unreacted prepolymers, and
then dried in a 50 °C oven until the mass remained constant.
■
ACKNOWLEDGMENTS
We acknowledge financial contribution from the National
Natural Science Foundation of China (grant nos. 21527812,
21676043, U1663223, and 21606035), Changjiang Scholars
Program (T2012049), and Fundamental Research Funds for
the Central Universities (DUT16TD19 and DUT17ZD203).

Mechanical property analysis results of the fabricated HCMs were
listed in Table S2.
Salt Solution Swelling-Crystallization Test. Four kinds of
HCMs (1:1, 3:1, 5:1, and pure PEGDA HCMs) were immersed into
20 mL of NaCl (1 M) for 7 days. The fully swollen HCMs were
placed in an environment chamber at room temperature, with
constant humidity and atmospheric pressure. After the crystal growth
was completed on the hydrogel interfaces, the crystals attached to the
hydrogel surfaces were observed by scanning electron microscopy and
the crystal size distribution was analyzed by nanomeasurer software.
■
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DOI: 10.1021/acsami.8b08381
ACS Appl. Mater. Interfaces 2018, 10, 26653−26661