Journal of Asian Ceramic Societies 2 (2014) 275–280

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Journal of Asian Ceramic Societies

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

Immunoprecipitation of bisphenol A by antibody–mesoporous silica

composites
Toru Orita a,b , Masahiro Tomita b , Kazuma Nakanishi b,c , Katsuya Kato c,∗
a
Taiyo Kagaku Co. Ltd., 800 Yamada-cho, Yokkaichi, Mie 512-1111, Japan
b
Division of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8570, Japan
c
National Institute of Advanced Industrial Science and Technology, 2266-78, Anagahora, Moriyamaku, Nagoya, Aichi 463-8560, Japan

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

Article history: Bisphenol A (BPA) is of global concern because of its disruption of endocrine systems and ubiquity in
Received 25 March 2014 aquatic environment. In this study, BPA antibody was successfully immobilised on novel mesoporous
Received in revised form 19 May 2014 silica (MPS) carriers that display unique properties such as high surface area, highly uniform pore distribu-
Accepted 22 May 2014
tion and high adsorption capacity. Mobil Crystalline Material MCM-41 (2.7 nm), Santa Barbara Amorphous
Available online 21 June 2014
SBA-15-1 (12.3 nm) and SBA-15-2 (24.0 nm) materials were used as supports for these antibodies. On
these carriers, the BPA antibody immobilisation reached 40 ␮g mg−1 . For each MPS, 15 ng of BPA antigen
Keywords:
was adsorbed on 1 mg of MPS–antibody composite, which resulted in an antibody activity of 30%. The
Antibody immobilisation
Bisphenol A
highest recovery rate of BPA antigen was observed for 80% acetonitrile in 10 mM phosphate buffer (pH
Mesoporous silica 7). After six repeated runs, BPA antibodies immobilised on SBA-15-1 and SBA-15-2 retained about 30%
Pore size of their initial activity. In contrast, these antibodies showed 13% lower residual activity on MCM-41 than
on SBA-15-1 and SBA-15-2. This result indicated that entire antibody molecules were adsorbed inside
SBA-15-1 and SBA-15-2 pores, stabilising their structural conformation.
© 2014 The Ceramic Society of Japan and the Korean Ceramic Society. Production and hosting by
Elsevier B.V. All rights reserved.

1. Introduction
Mesoporous silica (MPS) has attracted considerable research
interest since Mobil first proposed its use in 1992 [1]. In addition
to being easily functionalisable, this material has exhibited stability
for synthesis, high and specific surface areas and pore volumes [2,3],
well-defined channels, hydrothermal stability [4], adjustable pore
sizes [5] and narrow size distribution [6]. Therefore, MPS can find
applications in adsorption materials [7–9], catalyst carriers [10–12]
and separation sensors [13,14]. In addition, its implementation in
biological applications, such as drug delivery [15], transfection and
regenerative medicine [16], is expected.
The immobilisation of enzymes, antibodies and other proteins
on solid supports that can improve the biological stability and
∗ Corresponding author at: 2266-98, Anagahora, Shimoshidami, Moriyama,
Nagoya 463-8560, Japan. Tel.: +86 52 736 7551; fax: +86 52 736 7405.
E-mail address: katsuya-kato@aist.go.jp (K. Kato).
Peer review under responsibility of The Ceramic Society of Japan and the Korean
Ceramic Society.
2187-0764 © 2014 The Ceramic Society of Japan and the Korean Ceramic Society.
Production and hosting by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jascer.2014.05.010
activity under extreme conditions has proven quite useful for catal-
ysis, sensing and separation applications [17–19]. The creation
of MPS-based high-performance biosensors and protein separa-
tion systems hinges on a better understanding of the factors that
influence the pore size and morphology in these materials. Nakan-
ishi et al. [20] have previously described the relationship between
protein A molecules and MPS pore sizes, as well as the reusabil-
ity of antibodies immobilised on MPS. They observed that the
MPS-immobilised protein A showed higher binding activity to the
antigen (IgG) on MPSs with a smaller pore size (2.7 nm). Inter-
estingly, 8-anilino-1-naphthalenesulphonic acid (ANS) analysis
indicated that conformational changes during the immobilisation
process did not impact IgG binding activity. These findings sug-
gested that the optimisation of MPS pore size was extremely
important for protein A immobilisation [21–23].
Often present in environmental water, bisphenol A [2,2-bis(4-
hydroxyphenyl)propane, BPA] has now drawn attention because
of its harmful effects on the endocrine systems of humans and
wild animals [24–27]. Widely used for the production of epoxy
resins and polycarbonate plastics [28], this endocrine disrupter
is currently found in baby bottles, plastic drinking bottles, dental
seals, injection syringes, canned beverage drinks and metal food
containers. Several methods based on high-performance liquid
chromatography (HPLC) and gas chromatography/mass spectrom-
etry (GC/MS) [29–31] have been developed for the characterisation
of BPA. However, these methods are required to elaborate pretreat-
ments [32,33]. Trace amounts of BPA have also shown estrogenic
276 T. Orita et al. / Journal of Asian Ceramic Societies 2 (2014) 275–280
activity even at concentrations as low as 0.001 ng mL−1 [34–36]. In
this ultra-low concentration range, many factors, such as contami-
nation from glasswares, containers and injection syringes, interfere
with BPA characterisation. Furthermore, purified water contains
certain BPA levels as a result of leaching from resin parts in the
water purification system. Therefore, effective methods to concen-
trate and to detect low BPA concentrations need to be developed.
Because of their highly uniform channels, large surface area and
narrow pore-size distribution, MPS supports are expected to be
ideal sensing materials for enhancing the stability and sensitivity
of BPA detection.
To the best of our knowledge, few comparative studies have
addressed the adsorption of BPA antibody onto MPS, and its applica-
tion to microanalysis. In this study, various MPSs were used as BPA
antibody carriers and their activity was evaluated. Organic solvent-
tolerance and recyclability of the MPS immobilised antibody were
investigated because pore size plays a pivotal role in determin-
ing the immobilisation state of antibodies which is related to the
stability of antibodies.
The enhanced antibody stability and activity upon immobilisa-
tion on optimal MPS carriers will be advantageous in a wide range
of industrial scale applications such as microanalysis systems and
biosensors.
2. Experimental
2.1. Materials
All the materials were of analytical grade and were used
as received from the manufacturer. The triblock poly(ethylene
oxide)–poly(propylene oxide)–poly(ethylene oxide) copolymer
(Pluronic P123, Mw = 5800, EO20 PO70 EO20 ) was purchased from
Sigma–Aldrich (St. Louis, MO). Cetyltrimethylammonium bromide
(CTAB, [CH3 (CH2 )15 N(CH3 )3 ]Br) and 1,3,5-trimethylbenzene (TMB)
were procured from Wako Pure Chemical Industries (Tokyo, Japan).
Tetraethoxysilane (TEOS) was obtained from Shin-Etsu Chemical
Co. (Tokyo, Japan). The protein-free blocking buffer (No. 37570)
was acquired from Thermo Fisher Scientific Inc. (Waltham, MA).
Bisphenol A was obtained from Kanto Chemical Co. (Tokyo, Japan).
Bisphenol A polyclonal antibody was gifted by Japan EnviroChem-
icals (Osaka, Japan). The Bio-Rad Protein Assay Kit (500-0006) was
purchased from Bio-Rad Laboratories (Hercules, CA).
2.2. Synthesis of mesoporous silica materials
2.2.1. Synthesis of Mobil Crystalline Material MCM-41
MCM-41 was synthesised using CTAC as an organic template.
CTAB (2.6 g) was added to an aqueous HCl solution (pH 0.5, 30 mL)
and stirred for 2 h at room temperature. TEOS (3.5 g) was added to
the solution and the mixture was stirred for 5 h. Ammonia (3.0 g,
28 wt%, 14.7 M) was added at one time. After standing for 24 h, the
white precipitate was filtered, successively washed with deionised
water (50 mL) and ethanol (50 mL) and dried overnight at room
temperature. Finally, the sample was calcined at 500 ◦ C in air for
6 h (heating rate: 1 ◦ C min−1 ) to remove the organic template.
2.2.2. Synthesis of Santa Barbara Amorphous SBA-15-1
SBA-15-1 was synthesised using P123 as an organic template
[37,38]. P123 (1 g) was dissolved in 1.07 M hydrogen chloride (HCl,
35 mL) and the solution was stirred for 2 h at room temperature to
give SBA-15-1. n-Decane (7.6 g) was added to the solution and the
mixture was stirred for 2 h at room temperature. After addition of
NH4 F (11.5 mg) and TEOS (2.13 g), the solution was stirred for 1 h
at room temperature and kept at 40 ◦ C for 20 h using a tempera-
ture controlled bath. The solid product was collected by filtration,
washed and dried overnight at room temperature. Organic tem-
plate removals by calcination were performed through the same
procedure as for MCM-41.
2.2.3. Synthesis of SBA-15-2
P123 (1 g) was dissolved in 1.07 M HCl (35 mL) and the solution
was stirred for 2 h at room temperature. The solution was rapidly
mixed with TMB (5 g) to expand pore sizes and the mixture was
stirred for 3 d at 25 ◦ C [39]. NH4 F (11.5 mg) and TEOS (2.1 g) were
successively added to the mixture which was stirred for 1 h at room
temperature. The resulting solution was further stirred at 40 ◦ C in
a temperature controlled bath for 24 h before being transferred to
an autoclave and heated at 100 ◦ C for 48 h. The solid product was
collected by filtration, washed and dried at 70 ◦ C for 5 h. P123 and
TMB were removed by calcination using a procedure similar to that
used for MCM-41.
2.3. Material characterisation
The specific surface area and pore volume of materials were
measured by gas adsorption following the Brunauer–Emmett–
Teller (BET) method using a Tristar3000 micrometrics analyser
(Shimazu Co., Kyoto, Japan). Small-angle X-ray diffraction (XRD)
spectra were recorded using a Rigaku RINT-TTR diffractometer
(CuK␣, 50 kV, 300 mA) and wide-angle XRD spectra were measured
on a Rigaku RINT2000/PC (FeK␣, 40 kV, 30 mA). Sample morpholo-
gies were examined by field emission scanning electron microscopy
(FE-SEM; S-4300, Hitachi Co., Tokyo, Japan) with an accelerating
voltage of 10.0 kV. Before the FE-SEM observation, the samples
were coated with platinum using a Hitachi E-1020 ion sputtering
system (Hitachi High-Technologies Co., Tokyo, Japan). Transmis-
sion electron microscopy (TEM) images were acquired using JEOL
JEM 2010 electron microscope (JEOL Co., Tokyo, Japan) operating at
200 kV.
2.4. BPA antibody adsorption on MPS
MPS material (10 mg) was added to a solution of BPA antibody
(500 ␮g) in 10 mM phosphate buffer (1 mL, pH 7). This mixture was
kept at 4 ◦ C for 12 h to allow the BPA antibody to adsorb on the MPS
surface and centrifuged at 3000 rpm for 5 min. A Bio-Rad Protein
Assay reagent (200 ␮L) was mixed with the supernatant (10 ␮L)
for 20 min at room temperature to determine the protein concen-
tration, as recommended by the supplier. The amount of adsorbed
antibody was measured by a spectrophotometric method using
bovine serum albumin as the standard.
2.5. BPA antibody–antigen reaction on MPS
MPS (10 mg) was added to a solution of BPA antibody (500 ␮g)
in 10 mM phosphate buffer (1 mL, pH 7). The mixture was kept at
4 ◦ C for 12 h for BPA antibody adsorption. After solvent removal,
BPA (1 mL, 500 ng mL−1 ), used as an antigen, was added to the solid
and kept at 37 ◦ C for 3 h (Fig. 1). The reaction mixture was cen-
trifuged and the unbound antigen present in the supernatant was
isolated and analysed by high-performance liquid chromatography
(HPLC). HPLC was performed using a Shimadzu LC 10-AD instru-
ment equipped with an ultraviolet detector set at 210 nm and a
Wakosil-II 5C18HG column (Wako Pure Chemicals, Osaka, Japan)
eluted with an acetonitrile–water mobile phase (8.0/2.0; v/v) at a
flow rate of 0.15 mL min−1 . The retention time of BPA was 16.7 min.
Each reported value was the mean of at least three experiments.
 T. Orita et al. / Journal of Asian Ceramic Societies 2 (2014) 275–280
Fig. 1. Interactions between MPS-immobilised BPA antibody and BPA.
2.6. BPA recovery
To evaluate the solvent effects on BPA recovery rates, the
MPS–BPA antibody–BPA composite was kept in 70%, 80%, 90% or
100% acetonitrile solution (1 mL) in 10 mM phosphate buffer (pH
7) at room temperature for 3 h to cleave the antigen–antibody
Fig. 2. SEM images of the mesoporous silica materials: (a) MCM-41, (c) SBA-15-1, and (e) SBA-15-2. Scale bar: 500 nm. TEM micrographs of the mesoporous silica materials:
(b) MCM-41, (d) SBA-15-1, and (f) SBA-15-2. Scale bar: 20 nm.
277
interactions. The recovered BPA was measured through the pro-
cedure described in Section 2.5.
2.7. Recyclability of MPS immobilised BPA antibody
The reusability of immobilised antibodies is an important con-
sideration for industrial applications. To examine the residual
activity of BPA antibody, the procedures outlined in Section 2.5
were repeated up to three times using recovered MPS–BPA anti-
body composites.
2.8. BPA concentration
To enrich the BPA sample, a 50 ng mL−1 BPA solution was first
prepared. This BPA solution (1 mL) was mixed with the MPS–BPA
antibody composite and maintained at 37 ◦ C for 3 h. This process
was repeated four times to increase the BPA concentration. Next
an 80% acetonitrile solution (1 mL) in 10 mM phosphate buffer (pH
7) was added to the MPS–BPA antibody–BPA composites to release
BPA. The recovered BPA was measured through the same procedure
as mentioned in Section 2.5.
278 T. Orita et al. / Journal of Asian Ceramic Societies 2 (2014) 275–280

(a) at a relative pressure of ca. 0.3, and the volume adsorbed was
0.014 1.5 cm3 (STP) g−1 . Meanwhile, the isotherms of SBA-15-1 and SBA-
MCM-41
nm)

0.012 15-2 exhibited the same step at relative pressures of ca. 0.75
SBA-15-1 and 0.84, with adsorbed volumes of 2.0 and 2.7 cm3 (STP) g−1 ,
Pore volume (cm3/g

0.01 SBA-15-2 respectively. Fig. 3b shows the pore size distribution curves of
0.008 synthesised MCM-41, SBA-15-1, and SBA-15-2. The curves of MCM-
41, SBA-15-1, and SBA-15-2 displayed sharp peaks at 2.7, 12.3,
0.006 and 24.0 nm, respectively. Surface areas and pore volumes were
0.004 deduced using the BET model (Table 1). All the samples exhib-
ited relatively high surface areas (502–962 m2 g−1 ) and large pore
0.002 volumes (1.5–2.3 cm3 g−1 ). Moreover, pore sizes ranged from 2.7
to 24.0 nm, indicating that the materials consisted of mesopores.
0
0 5 10 15 20 25 30 All the samples were synthesised according to the methods in our
previous reports [41] and XRD patterns of the products showed
Pore diameter (nm)
similar profiles. The wide-angle XRD patterns of MCM-41 displayed
(b) 1800
three reflection peaks corresponding to (1 0 0), (1 1 0) and (2 0 0)
planes, confirming the presence of ordered hexagonal mesoporous
STP)

1600 MCM-41
structures [41,42]. Small-angle XRD patterns showed three peaks
SBA-15-1
1400 corresponding to the (1 0 0), (1 1 0) and (2 0 0) reflections of two-
Quantity adsorbed (cm3/g

SBA-15-2
1200
1000
800
600
400
200
0 of the MPS-immobilised BPA antibody
0 0.2 0.4 0.6
Relative Pressure (P/P0)
Fig. 3. (a) Nitrogen adsorption–desorption isotherms and (b) pore size distributions
of MCM-41, SBA-15-1, and SBA-15-2.
3. Results and discussion
3.1. Characterisation of MPS
MPS morphologies and structures were characterised through
SEM, TEM and nitrogen (N2 ) adsorption/desorption analyses. Shape
is known to play an important role in the behaviour of MPS beads as
protein carriers [40]. SEM images showed that MCM and SBA mate-
rials formed aggregated polyhedral particles with average particle
sizes of 50 × 200 nm (MCM-41, Fig. 2a), 400 × 200 nm (SBA-15-
1, Fig. 2c) and 1000 × 800 nm (SBA-15-2, Fig. 2e). TEM analysis
showed that the MPS material displayed a high degree of structural
order (Fig. 2). Surface area and average pore diameter are known
to impact the immobilisation and activity of supported antibod-
ies. Therefore, N2 adsorption/desorption measurements (Fig. 3a)
were performed for each MPS and the Barrett–Joyner–Hallenda
model (Fig. 3b) was applied to the adsorption isotherm for MCM-41,
SBA-15-1, and SBA-15-2. This approach typically underestimates
the surface area, pore volume and diameter of mesopores but is
widely used. All of the isotherms were type IV curves. The isotherm
of MCM-41-1 indicated a reversible adsorption–desorption pro-
cess with a characteristic nitrogen condensation evaporation step
dimensional hexagonal mesostructures for SBA-15-1 [43–45] but a
broad peak for SBA-15-2. This suggests that the addition of TMB to
P123 formed a mesocellular silica foam [46].
3.2. Immobilisation study
3.2.1. BPA antibody adsorption on MPS and BPA binding activity
0.8 1 Antibodies molecules are Y-shaped and consist of four polypep-
tide chains: two identical heavy chains and two identical light
chains connected by disulphide bonds. The under part of the Y is
called fragment crystallisable (Fc fragment), which has a molecular
size of 3–5 nm. The upper part of the Y is called fragment antigen
binding (Fab fragment), which has a molecular size of 9–11 nm and
recognises specific foreign objects [41,47]. The relative adsorbed
rate of BPA antibody was determined for MCM-41 (2.7 nm), SBA-
15-1 (12.3 nm) and SBA-15-2 (24.0 nm) using the Bradford assay
(Fig. 4, solid bar). In this assay, MPS (10 mg) was added to a solution
of BPA antibody (500 ␮g mL−1 , 1 mL). The absorbance of BPA anti-
body (50 ␮g) was assumed to be the adsorption rate of the antibody
100%. We analysed the amount of BPA antibody in the supernatant
and decided the relative adsorption rate. All MPSs adsorbed over
80% (40 ␮g mg−1 ) of the BPA antibody. In the case of the MCM-41
(2.7 nm), the Fc fragment which has a molecular size of 3–5 nm, fit
into the pore. On the contrary, the Fab fragment which has a molec-
ular size of 9–11 nm absorbed inside the SBA-15-1 (12.3 nm) and
SBA-15-2 (24.0 nm); therefore, the adsorption amount of antibod-
ies was a similar to three MPS.
Subsequently, the relative binding ratio between the MPS-
immobilised antibody and BPA was analysed (Fig. 4, open bar).
In this assay, MPS–BPA antibody composite (10 mg) was added to
a solution of BPA (500 ng mL−1 , 1 mL). The HPLC peak height of
BPA (50 ng mL−1 ) was set as 100% binding activity. We analysed
the amount of BPA in the supernatant by HPLC and decided the
relative binding rate. BPA amounts of 14, 17 and 16 ng adsorbed
on 1 mg of MCM-41, SBA-15-1 and SBA-15-2-based MPS–antibody

Table 1
Structural properties of mesoporous silicates prepared in this manuscript.

Mesoporous silica Pore diametera (nm) Surface areab (m2 g−1 ) Pore volumea (cm3 g−1 ) XRD patternsc Average particle sized (nm × nm)

MCM-41 2.7 962 1.5 2D-hexagonal 50 × 200

SBA-15-1 12.3 757 2.0 2D-hexagonal 400 × 200
SBA-15-2 24.0 502 2.3 Disordered 1000 × 800
a
Calculated according to BJH analysis (adsorption branch between 1.7 and 300 nm diameters).
b
Specific surface area according to BET.
c
Determined by small-angle XRD [43–47].
d
Average particle size is determined by SEM measurements.
 T. Orita et al. / Journal of Asian Ceramic Societies 2 (2014) 275–280
Fig. 4. Relative BPA antibody adsorption on MPS and BPA binding activity of MPS-
immobilised BPA antibody.
Relative adsorption rate (%) = 100 × (absorbance of BPA antibody − absorbance of
unbound antibody in the supernatant)/absorbance of BPA antibody.
Relative binding rate (%) = 100 × (absorbance of unbound antigen in the supernatant
on the MCM-41-BPA antigen system − absorbance of unbound antigen in the super-
natant on the MPS–BPA antibody–BPA antigen system)/absorbance of unbound
antigen in the supernatant on the MCM-41–BPA antigen system.
composites, respectively. Therefore, the immobilised antibody
exhibited BPA binding activities of 28%, 33% and 32% on MCM-
41, SBA-15-1 and SBA-15-2, respectively. These results indicate
that MPS pore size and morphology did not significantly affect
the BPA antibody adsorption and binding activity of the resulting
MPS–antibody composites.
3.2.2. Effects of acetonitrile concentration on BPA recovery
Organic solvents strongly affect the BPA recovery rates of the
BPA–MPS system. Therefore, the MPS–BPA antibody–BPA com-
posites were maintained in 70%, 80%, 90% and 100% acetonitrile
solutions in 10 mM phosphate buffer (pH 7). As shown in Fig. 5,
the 80% acetonitrile solution provided relative BPA recovery rates
of almost 100% for all the MPSs. Relative recovery rates were high
only for MCM-41 (90%) and SBA-15-2 (100%) with 70% acetonitrile.
In contrast, for 90% and 100% acetonitrile solutions, these relative
recovery rates decreased to 70–90%. Relative recovery rates were
found to strongly depend on the acetonitrile concentration. The Fab
fragment in the BPA antibody displayed a greatly reduced activity
in 90% and 100% acetonitrile solutions compared to 70% and 80%
Fig. 5. Relative BPA recovery rate at various acetonitrile concentrations.
Relative recovery rate (%) = 100 × recovery amount of BPA antigen at various ace-
tonitrile concentrations/binding amount of BPA antigen.
279
Fig. 6. Residual BPA antibody activity on MPS taken after several reuse cycles.
Relative activity (%) = 100 × recovery amount of BPA antigen after recycles/recovery
amount of BPA antigen before reuse.
solutions because its structure changed at high acetonitrile con-
centrations. Reactions were therefore performed under optimum
conditions (80% acetonitrile) in the following recyclability and BPA
enrichment experiments.
3.2.3. Effects of reuse on BPA antibody activity
The residual activity of the MPS-immobilised BPA antibody was
examined after multiple antigen binding–recovery cycles. Fig. 6
shows the residual activities of MPS–BPA antibody composites for
BPA after three recycles relative to the original activity before reuse
(taken as 100%). The residual activities after three recycles for SBA-
15-1 and SBA-15-2 amounted to 32% and 30%, respectively. BPA
antibody immobilised on MCM-41 displayed a lower residual activ-
ity (13%) after three recycles. It is understood that the immobilised
antibodies in the extremely large pores did not get easily deac-
tivated by the acetonitrile treatment because entire molecules of
antibodies absorbed inside the pores; therefore, the structural con-
formation of the antibody was greatly stabilised.
3.2.4. Effect of MPS on the enrichment of BPA traces
To concentrate BPA, the antigen–antibody reaction was
repeated four times using 50 ng mL−1 BPA for a total adsorption
of 200 ng on the MPS–BPA antibody composites. Relative recov-
ery rates for SBA-15-1 and SBA-15-2 were equal to 82.4% (165 ng)
and 79.3% (159 ng), respectively (Fig. 7). On the other hand, the
rate was markedly reduced for MCM-41 (42.3%). These results
suggest that multiple soakings, separations and washing steps
involved in recycling easily strip the BPA antibody from MCM-41
pores, because only the Fc fragment of the antibody adsorbed on
Fig. 7. Relative recovery rate of concentrated BPA.
Relative recovery rate (%) = 100 × recovery amount of BPA antigen/total adsorption
amount of BPA antigen (200 ng).
280 T. Orita et al. / Journal of Asian Ceramic Societies 2 (2014) 275–280
the support. BPA antibody presented relatively weaker interaction
forces with MCM-41 compared with SBA-15-1 and SBA-15-2 con-
sisting of larger pores, which resulted in a reduced relative recovery
rate for MCM-41.
4. Conclusions
Three types of MPS – MCM-41 (2.7 nm), SBA-15-1 (12.3 nm)
and SBA-15-2 (24.0 nm) – were used for BPA binding exper-
iments. Investigations of BPA antibody immobilisation and
antibody–antigen reaction showed that all MPSs adsorbed almost
the same amount of BPA antibody (approximately 80%) and exhib-
ited similar binding rates (approximately 30%). BPA recovery rates
from 70%, 90% and 100% acetonitrile solutions were significantly
reduced, but were the highest when 80% acetonitrile was used.
After three binding–recovery cycles, the immobilised BPA antibody
retained over 30% of its initial activity on SBA-15-1 and SBA-15-2.
This result indicated that entire antibody molecules were adsorbed
inside the pores, greatly stabilising their conformational orienta-
tion.
These findings open new and interesting prospects for ordered
mesoporous materials, and provide important information on their
ability to detect and recover ultra-low concentration compounds in
microanalysis systems.
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