Journal of Colloid and Interface Science 507 (2017) 145–153

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Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Regular Article

Synthesis of silica nanoparticles using biomimetic mineralization with

polyallylamine hydrochloride
Kyoung-Ku Kang, Hyun-Seok Oh, Dong-Young Kim, Gyurak Shim, Chang-Soo Lee ⇑
Department of Chemical Engineering, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Spherical and monodispersed silica

nanoparticles were synthesized by
using PAH. #EKFKEOGFKWO
 PAH is strongly involved in nucleation
and condensation reactions of silica.
 PAH can accelerate the nucleation
and condensation of the hydrolyzed 12 mM
TEOS. PAH
 PAH has a relatively very weak
Low conc. of PAH pH7 High conc. of PAH
interaction with aqueous silicates. HCl n

NH2

$CUKEOGFKWO

a r t i c l e i n f o a b s t r a c t

Article history: To synthesize silica particles under mild conditions, we proposed a biomimetic synthesis method. The
Received 7 April 2017 synthesis process was carried out based on a biphasic sol-gel synthesis method using TEOS (tetraethyl
Revised 11 July 2017 orthosilicate) as a silica source and PAH (polyallylamine) as a substitute for proteins of marine microor-
Accepted 29 July 2017
ganisms for biosilicification. The function and activity of the PAH, used as a replacement for bioactive
Available online 31 July 2017
substances, were confirmed through comparisons between control experiments and designed experi-
ments. The PAH exhibited the ability accelerate condensation with hydrolyzed TEOS in aqueous solutions.
Keywords:
The PAH also exhibited high condensation activity in acidic and neutral conditions to produce silica par-
Silica nanoparticle
PAH
ticles. Moreover, PAH also created the nuclei of the silica particles, and the number of nuclei could be con-
Biomimetic trolled by the concentration of PAH. In addition to exhibiting these unique capabilities, PAH did not
Biosilicification generate any complexes or composites with the silica species. Depending on the synthesis conditions,
Biomineralization the synthesized silica particles exhibited various shapes, such as sponge-like, self-assembled, irregular
spherical and completely spherical shapes. The sizes of the primary particles were diverse, with a range
from 10 nm to 50 nm. In particular, by adjusting the PAH concentration, it was possible to obtain nearly
perfect spherical-shaped silica nanoparticles with uniform sizes, which has rarely been reported. Above
all, using this paper, we can get closer to understanding the principles of silica formation using PAH as a
replacement for the bioactive proteins of microorganisms.
Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction

⇑ Corresponding author. Silicon oxide, which is called silica, is one of the most abundant
E-mail address: rhadum@cnu.ac.kr (C.-S. Lee).
elements on earth [1,2]. For this reason, silica has been widely used

http://dx.doi.org/10.1016/j.jcis.2017.07.115
0021-9797/Ó 2017 Elsevier Inc. All rights reserved.
146 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153
to manufacture glass, ceramics, and silicones, traditionally [2]. Due
to its excellent physicochemical stability, silica is also extensively
used in many industries to date. Moreover, various forms of silica
can be used for the required application, and these various forms
include glass frits, powders, and colloids, depending on the synthe-
sis method and processing method [3–10]. Among these different
types of silica, colloidal silica has useful advantages, such as large
surface areas, low toxicity, high biocompatibility, optically high
transparency, high chemical stability, and high thermal stability
[11–13]. Moreover, colloidal silica has an another advantage due
to the ability to easily modify the free silanol groups on its surface
using various modification techniques [13–18]. Because of these
useful and diverse unique properties, colloidal silica is used as a
raw material for numerous industrial and research fields world-
wide, and a variety of synthesis methods and applications have
been researched.
The conventional synthesis methods for colloidal silica can be
classified into two big categories. One category includes gas phase
syntheses, and the other includes liquid phase syntheses
[2,11,13,19–26]. In general, it is known that liquid phase synthesis
methods are more advantageous for controlling the size, shape and
fine structure of the particles [2,13,22,25]. However, conventional
liquid phase synthesis methods, which are based on the sol-gel
process, have been carried out under harsh processing conditions.
In particular, the reactant mixtures contain many highly toxic
chemicals, and the pH of these mixtures can be extremely high
or low. Moreover, the processing temperatures, which are over
100 °C, are also too high [2,13,25].
The discovery of biosilicification using marine microorgan-
isms, such as diatoms and sponges, has opened a new era of
synthesis methods for precipitating silica under mild conditions
[27–43]. Unlike sol-gel-based synthesis methods, biosilicification
is a marine microorganism-based silica production system,
which proceeds at ambient temperatures and neutral pH condi-
tions. These mild processing conditions can lower process costs
and are environmentally friendly. As research on the silica pro-
duction mechanism of marine organisms has progressed, the
key biomolecules, such as proteins and peptides, that can par-
ticipate in the silicification of microorganisms have been found
[36,42,44]. Well-known examples of such silica-forming biomo-
lecules include silicateins, which are found in sponges, and a
silaffin R5, which is found in diatoms [29,37,44,45]. Silicification
using biomimetic synthesis methods has been reported for var-
ious similar silicateins, silaffins (R5), and biomolecules that
were prepared using recombination, readily available proteins
and synthetic polymers containing functional groups similar to
those of biomolecules [29,33,37,39,40,45–48]. Furthermore,
these biomolecules exhibited silica forming abilities, and the
synthesized silica exhibited various morphologies, such as
spherical, branched and agglomerated shapes. Particularly, such
biomolecules have been reported to perform similar roles as
sol-gel catalysts in specific regions [37,38,40,41,46,49]. However,
these studies have had several limitations. The silica sources
(tetramethyl orthosilicate or tetraethyl orthosilicate) must be
prehydrolyzed, and many of these studies used buffer solutions
as solvents. In other words, most of the previous studies show
that the study of the function of physiologically active sub-
stances is limited to the condensation reaction and particle
growth of aqueous silicates. In addition, when the silicon alkox-
ide is used as a silica source, it must be hydrolyzed using a
strong acid such as hydrochloric acid in advance. In other cases,
if sodium silicate derivatives can be used as the silica source,
they become strong alkaline during the hydration process. In
both cases, it would be difficult to avoid the use of acids or
bases, and it would be cumbersome to operate the reactant
manufacturing process separately, rather than the silica synthe-
sis process, the main process. Ultimately, the pre-preparation of
hydrated silica source will be an invisible weakness when con-
sidering the commercialization of eco-friendly biosilicification
processes.
In this paper, we report the synthesis of silica in aqueous solu-
tions using a biomimetic synthesis with PAH as a replacement
silica-forming biomolecule. In order to extend the investigation
of the function of PAH to the whole sol-gel process, all the synthe-
sis processes were carried out using pure TEOS as a silica source.
For a comparison study of the reaction rate of both hydrolysis
and condensation, the synthesis processes were carried out using
a biphasic synthesis system, except for the separated prehydrolysis
reaction. The effects of the synthesis parameters on the silica for-
mation and growth were also investigated under various pH condi-
tions and PAH concentrations of the mother liquors. It was possible
to control the particle size and shape of the silica produced by
changing the pH and PAH concentrations in the mother liquors.
The prepared silica particles exhibited sizes from 15 nm to 50 nm
and various shapes, such as irregular shapes, monodisperse
spheres, and interconnected spherical and branched structures.
Furthermore, it was experimentally proven that PAH participated
in condensation reactions related to the growth of the silica parti-
cles. These synthetic methods using artificial molecules that imi-
tate biomolecules can be employed for the synthesis of silica
nanoparticles in non-toxic solvent systems, and the synthesized
silica can be applied to medical, pharmaceutical and biotechnolog-
ical studies and industries. In addition, this study suggests basic
information about the possibility of eliminating the pre-
hydrolysis step.
2. Material and methods
Tetraethyl orthosilicate (TEOS, 98%), polyallylamine hydrochlo-
ride (PAH, average Mw = 15,000) and sodium hydroxide (NaOH,
>98%) were purchased from Sigma-Aldrich. Aqueous hydrochloric
acid (HCl, 35–37 wt%) was obtained from Samchun Pure Chemical.
Deionized water (>18 k ohm) was obtained using Milli-Q water.
Absolute grade ethanol for washing was obtained from Sigma-
Aldrich. All chemicals were used as purchased without further
purification.
The effect of PAH at various pH values from acidic to basic con-
ditions was investigated. The concentration of PAH was fixed to 12
mM. Various concentrations of the PAH aqueous solutions were
prepared at pH 7 and adjusted with NaOH aqueous solutions. All
of the aqueous PAH solutions were prepared as stock solutions in
advance. The silica synthesis was carried out using a modified
sol-gel method. The rate of hydrolysis and condensation was inves-
tigated using a biphasic synthesis system. The particle growth was
also induced by increasing the synthesis temperature.
TEOS was used as the silica source. The silica synthesis was car-
ried out by adding TEOS to an aqueous solution containing PAH
prepared in advance. For maintaining biphasic conditions, TEOS
was added without agitation at room temperature. The biphasic
solution consisted of TEOS as the upper phase and an aqueous
solution of PAH as the lower phase. The synthetic mother liquor
was then completely sealed. After, the mixture was stirred at
500 rpm using a magnetic stirrer, the temperature was increased
to 50 °C for 1 h, and the mixture was stirred for 24 h at 50 °C.
The total synthesis time was limited to 24 h for comparing the
reaction rates. Then, the resultant mixture was cooled to room
temperature. After the preparation procedure, the silica was
obtained after centrifugation and washed three times with deion-
ized water. Finally, the washed samples were further rinsed with
ethanol. Table 1 summarizes all the experimental conditions dur-
ing the silica synthesis, including the control experiment.
 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153 147

Table 1
Composition of the reactants for synthesizing silica using the biomimetic method with PAH.

Catalyst pH of the catalyst solutiona Molar ratio

Reagent Concentration (mM) Si Catalyst
HCl 12.00 2.40 1.00 0.041
– 0.00 6.80 – –
NaOH 12.00 11.70 1.00 0.041
NH4OH 12.00 10.80 1.00 0.041
PAH 12.00 4.00 1.00 0.041
PAH 12.00 5.00 1.00 0.041
PAH 12.00 6.00 1.00 0.041
PAH 12.00 7.00 1.00 0.041
PAH 12.00 8.00 1.00 0.041
PAH 12.00 9.50 1.00 0.041
PAH 1.00 7.00 1.00 0.003
PAH 5.00 7.00 1.00 0.017
PAH 10.00 7.00 1.00 0.034
PAH 20.00 7.00 1.00 0.068
PAH 30.00 7.00 1.00 0.103
PAH 40.00 7.00 1.00 0.137
a
The catalyst solution was prepared in advance with distilled water, the catalyst and the pH adjusting reagent.

The synthesized silica was characterized using FE-SEM (field-
emission scanning electron microscopy: S-4800, Hitachi), TEM
(transmittance electron microscopy: Tecnai G2 F30 S-TWIN, FEI)
and EDX (energy dispersive X-ray spectroscopy). The FE-SEM spec-
imens were prepared by rinsing them with ethanol, dropping the
samples dispersed in ethanol onto a silicon wafer, and drying them
at 50 °C. The TEM specimens were prepared by dropping a sample
dispersed in ethanol on a TEM grid and then drying at 50 °C. The
distribution of the synthesized silica was analyzed using ImageJ
software.
3. Results and discussion
Fig. 1 shows SEM images of samples synthesized using the con-
ventional sol-gel process as a control experiment to observe the
effect of PAH. These experiments proceeded using the biphasic
synthesis method and did not contain PAH in the mother liquors,
and each synthesis process was carried out under acidic, neutral
and basic conditions. HCl was used as a catalyst under acidic con-
ditions. Under neutral conditions, the synthesis of silica was car-
ried out using only water and TEOS without adding any catalyst.

Fig. 1. FE-SEM images of the silica synthesized from the control experiments excluding PAH. (a) pH 2.4, 12 mM HCl; (b) pH 6.8, deionized water only; (c) pH 11.7, 12 mM
NaOH; and (d) pH 10.8, 12 mM NH4OH.
148 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153
Under basic conditions, the sol-gel process was carried out using
aqueous NaOH and ammonia as catalysts. According to the SEM
analysis, for the samples synthesized under acidic and neutral con-
ditions, a silica thin film was observed, and silica particles did not
appear. The formation of the silica thin film was attributed to the
preparation of the SEM specimens and favorable growth patterns
of silica under acidic conditions.
When the sol-gel process was carried out using silicon alkoxide
as the silica source, its hydrolysis and condensation reactions were
significantly important [13,25]. Generally, under acidic conditions,
hydrolysis proceeds faster than condensation [50,51]. For pH con-
ditions of 4 or less, it is well known that fast hydrolysis can be
attributed to the ability of the alkoxy groups to stabilize the tran-
sition state of hydrolyzed silicon alkoxide [25,50]. Moreover, con-
densation occurs between a silicon having a neutral nucleophilic
silanol group and a protonated silicon having a silanol group
[25]. Therefore, the silica prepared under acidic conditions exhib-
ited polymeric species with various branched networks with sila-
nol groups. However, under basic conditions, it is known that
hydrolysis and condensation reactions occur at the same time
[25,50]. Silica synthesized under base conditions usually grows
after the particle nuclei are formed. Although this process differs
depending on the synthesis conditions, it is common that sec-
ondary growth occurs due to aggregation of primary particles
grown from the nuclei [2,25]. Additionally, the particles produced
were obtained in the form of colloids or precipitated solids in the
mother liquor, and these had extremely diverse morphologies
and sizes. Thus, the silica synthesized under basic conditions was
entirely different from the silica synthesized under acidic condi-
tions. The silica thin films in Fig. 1 a and b were attributed to the
typical sol-gel process as described above. In other words, these sil-
ica thin films originated from the polymeric silica species synthe-
sized under acidic and neutral conditions and drying process of
the SEM specimen preparation. The results also show that the
sol-gel reaction for polymer formation under acidic and neutral
conditions was more dominant than the sol-gel reaction for parti-
cle formation.
In contrast, the silica produced under basic conditions was dif-
ferent from the silica produced under acidic and neutral condi-
tions. In the case of silica synthesized using NaOH as the basic
catalyst, silica nanoparticles were observed. Additionally, when
ammonia with the same molar composition was used as the basic
catalyst, the formation of silica nanoparticles was also observed.
These results indicate that basic catalysts have an advantage for
forming precipitated silica in aqueous solutions [2,25]. However,
the shapes of the silica nanoparticles synthesized using two dif-
ferent basic catalysts were completely different. The silica synthe-
sized with NaOH as a catalyst showed agglomerates that were
composed of at least over three primary particles with a relatively
uniform size of 20 nm or more. However, in the case of silica pre-
pared using ammonia as the catalyst, it was possible to observe
the generation of spherical particles with a quite uniform size
of 15 nm or less. In addition, unlike the acidic and neutral condi-
tions during the synthesis process, the shape of the precipitated
silica after centrifugation could be easily observed with the naked
eye. Therefore, it was relatively easy to wash and separate the
precipitated products, as compared to the silica synthesized
under acidic and neutral conditions. Based on these results, the
synthesis of silica particles using TEOS under aqueous basic con-
ditions is advantageous for the production of silica particles com-
pared to acidic and neutral conditions. In particular, the basic
catalysts play a role in increasing the condensation reaction rate
during the particle formation. Finally, even when silica was syn-
thesized using different basic catalysts, the sizes and morpholo-
gies of the particles produced were greatly influenced by the
kind of catalyst used, even if the amount of the catalyst was
the same. These results indicate that the degree of activity of
the catalyst can have a significant influence on the generation
and growth of the particles.
To investigate the role of PAH in the formation of silica, the pH
was changed from acidic to basic conditions. Fig. 2 shows the SEM
images of the silica produced under each condition. The particle
size distribution obtained from the image analysis when the silica
synthesis was carried out at pH 7 is also shown in Fig. 2. Due to the
hydrochloric acid contained in the PAH, the pH value was 4.1. Par-
ticle formation was observed, in contrast to the previous results.
The silica synthesized using only PAH had a different morphology,
which was significantly different from the silica synthesized under
the absence of PAH under acidic and neutral conditions. The result-
ing particles were in the form of aggregates that consisted of many
primary particles. The size of the primary particles was approxi-
mately 10 nm. At pH 5 and 6, an aggregated form of primary par-
ticles was also observed, and this morphology was similar to that
of the silica synthesized using only PAH. As the pH was increased
under acidic conditions, the primary particles gradually grew to
over 20 nm in size. The synthesized silica changed dramatically
under neutral and basic conditions. First, under neutral conditions,
the primary particles changed to sphere-like shapes, and their sizes
grew to nearly 45 nm. Moreover, the synthesized silica particles
were well-dispersed and were in a different form than the
observed aggregates formed under acidic conditions. When the
pH of the mother liquor was increased under basic conditions, sev-
eral aggregations of primary particles were also observed. How-
ever, the shapes of the aggregated particles and sizes of the
primary particles were different from those of the formerly synthe-
sized silica under basic conditions in the absence of PAH, and the
aggregation morphology was similar to that of a three-
dimensional interconnected network. These results indicate that
PAH promoted silica formation [52,53]. More precisely, the con-
densation of hydrolyzed TEOS was accelerated by PAH in the aque-
ous solution, which was attributed to the amine groups contained
in PAH [37,54]. Moreover, this acceleration in the condensation
rate made it possible to generate silica particles under acidic con-
ditions, in which the condensation rate was relatively slow com-
pared to the hydrolysis rate. This result became even more
evident when compared with the previous control experiments.
Moreover, this increase in the condensation rate was revealed by
the dispersion and aggregation of the silica particles. As the con-
densation rate increased, all the aqueous hydrolyzed TEOS actively
participated in the condensation for growing the silica particles,
and then, the residual hydrolyzed TEOS was fully consumed in
the mother liquor. Thus, the possibility of the presence of residual
hydrolyzed TEOS species acting as a coagulant was very low. The
presence of well-dispersed primary particles without any aggrega-
tion under neutral conditions also provided indirect evidence for
this phenomenon.
The aggregation of silica nanoparticles under basic conditions in
the presence of PAH was due to the conventional sol-gel synthesis
[55–57]. The silica nanoparticles synthesized and dispersed in the
basic solution could dissolve again, and this redissolved silica could
participate in recondensation again. Redissolution and recondensa-
tion are well-known phenomena for the synthesis of silica
nanoparticles under basic conditions. When the silica was synthe-
sized using PAH under basic conditions, this phenomenon might
have occurred in the similar manner of a conventional sol-gel syn-
thesis, and the redissolved silica species seemed to act as a coagu-
lant for particle aggregation. However, in this state, the redissolved
silicate concentration was low, and the silica particles were already
sufficiently fully grown. It is believed that PAH as a catalyst can
influence the entire formation process of silica from nucleation
to growth. Therefore, at the end of the silica particle growth pro-
cess, the silica formation reached an equilibrium between the
 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153 149

(a) (b)

(c) (d)

(e) (f)

(g) 30

25

20
Probability (%)

15

10

0
0 10 20 30 40 50 60 70 80 90 100
Particle size (nm)

Fig. 2. FE-SEM images of the silica samples synthesized under various pH conditions and the particle size distribution synthesized at pH 7. SEM images of the silica
synthesized at (a) pH 4, (b) pH 5, (c) pH 6, (d) pH 7, (e) pH 8, and (f) pH 9.5. The concentration of PAH in the mother liquor was equal to 12 mM. (g) The particle size
distribution of the silica synthesized at pH 7 and obtained from SEM images using image analysis software.

redissolution and recondensation processes. In other words, the recondensation processes seemed to be dominated by the presence
silica formation was dominated by the presence of PAH. However, of NaOH, which acted as a basic catalyst. At this stage, the growth
after the silica particle growth was complete, the redissolution and of the particles due to the presence of PAH was stagnated,
150 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153

(a) (b)

(c) (d)

(e) (f)

(h)
PAH 40 mM

PAH 30 mM

PAH 20 mM

25
Probaility (%)

20 PAH 10 mM
15
10
5
0
0 10 20 30 40 50 60 70 80 90 100
Particle size (nm)

Fig. 3. FE-SEM images and particle size distributions of the silica particles synthesized under the same conditions, except for the changing PAH concentration. The pH was
adjusted to 7 with aqueous NaOH, and the concentration of TEOS in each mother liquor was equal to 200 mM. SEM images of the silica synthesized with (a) 1 mM, (b) 5 mM,
(c) 10 mM, (d) 20 mM, (e) 30 mM, and (f) 40 mM. (h) The particle size distributions of the silica synthesized using 10 mM, 20 mM, 30 mM, and 40 mM.

and redissolution and recondensation due to NaOH became The role of PAH during the synthesis of the silica nanoparticles
predominant. It is believed that the three-dimensional network was also investigated using various concentrations of PAH in the
shape of the particles was generated due to the influence of NaOH. neutralized mother liquor. Fig. 3 shows SEM images and particle
 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153 151

size distributions of the synthesized silica nanoparticles. As
expected, the formation of silica nanoparticles could be observed
at low PAH concentrations. In the case of 1 mM and 5 mM PAH,
the generated silica exhibited unique branch-like interconnected
shapes [49]. The thickness of the branches was similar to the size
of the primary particles, which was approximately 30 nm. As the
PAH concentration increased, the thickness of the branches seemed
to be slightly increased. This morphology was different from that of
the silica previously synthesized under acidic and basic conditions
in the presence of PAH and appeared to have a sponge-like form.
These results indicate that even very small amounts of PAH could
promote silica formation and influence the fabricated silica struc-
ture. These results also provide indirect evidence that PAH was
capable of creating silica nuclei capable of growing into particles.
It is well known that the growth of silica starts and occurs from
the generated nuclei. In general, when a large number of nuclei
were generated in the mother liquor, the synthesized silica was
spherical because the presence of a large number of nuclei induced
uniform silica particle growth. However, when the concentration
of PAH was low, the silica particles exhibited a branch-like struc-
ture. Hence, PAH was involved in the production of the nuclei.
According to the results from the previous control experiments, sil-
ica particle generation was not observed under neutral conditions.
However, the formation of silica particles was observed by adding
only a small amount of PAH under neutral conditions. This shows
that PAH could induce the creation of particulate silica nuclei
and that the concentration of PAH could be related to the number
of nuclei generated. Consequently, the PAH is capable of creating
silica nuclei that are capable of growing into particles, and the
number of nuclei created can be controlled by the concentration
of PAH.
The morphology and size of the silica nanoparticles synthesized
using 10 mM PAH were similar to those synthesized using 12 mM
PAH and, they were well dispersed. A remarkable change in the
morphology, however, was observed when the concentration of
PAH was increased to over 20 mM. In particular, as the concentra-
tion of PAH increased, the particles gradually became perfect
spherical shapes, and the particle size distribution became nar-
rower without changing the mean particle size, which was approx-
imately 45 nm. These results were due to the concentration of PAH
in the mother liquor. As the concentration of PAH increased, simul-
taneously, the number of amine groups also increased [58],

(a) (b)

40 40
C C
Si Si
O O
30 30
Counts

20 20

10 10

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Position (nm) Position (nm)

Fig. 4. TEM images and EDX spectra of the silica particles synthesized at pH 7 using 12 mM PAH before and after washing. (a) TEM images and EDX spectra of the silica
particles before washing and (b) TEM images and EDX spectra of the silica particles after washing.
152 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153

increasing the formation of silica nuclei in the mother liquor. This quently, PAH can be used as a substitute for the proteins of microor-
sequential phenomenon was attributed to the morphological ganisms for biosilicification, and the role of PAH is to accelerate the
change of the silica particles and change of the particle size distri- condensation of hydrolyzed TEOS without changing itself.
bution. A large number of nuclei generated promoted simultane-
ous condensation throughout the entire mother liquor, and these
4. Conclusions
particles became more homogeneous than those synthesized
under low concentrations of PAH. This homogeneity was the most
Silica particles were synthesized using a biomimetic synthesis
important factor for the formation of spherical silica nanoparticles
method. To compare to the reaction rates between hydrolysis
with uniform size, and accelerating the condensation rate of PAH
and condensation under experimental conditions, the synthetic
seemed to help the growth of these unique shapes. In addition,
process was designed as a biphasic process. TEOS was used as
the reproducibility of the synthetic method and the stability of
the silica source, and PAH was used as a substitute for amine
synthesized silica nanoparticles were examined (see Figs. S2 and
group-containing proteins of marine microorganisms with the
S3). The reproducibility of the synthetic method was verified for
capability of biomineralization. To observe the influence of PAH
three times and confirmed to have excellent reproducibility. Inter-
on the formation of the silica particles in an aqueous solution, a
estingly, the silica nanoparticles synthesized maintain pristine
typical sol-gel method was carried out under acidic, neutral and
their morphology without any additional treatment for the
basic conditions in the control experiments. According to the
increase of stabilization.
results of the control experiments, for the cases under acidic and
Fig. 4 shows TEM images and EDX (energy-dispersive X-ray)
neutral conditions, the formation of silica particles did not occur.
spectra of the synthesized silica nanoparticles. The samples were
However, when PAH was added, silica particles were formed, even
observed using TEM analysis to investigate the presence of PAH
under acidic and neutral conditions, and as the pH increased, the
on the silica particles. For a more precise analysis, two different sil-
growth of primary particles up to a size of 45 nm could be
ica particles were prepared and subjected to TEM analyses. The
observed. Particularly, under neutral and basic conditions, particles
synthesis of these two silica nanoparticles proceeded in the same
with well-developed shapes were obtained. To prove the influence
manner, except for the post treatments. The mother liquor was
of the concentration of PAH, experiments were conducted using
prepared using 12 mM PAH and TEOS under neutral conditions.
various PAH concentrations under neutral conditions. Branch-
After the same synthesis process, one sample was obtained only
like-shaped silica particles were formed, even at low concentra-
after centrifugation without washing, and the other was obtained
tions of 1 mM and 5 mM PAH. Moreover, when the concentration
after three washing steps were performed. In the case of the
of PAH was 20 mM or more, it was possible to obtain nearly perfect
unwashed silica particles, the surface of the particles was covered
spherical particles with an approximate size of 45 nm, and the
with a substance that was presumed to be a polymer. In contrast,
sizes of the particles became gradually more uniform. Observations
the washed silica particles did not have any substance on the sur-
of the presence of PAH in the silica particles from the EDX analysis
face. An EDX analysis was performed to confirm the distribution of
using TEM revealed that most of the PAH was distributed on the
PAH in the silica nanoparticles. The black lines indicate the pres-
surface of the particles. Also, the PAH and silica particles had very
ence of carbon atoms in the EDX spectrum, and the silicon and oxy-
weak interactions. Based on these results, the function and activity
gen atoms are indicated in red1 and green, respectively. The carbon
of PAH were hypothesized. It was confirmed that the role of PAH
atoms were found to be uniformly distributed throughout the parti-
during silica synthesis in an aqueous solution was to accelerate
cles, regardless of their shape. However, the spectra for the presence
the condensation rate of hydrolyzed TEOS, and its activity was also
of silicon and oxygen atoms indicated an increase and then a
very high. In addition, PAH could create nuclei for the growth of the
decrease according to the shape of the silica particles. The TEM
silica particles. While PAH exhibited both condensation rate
and EDX results provided meaningful information regarding the pos-
enhancement and nucleation capabilities, it also exhibited high
sible interactions between PAH and silica from silica nucleation to
activity without strong interactions with the silicate in the aque-
particle growth. As mentioned above, PAH has the ability to acceler-
ous solution during the synthesis process.
ate the condensation of hydrolyzed TEOS. Hence, several interactions
The silica generation principle for proteins of marine microor-
between PAH and the hydrolyzed TEOS must have existed. Addition-
ganisms can be indirectly explained using these results. Most of
ally, it was considered that these interactions would occur through-
the marine organisms capable of synthesizing silica have unique
out the entire process. Furthermore, if these interactions were
proteins that include amine groups. These marine microorganisms
strong, the resulting silica particles would necessarily contain PAH.
can utilize their proteins. Furthermore, it can be supposed that
According to these predictions, the resulting silica nanoparticles
these proteins can accelerate the condensation rate of a trace
should have a certain level of PAH both inside and outside. However,
amount of aqueous silicate contained in sea water. It is also consid-
the results from the TEM and EDX analyses indicated completely dif-
ered that these abilities can generate uniquely structured silica.
ferent results. The TEM analysis revealed that the PAH, which was
However, these proteins are not incorporated into the generated
distributed on the surface, weakly interacted with the silica particles
silica. They are likely to remain in the microorganisms, and it is
and was easily removed by simple washing. As mentioned above, the
expected that the microorganisms can continuously utilize such
EDX results did not indicate the presence of PAH. From these results,
proteins for silica generation. In conclusion, these biomimetic
the role of PAH during the synthesis of silica in aqueous solutions is
experiments enhance not only the understanding of the basic func-
to accelerate the condensation rate of hydrolyzed TEOS, and its activ-
tions of biomaterials but also the field of biomineralization, includ-
ity is very high. However, the manifestation of these properties does
ing research for the substitution of bioactive materials and
not include any strongly physiochemical interactions. Furthermore,
synthesis of new materials for applications.
the PAH could remain in the aqueous solution continuously and par-
ticipate in the growth of silica particles again. Based on these results,
it is assumed that proteins contained in microorganisms during Acknowledgement
biomineralization will show similar activity and behavior. Conse-
This work was supported by the Basic Core Technology Devel-
opment Program for the Oceans and the Polar Regions (NRF-
1
For interpretation of color in Fig. 4, the reader is referred to the web version of 2015M1A5A1037054) through the National Research Foundation
this article. of Korea (NRF), funded by the Ministry of Science, ICT & Future
 K.-K. Kang et al. / Journal of Colloid and Interface Science 507 (2017) 145–153
Planning, and from the Global Research Laboratory (NRF-
2015K1A1A2033054) through the National Research Foundation
of Korea (NRF), funded by the Ministry of Science, ICT (Information
and Communication Technologies) & Future Planning.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jcis.2017.07.115.
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