pubs.acs.org/journal/ascecg
Research Article
Cellulose Nanofiber and Magnetic Nanoparticles as Building Blocks
Constructing Biomass-Based Porous Structured Particles and Their
Protein Adsorption Performance
Annie M. Rahmatika, Youhei Toyoda, Tue T. Nguyen, Yohsuke Goi, Takeo Kitamura, Yuko Morita,
Kazunori Kume, and Takashi Ogi*
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Cite This: ACS Sustainable Chem. Eng. 2020, 8, 18686−18695
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ABSTRACT: Nanostructured fine particles have attracted attention
as next generation materials because of their unique features and ease
of handling compared with those of nanoparticles. However, most
previously reported studies are limited to using nanoparticles or
precursor solutions (e.g., atoms or molecules) as building blocks. In
this study, we successfully developed a new type of porous structured
fine particles via self-assembly of TEMPO-oxidized cellulose
nanofibers (TOCNs) and magnetic nanoparticles (Fe3O4 NPs) as
building blocks by spray-drying followed by template removal
method. The resulting porous structured TOCN−Fe3O4 particles
possessed unique macro−meso−microporous structures with a
highly negative charge (ζ potential = −55 mV) and sufficient
magnetization (Ms = 15 emu/g). The Fe3O4 NPs played an
important role not only in enabling effective collection through magnetic separation but also in increasing the specific surface area by
inhibiting aggregation of the TOCNs during the drying process while maintaining the intrinsic ζ potential value of the TOCNs. The
porous structured TOCN−Fe3O4 particles allowed excellent mass transfer of lysozyme (a model protein adsorbate), which led to
high adsorption capacities of >950 mg/g, rapid equilibrium (<10 min), magnetic separation capability, good reusability, and
excellent selectivity in a binary solution of lysozyme and bovine serum albumin.
KEYWORDS: TEMPO cellulose nanofibers, Iron oxide nanoparticles, Nanostructured particle, Spray drying,
Template assisted aerosol process, Lysozyme
■
INTRODUCTION
Nanostructuring of submicrometer-sized particles, such as
porous, hollow, and core−shell structured particles, is a
promising approach to develop sustainable particulate
materials. Because of the unique characteristics of nanostructured fine particles, such as high specific surface area,
lightweight, unique optical properties, excellent mass transfer,
and high durability, they are expected to be applied to a wide
range of fields, such as catalysts, adsorbents, carrier agents,
sensors, and pharmaceuticals.1−3 Many studies on nanostructured fine particles have been reported.4−6 However, the
materials (building blocks) used to construct the nanostructured particles are limited to nanoparticles and precursor
solutions (atoms or molecules). In other words, utilization of
cutting-edge nanomaterials (e.g., nanofibers, nanotubes,
molecular organic frameworks, and quantum dots) as building
blocks is challenging, although it has the potential to lead to
creation of new materials.
Another important factor for sustainable particulate
materials development is the selection of raw materials.
© 2020 American Chemical Society
Because cellulose is the most abundant renewable biomass in
the world, it is highly attractive as a sustainable material for
industrial scale-up.7 In particular, TEMPO-oxidized cellulose
nanofiber (TOCN) is an emerging biobased nanomaterial with
great potential in adsorption of positively charged compounds
owing to its abundant carboxylate groups, highly negative ζ
potential with amorphous and crystalline regions in uniform
nanoscale diameter, and few micrometer fiber lengths.8
Previous studies on TOCN-based nanomaterials have shown
that they have high adsorption capacities for heavy metal
cations (Cu(II), Fe(II), Zn(II), and As(III)), dye molecules,
and organic solvents.9−11 The TOCN surface contains
carboxyl and hydroxyl groups that can effectively coordinate
Received: October 14, 2020
Revised: November 20, 2020
Published: December 8, 2020
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ACS Sustainable Chem. Eng. 2020, 8, 18686−18695
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Research Article
Figure 1. Schematic of preparation of porous structured TOCN−Fe3O4 (TFP-114) particles.
and capture cationic molecules.12 However, in practical
application, it is difficult to develop TOCN as nanostructured
fine particles or composite particles while maintaining the
excellent intrinsic properties of TOCN (high specific surface
area and high negative charge), especially in the dried state.13
TOCNs easily integrate and aggregate through loss of the
repulsive forces by capillary effect during water evaporation in
a simple drying process.14 As a result, dried TOCN possesses a
dense structure, low specific surface area, low porosity, and
small pore size, which decrease the intraparticle diffusion rate
of an adsorbate. The low porosity and small pore size are
acceptable for cationic molecule adsorption owing to the small
molecule size, but they are a drawback for adsorption of
macromolecules, such as protein. The control process of
protein adsorption on the surface of the adsorbent material
following the specific requirements is needed for various
applications, such as separation, purification, drugdelivery
agents, biosensors, and hemodiafiltration filtration. In recent
years, some researchers have shown that macromolecule
adsorption requires particles with specific functionalization,
relatively high porosity, and large pore size (>100 nm).15,16
Therefore, to expand the application range of TOCN in the
dried state, developing TOCN materials with high porosity
and large pore size without losing the high negative charge is
required, but this is challenging.
Recently, to maintain the dispersed state (high specific
surface area) of TOCN, our group proposed a method to
support TOCN on nanostructured particles, that is, formation
of TOCN-decorated macroporous SiO2 particles. We found
that the macroporous structure improves the specific surface
area in conjunction with outstanding protein adsorption
performance.17,18 Furthermore, we successfully changed the
pore size of the SiO2 particles in the range 100−500 nm and
revealed the optimum structure of the TOCN-decorated SiO2
particles to achieve significant and high performance protein
adsorption. However, this method has some drawbacks. First,
it requires complicated and multistep routes for (i) synthesis of
the macroporous SiO2 particles, (ii) surface modification of the
macroporous SiO2 particles, and (iii) loading TOCN on the
macroporous SiO2 particles. Second, after the protein adsorbs,
a centrifugation process is necessary to recycle the particles,
causing high product loss and production costs.19
From the viewpoint of construction of a sustainable society,
it is desirable to develop an environmentally friendly, energy
saving, and cost-effective method. Therefore, in this research,
we propose a new concept to solve the issues mentioned
above: proposed concept for producing protein adsorbents
with excellent mass transfer, good adsorption properties, and
magnetic separation ability. A starting precursor composed of
cellulose nanofibers (TOCNs), template particles (poly(methyl methacrylate) (PMMA) particles), and magnetic
nanoparticles (Fe3O4 NPs) is sprayed into the heating zone
(<200 °C). During solvent evaporation, self-assembly of
TOCN, PMMA, and Fe3O4 NPs forms TOCN−Fe3O4−
PMMA composite particles. The PMMA particles are removed
to produce porous structured particles with TOCN and Fe3O4
NPs as a building block.
The proposed concept enables fewer downstream processing
steps and reagents (e.g., surface charge control agent)
compared with the previously reported process. In addition,
the mixed magnetic nanoparticles are expected to provide
other advantages: (1) adsorbent separation can be performed
in a magnetic field and (2) increased specific surface area with
formation of micropores to mesopores by inhibiting
aggregation of the TOCNs during the drying process.
Formation of micropores and mesopores inside the particles
is expected to create interconnected pore channels, contributing to promotion of mass transfer of proteins. In this study,
the adsorption performance (adsorption rate, selectivity, and
reusability) of lysozyme protein on the obtained porous
structured TOCN−Fe3O4 particles was evaluated. This is the
first report of synthesis of nanostructured fine particles
composed of TOCN and Fe3O4 building blocks with
micropores to macropores and characterization of protein
adsorption.
■
EXPERIMENTAL SECTION
Materials. TEMPO Oxidized Cellulose Nanofiber (TOCN)
(Rheocrysta I-2SX, 2 wt %) was supplied from DKS Co., Ltd.,
Japan. Poly(methyl methacrylate) (PMMA) (Techpolymer grade
MAS-5GX, powder, particle size 503 nm) was supplied from Sekisui
Chemical Co., Ltd., Japan. Iron oxide nanoparticles (Fe3O4 NPs,
particle size 10 nm) were provided from Toda Kogyo Co., Ltd., Japan.
The model protein, lysozyme (Lys), was purchased from MP
Biomedicals, Solon, OH, and Bovine Serum Albumin (BSA) was
purchased from FUJIFILM Wako Pure Chemical Co, Japan.
Ammonia chloride buffer solution (pH 9) was prepared by dissolving
1.07 g of ammonium chloride in 50 mL of 0.1 M ammonia. This
solution was diluted with 50 mL NaOH solution 0.02 M. NaOH
solution (0.1 M) was purchased from Sigma-Aldrich.
Synthesis of Porous Structured TOCN−Fe3O4 (TFP-114)
Particles. The porous structured TOCN−Fe3O4 (TFP-114) particles
were synthesized using an aqueous TOCN dispersion, PMMA
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particles as a template, and Fe3O4 NPs. A schematic of the route for
preparation of the TFP-114 particles is shown in Figure 1. First, 2.0 g
of 2 wt % TOCN dispersion and 0.16 g of PMMA fine particles were
dispersed in 50 mL of distilled water using an ultrasonic device (IKA,
T 10 basic ULTRA-TURRAX S004, Japan) at 30000 rpm for 20 min.
Fe3O4 NPs were then added at a weight ratio of 1 to TOCN, and the
mixture was further stirred for 40 min. The mixed aqueous precursor
suspension was then sprayed through a two-fluid nozzle system (mini
spray-dryer B-290, BÜ CHI, Switzerland) at an inlet temperature of
180 °C, a liquid flow rate of 2.5 mL/min, and a hot air flow rate of 6
L/min. To obtain a macroporous structure, the collected dried
particles were etched with toluene to remove the PMMA particles as
the template. The etched particles were collected from the toluene
with a magnet (1.0 T), and toluene was removed. Finally, the sample
was washed with ethanol and dried at 80 °C for 30 min to obtain
TFP-114 particles.
To better understand of characteristics of the TFP-114 particles
and for adsorption performance comparison, porous structured
TOCN (TFP-104) particles and TOCN particles (TFP-100) particles
were prepared by the same method. The detailed concentrations of
each prepared precursor sample are given in Table 1. The experiments
were repeated more than two times to check the reproducibility of the
results, and identical results were obtained.
phoresis (SDS-PAGE), followed by Coomassie brilliant blue (CBB)
gel staining. The protein amount was quantified using ImageJ software
(NIH).
Characterization of TFP Particles. Scanning electron microscopy (SEM) (S-5000, Hitachi Ltd., Japan) was used to observe the
morphology of the particles at 3.0 to 20 kV under a nitrogen
atmosphere. Before SEM analysis, the particles were dispersed in
ethanol, and then the ethanol solution was dropped onto an
aluminum plate and heated at 60 °C. A thin layer of platinum was
then sputtered on the dispersed particles. The particle size
distribution and ζ potential in liquid suspension were measured at
25 °C using a Zetasizer Nano analyzer (Malvern Instrument Inc., UK)
after dispersing 1% w/v particles in water for 30 min. The particle size
distribution of TFP particles was taken by measuring the Feret’s
diameter of more than 300 particles from the SEM images. The
presence of Fe3O4 NPs was determined by X-ray diffraction (XRD;
Bruker D2 Phaser, Bruker AXS GmbH, Germany). Fourier transform
infrared spectroscopy (IRAffinity-1S, Shimadzu, Japan) was performed to investigate the characteristics of the functional groups on
the particles. The specific surface area (SSA) was determined by N2
adsorption using the Brunauer−Emmett−Teller (BET) model. The
N2 adsorption/desorption of isotherms were measured after heating
the sample at 180 °C for 3 h (BELSORP-max, BEL, Japan). The pore
size distribution was calculated using the Innes and Horvath−
Kawazoe (HK) models for the mesopore and micropore size
distributions, respectively.
Table 1. Precursor Concentrations of Various TFP
Particlesa
Sample Name
TFP-100 (TOCN
particles)
TFP-104 (porous
structured TOCN
particles)
TFP-114 (porous
structured TOC
N−Fe3O4 particles)
TOCN
(T) (wt
%)
Fe3O4(F)
(wt %)
PMMA
(P) (wt
%)
Wt
ratio of
T:F:P
Total
vol
(mL)
1:0:0
50
0.32
1:0:4
50
0.32
1:1:4
50
0.08
0.08
0.08
0.08
Research Article
■
RESULTS AND DISCUSSION
Morphology of TFP Particles. Here, we report fabrication
of porous structured TOCN−Fe3O4 (TFP-114) particles by
spray-drying followed by a template removal process. The welldefined micro−meso−macroporous structure of the TFP-114
particles was achieved by self-assembly of TOCN, PMMA, and
Fe3O4 as building blocks during droplet evaporation. SEM
images of the obtained TFP particles and the possible particle
formation mechanisms are shown in Figures 2 and 3,
respectively.
The morphologies of the TFP-100 particles are shown in
Figure 2a−c. The TOCNs ultimately aggregated and formed
wrinkled particles with a geometric mean diameter (Dp) of 1.9
± 0.9 μm. The TOCNs used in this study had widths of 2−5
nm and lengths of ≥1 μm (see Figure S1). The particle
formation mechanism is shown in Figure 3a. In the suspension
state, TOCNs were individually dispersed well in water due to
their surface charge (ζ potential of −66 mV). When the
atomized droplets containing TOCNs contacted hot air, the
free water molecules on the surface of droplets rapidly
evaporated. Rapid solvent evaporation led to the accumulation
of nanofibers (TOCNs) on the interface of droplets. When the
droplets shrank in association with solvent evaporation, the
TOCNs lost their stability (repulsive forces) by the capillary
effect, hydrogen bonding, and van der Waals forces.20,21 As a
result, a shell composed of nanofibers formed on the surface of
the droplets. However, due to the high aspect ratio (>150) of
the TOCN, in the further evaporation process, some TOCNs
experience entangled, and others are rearranged in the droplet,
which eventually deformed the droplet shape due to uneven
shrinkage forces and finally tended to buckle. Therefore,
wrinkled particles are formed with a cavity size of 100−250 nm
at several meeting points of deep grooves on the surface of the
particles (Figure 2c). This phenomenon is in accordance with
previous spray-dried cellulose nanofibers.22,23
Addition of PMMA particles to the spray suspension
containing TOCN led to formation of porous structure on
TFP-104 particles (Figure 2d−f). Self-assembly of TOCN and
a
Distilled water was used as the solvent.
Evaluation of Equilibrium Adsorption Performance. To
investigate the adsorption performance of the TFP particles (TFP100, TFP-104, and TFP-114), 50 mL of aqueous Lys solution was
prepared at a total concentration of 0.2 mg/mL in ammonium
chloride buffer solution (pH 9). Next, 0.01 g of each of the TFP
particles was added to the Lys solution and collected several times by
decantation using a magnet for the TFP-114 particles and
centrifugation (15000 rpm, 5 min) for the TFP-100 and TFP-104
particles. The supernatant was then analyzed by ultraviolet−visible
(UV−Vis) spectrophotometry (UV-3150, Shimadzu, Japan) to
measure the absorbance intensity change at a wavelength of 281 nm.
Reusability Evaluation of TFP-114 Particles. The reusability of
the TFP-114 particles was investigated by repeating the adsorption−
desorption procedure four times using the same particles. Typically,
4.0 mg of the TFP-114 particles was added to 20 mL of 0.2 mg/mL
Lys solution (pH 9) and ultrasonicated at room temperature for 2 h.
The TFP-114 particles were then removed with a magnet, and the
change of the Lys amount in the supernatant was quantified by UV−
vis spectrophotometry. The desorption process was performed by
dispersing the reused TFP-114 particles in 20 mL of 1 M NaOH for 2
h. After decantation, the amount of Lys in the NaOH solution was
determined.
Evaluation of Selective Adsorption Performance in a Binary
Mixture. To evaluate application of the TFP-114 particles to selective
adsorption, a binary protein mixture of BSA and Lys was prepared in
50 mL ammonium chloride buffer solution (0.2 mg/mL for BSA and
Lys). Next, 0.01 g of the TFP-114 particles was added, and the
mixture was ultrasonicated for 120 min. The TFP-114 particles were
removed by magnetic separation, and the supernatant was collected
and analyzed by sodium dodecyl sulfate polyacrylamide gel electro18688
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Figure 2. SEM images of the TFP particles at low and high magnification: (a−c) TFP-100, (d−f) TFP-104, and (g−i) TFP-114. Dp is a geometric
mean diameter.
both the outer skeleton surface and inner surface of the porous
structure of the TFP-114 particles (Figure S6). As another
comparison, TOCN-Fe3O4 particles (TFP 110) were also
prepared to observe the result of self-assembly between TOCN
and Fe3O4 NPs in spray-drying process (Figure S7). The result
also shows that Fe3O4 NPs were clearly well distributed in
TOCN setwork. This indicates that there was good selforganization among the building block constituents (Fe3O4
NPs and TOCN) during the spray-drying process. From the
similar morphology to the TFP-104 particles and the good
dispersibility of the Fe3O4 NPs, the possible particle formation
process is shown in Figure 3c. Generally, it is difficult to
disperse NPs in a solvent because of their high surface energy.
However, several studies have shown that well-dispersed
TOCNs can effectively disperse nanomaterials through
electrostatic repulsion.26,27 Thus, after the droplet containing
PMMA, TOCN, and Fe3O4 NPs was sprayed and solvent
evaporation started, self-assembly occurred in the drying
process because of the repulsive interactions among all of
the components. As solvent evaporation occurred, the PMMA
particles, as the largest component, then became the main
backbone for formation of a porous structure. The nanomaterials, in this case, the Fe3O4 NPs and TOCN, then acted as a
lubricant that filled the space between the template particles to
form building blocks of dried composite particles while
maintaining their respective positions. Even though the TFP104 particles had a porous structure that increased the surface
area, it is still believed that the skeleton of the particles
consisted of aggregated TOCNs. Thus, we expect that addition
of Fe3O4 NPs will further expand the TOCN network because
the equilibrium for mutual dispersion between the TOCNs
PMMA in the spray droplet is shown in Figure 3b. The PMMA
used in this study had a spherical form with a particle size of
503 nm and a ζ potential of −56 mV (see Figure S2). A strong
repulsive interaction successfully acted between PMMA and
the highly negatively charged TOCNs (ζ = −66 mV), and they
were individually distributed in the droplet. Most of the
TOCNs then filled the voids between the closed packed
structure of PMMA as water evaporated.24,25 As a result, the
PMMA template particles were homogeneously distributed in
the TOCN network of the compact dried composite particles.
Subsequently, the PMMA particles could be simply removed
through an etching process using toluene to form macroporous
structured particles with a pore size of 445 nm and TOCN as
the building block component (Figure 2f). Because the
diameters of the pores corresponded to those of the PMMA
particles, etching treatment did not affect the structure of the
dried particles (Figure S3). In addition, even though the same
amount of TOCN was used, the size of the TFP-104 particles
(Dp = 2.8 μm) was significantly larger than that of the TFP-100
particles (Dp = 1.9 μm). This indicates that the TFP-104
particles possess a higher specific surface area because the
larger particle size is attributed to the pore volume owing to
removal of PMMA. Optimization of the porous structure was
performed by variation of the TOCN/PMMA concentration
(Figure S4).
To enhance the separation efficiency, Fe3O4 NPs with a
particle size of 10 nm and a ζ potential of −39 mV (Figure S5)
were added into the TOCN−PMMA precursor suspension to
prepare TFP-114 particles. Porous structured particles with a
macropore size of 442 nm and a particle size of 2.9 μm were
formed with a similar morphology to the TFP-104 particles
(Figure 2g−i). However, Fe3O4 NPs were clearly observed at
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Research Article
Figure 3. Mechanisms for formation of various TFP particles: (a) TFP-100, (b) TFP-104, and (c) TFP-114.
nonporous or macroporous materials.30 However, porous
structured particles exhibited hysteresis type H3 for P/P0 of
0.8−0.95 that suggests more mesoporous structures than the
dense TOCN particles. In the low relative pressure range (P/
P0 < 0.05), N2 adsorption on the TFP-114 particles was about
3 times higher than on the TFP-100 and TFP-104 particles.
This results indicates the formation of microporous structures
in the TOCN network during the drying process.
To investigate the influence of PMMA and Fe3O4 NPs on
formation of porous structures, we determined the pore size
distributions of the TFP-100, TFP-104, and TFP-114 particles
(Figure 5a,b). The mesopore size distribution was analyzed in
the range 2−100 nm by Innes theory. The micropore size
distribution was examined in the range 1−2 nm by the HK
method. As a result, introduction of PMMA and Fe3O4 NPs
into the TOCN network through the spray-drying process
significantly increased the specific surface area by forming
various sizes of pores. (1) The kissing effect on the PMMA
particles formed interconnected channels in the range of
mesopores to macropores. The pore size distribution curves for
the porous structured particles showed significant improvement compared with the dense structure (TFP-100) with
distinct peaks at <7, 13, and >50 nm. In addition, the Fe3O4
NPs shifted the macropore size from a peak of >80 to 60 nm
(determined from the mesopore size distribution in Figure 5b),
because the existence of Fe3O4 NPs, in addition to TOCN,
and Fe3O4 NPs will prevent formation of TOCN dense
packing during solvent evaporation.
Characteristics of TFP Particles. The presence of Fe3O4
NPs was confirmed by XRD analysis. The XRD pattern of the
TFP-114 particles is shown in Figure 4a. The XRD pattern of
the TFP-114 particles showed six characteristic peaks at 30.2°,
35.5°, 43.2°, 53.8°, 57.3°, and 63°, which correspond to the
(220), (311), (400), (422), (511), and (440) crystal planes of
magnetite (Fe3O4, JCPDS No. 1011084).28
Despite the presence of Fe3O4 NPs and the PMMA removal
process, all the resulting TFP particles could maintain the high
ζ potential of the TOCN (Figure 4b) and showed an
absorption band at 1610 cm−1 attributed to the carboxyl
groups (Figure 4c).29 The TFP-100 particles maintained a high
negative ζ potential of −65 mV. This means that spray-drying
successfully constructed a nanostructure particle without
changing the chemical functionalization. The ζ potentials of
the porous structured particles (TFP-104 and TFP-114)
slightly decreased owing to presence of the carbonyl group
(C=O, absorption band at 1725 cm−1), which belongs to
PMMA remaining in the particles. However, the ζ potential
was still from −52 to −55 mV.
The specific surface area of the TFP particles increased with
the presence of porous structure and Fe3O4 NPs. From N2
adsorption−desorption isotherms graph (Figure 4d), all of
TFP particles showed a type II isotherm which associated with
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Figure 4. (a) XRD spectrum of the TFP-114 particles. (b) Specific surface areas (SSABET) and ζ potentials of the TFP particles. (c) FTIR spectra
of the TFP particles. (d) N2 adsorption−desorption isotherms of the TFP particles.
interaction between the negatively charged TFP particles and
positively charged Lys. In step II, the adsorbed amount
significantly decreased in the dense structure of the TFP-100
particles, while a slight reduction occurred for the porous
structured particles. This phenomenon can be attributed to the
insufficient adsorption sites of the dense structure. The
competition between occupation of the adsorption sites
makes more protein migrate than adsorb.31 Conversely, the
macroporous structures provided abundant open sites for the
adsorption process, which prevented migration of protein. In
addition, our previous research showed that adsorption of Lys
on TOCN network structure decorated porous structured SiO2
particles occurred more heterogeneously and consisted of
more multilayer adsorption than that of the dense structure.18
After 30 min, the equilibrium adsorption capacity (step III)
was achieved in the adsorption process of the TFP-100
particles. For porous structured particles, the equilibrium
adsorption capacity was reached in less than 10 min.
To understand the effect of a typical structure created by the
presence of Fe3O4 NPs, the adsorption capacities of the TFP
particles for the same base amount of TOCN are shown in
Figure 6c. First, the concentration of TOCN in the TFP
particles was approximated by thermogravimetric analysis (see
Figure S10 and Table S2). The weight loss owing to the
decomposition process at high temperature was assumed to be
the weight of TOCN. The details of the measurement and the
estimated weight concentration are described in the Supporting Information. The adsorption capacity from Figure 6a was
then divided by the weight of TOCN in the TFP particles.
Interestingly, the adsorption capacity of the TFP-114 particles
was more than 2 times the capacity of the TFP-104 particles
and more than 3 times the capacity of the TFP-100 particles.
To clarify whether this increase was because of the adsorption
increased the distance between the PMMA particles, and,
finally, the attachment area of two PMMA particles became
smaller owing to the kissing effect (Figure 5c). (2) The
presence of Fe3O4 NPs in TFP particles significantly increased
the pore volume of microporous at a size of <1.3 nm and
mesoporous at a wide variation in size (Figure 5a,b and Figure
S8 a,b). These results clearly indicate that Fe3O4 NPs
enhanced the good interconnected channel by splitting the
TOCN aggregates, and micropores and mesopores formed
between the TOCNs and NPs. These results indicate that the
structure of TFP-114 particles will provide good penetration
by retaining a lot of space between the TOCNs.
Protein Adsorption Behavior on TFP Particles. Macromolecule penetration into the TFP-114 particle structure was
investigated by protein adsorption experiments. The protein
adsorption performance was investigated by adsorption of Lys
at pH 9 and 25 °C several times. The TFP-114 particles
showed the highest adsorption capacity, following by the TFP104 and TFP-100 particles (Figure 6a). In other words, the
porous structure successfully increased the Lys adsorption
performance from 600 mg/g for the TPF-100 particles to 844
mg/g for the TFP-104 particles and 955 mg/g for the TFP-114
particles. The porous structures provided excellent mass
transfer of protein into the internal structure of the particles,
which contained abundant active sites. We found that TFP-100
particles showed a different trend in the adsorption process
before reaching the equilibrium state. Overall, the adsorption
process was divided into three steps (Figure 6a) with the
adsorption behavior followed a monolayer-type (Figure S9 and
Table S1). The mechanism of each step is shown in Figure 6b.
First, the amount of Lys adsorbed on the TFP particles rapidly
increased within 5 min. In this step, the adsorption process
resulted in a large adsorbed amount owing to electrostatic
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Figure 5. (a) Pore size distributions of 2−100 nm pores calculated by the Innes model. (b) Pore size distributions of 1−2 nm pores calculated by
the HK model. (c) Illustration of self-assembly of TOCNs, PMMA particles, and Fe3O4 NPs.
Reusability and Selectivity of TFP-114 Particles. The
reusability of the TFP-114 particles is shown in Figure 7a. The
adsorption and desorption cycles were performed at pH 9 and
pH 11.5, respectively. The relative adsorption capacity of Lys
in each cycle was evaluated by the first adsorption capacity
(Figure 6a). The relative desorption capacity was assessed by
the capacity for Lys desorption in each cycle relative to the first
desorption capacity. The results showed that the TFP-114
particles can maintain their adsorption and desorption
capacities for up to four cycles. This also indicates that the
adsorption and desorption processes can be easily tuned by
changing the pH of the solution because the TFP-114 particles
are negative charged in a wide pH range while Lys is positively
charged for pH > 10.5 (Figure S13).
The selectivity of the TFP-114 particles was assessed by Lys
adsorption from a binary solution of Lys and BSA. We
prepared a binary protein solution containing Lys (pI 10.5,
14.3 kDa) and BSA (pI 5.3, 68 kDa) at the same
concentrations. The concentrations of the proteins in the
ability of the Fe3O4 NPs used in this study, Fe3O4 particles
(TFP-010) and TOCN-Fe3O4 particles (TFP-110) were used
to remove Lys under the same conditions (Figure S11). The
results showed that adsorption of Lys on the Fe3O4 NPs was
very small compared with TOCN, so this effect can be ignored.
This result clearly shows that the unique structure formed by
combining the porous structure and Fe3O4 NPs successfully
promotes dispersion of TOCNs without loss of their surface
charge for the adsorption process. Unlike the TFP-104
particles, the presence of the Fe3O4 NPs increased the number
of internal open channels in the TFP-114 particles. As a result,
this increased number of available channels allowed the
solution to easily penetrate into the particles in which there
were abundant adsorption sites for the protein. In addition, the
TFP-114 particles have another advantage that they can be
easily collected from the solution by centrifugation or magnetic
separation (Figure 6d) with a saturated magnetization value of
15 emu/g (Figure S12).
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Research Article
Figure 6. (a) Adsorption performance of the TFP particles. (b) Illustration of the steps of Lys adsorption on the TFP particles. (c) Adsorption
capacities based on the amount of TOCN. (d) Mixed solution before and after decantation of TFP-114 particles.
Figure 7. (a) Reusability of the TFP-114 particles. (b) SDS-PAGE of Lys and BSA before adsorption (lane 1) and after adsorption on the TFP-114
particles (lane 2). (c) Calculated selectivity for Lys based on SDS-PAGE.
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binary solution before (Figure 7b, lane 1) and after adsorption
(Figure 7b, lane 2) were quantified by SDS-PAGE (Figure
7b,c). In comparison with the Lys band below 15 kDa in lane 1
(Figure 7b), the Lys in solution was almost completely
removed by the TFP-114 particles after the adsorption process
(lane 2 in Figure 7b). The disappearance of the Lys band
suggested that Lys adsorbed to the TFP-114 particles. The
relative intensities of Lys showed that only 7% of the initial Lys
concentration remained in the solution. By contrast, BSA was
only slightly removed by the TFP-114 particles because a
strong band was still present at around 70 kDa after
adsorption. On the basis of the amount of BSA from lane 1,
89% of BSA remained in the solution. Thus, the TFP-114
particles showed strong selectivity for Lys based on electrostatic interaction in this binary solution.
orcid.org/0000-0003-3982-857X; Email: ogit@
hiroshima-u.ac.jp
Authors
Annie M. Rahmatika − Department of Chemical Engineering,
Graduate School of Advanced Science and Engineering,
Hiroshima University, Higashihiroshima 739-8527, Japan;
Department of Bioresources Technology and Veterinary,
Vocational College, Gadjah Mada University, Depok 55281,
Indonesia; orcid.org/0000-0003-3622-0606
Youhei Toyoda − Department of Chemical Engineering,
Graduate School of Advanced Science and Engineering,
Hiroshima University, Higashihiroshima 739-8527, Japan
Tue T. Nguyen − Department of Chemical Engineering,
Graduate School of Advanced Science and Engineering,
Hiroshima University, Higashihiroshima 739-8527, Japan
Yohsuke Goi − R&D Headquarters, DKS Co. Ltd., Kyoto
601-8391, Japan
Takeo Kitamura − R&D Headquarters, DKS Co. Ltd., Kyoto
601-8391, Japan
Yuko Morita − R&D Headquarters, DKS Co. Ltd., Kyoto
601-8391, Japan
Kazunori Kume − Graduate School of Integrated Sciences for
Life, Hiroshima University, Higashihiroshima 739-8530,
Japan
■
CONCLUSIONS
Porous structured TOCN−Fe3O4 (TFP-114) particles have
been successfully prepared by self-assembly of TOCN and
Fe3O4 NPs as building blocks through spray-drying followed
by a template removal process. The combination of a porous
structure and Fe3O4 NPs successfully dispersed in TOCN in
the dried particles resulted in a specific surface area of nearly 4
times that of the TOCN particles. Removal of the template
(PMMA) particles caused formation of porous structures with
interconnected channels within the particles in the range of
micropores to macropores. The presence of Fe3O4 NPs
enhanced formation of many meso−micropores because their
presence among the TOCN networks inhibited aggregation of
TOCNs during the drying process. The intraparticle structure
in the TFP-114 particles showed a good mass transfer process
through macro−meso−micropore coordination but maintained a highly negative ζ potential of more than −55 mV.
The adsorption properties of the TFP-114 particles were
analyzed and compared with those of porous structured
TOCN (TFP-104) and TOCN (TFP-100) particles prepared
by spray-drying under the same conditions. The TFP-114
particles showed the highest adsorption capacity (>950 mg
Lys/g adsorbent) for the same amount of adsorbent particles
and about 3 times higher adsorption capacity (>2000 mg Lys/
g TOCN) than the TFP-100 particles for the same amount of
TOCN. In addition, the TFP-114 particles provided rapid
equilibrium adsorption (<10 min), maintained adsorption−
desorption performance for up to four cycles, showed good
selectivity in a binary solution, and were easy to collect by
centrifugation or magnetic separation.
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acssuschemeng.0c07542
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. All images in the manuscript including TOC
graphic were created by one of the authors (A.M.R.)
Funding
JSPS KAKENHI Grant Number 19H02500.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grant Number
19H02500. This work was partly supported by the JSPS Coreto-Core Program, Hosokawa Powder Technology Foundation,
the Mazda Foundation, and the Electric Technology Research
Foundation of Chugoku. The authors would like to thank the
Japanese Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) for providing a doctoral scholarship
(A.M.R.).
ASSOCIATED CONTENT
■
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acssuschemeng.0c07542.
AFM images of TOCNs, SEM image of PMMA
particles, SEM images of particles before and after
etching, SEM image of Fe3O4 NPs, TG measurements,
adsorption performance of Fe3O4, magnetization result,
and ζ potential of TFP 114 results (PDF)
■
Research Article
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