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Biosensors and Bioelectronics 54 (2014) 8590

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Biosensors and Bioelectronics


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

A novel pH-controlled immunosensor using hollow mesoporous silica


and apoferritin combined system for target virus assay
Xindong Wang, Jing Dong, Xinyu Liu, Yanzhen Liu, Shiyun Ai n
College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China

art ic l e i nf o

a b s t r a c t

Article history:
Received 21 August 2013
Received in revised form
9 October 2013
Accepted 22 October 2013
Available online 6 November 2013

A novel pH-controlled immunosensor using hollow mesoporous silica and apoferritin combined system
has been reported for the rst time. The goal of this study was to introduce more electroactive probes
into the electrochemical immunosensor. Under such background, we focused our attention on hollow
mesoporous silica and apoferritin, both of which have admirable accommodating performances and can
be applied for encapsulating electroactive probes. Based on the pH-controlled disassociationreconstitution process of apoferritin and the mesoporous channels of silica, after the appropriate chemical
modication, apoferritin could be fabricated with silica. The results showed that the hollow mesoporous
silica and apoferritin combined system was successfully assembled. The excellent performance of the
combined system can be applied for ultrasensitive detection of a target virus.
Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.

Keywords:
Hollow mesoporous silica
Apoferritin
Electrochemical immunosensor
Ultrasensitive detection

1. Introduction
Hollow structures have attracted considerable interest in both
fundamental research and practical applications (An and Hyeon,
2009; Hu et al., 2011; Lou et al., 2008; Zhu et al., 2005b) due to
their well-dened morphology, low density, and large surface
area. In past few decades, hollow structured nanomaterials with
tailored physical and chemical properties have been widely
applied in catalysis (Huang et al., 2009), controlled release (Zhu
et al., 2005b), and energy storage (Jayaprakash et al., 2011). Among
them, hollow mesoporous silica (HMS) is highly attractive owing
to its outstanding properties, such as the non-toxic nature,
excellent mechanical and thermal stability, easy modication
and very high specic surface area (Fang et al., 2013). Some
research groups have explored the storage and delivery properties
of HMS (He et al., 2011; Zhu et al., 2005a, 2005b), the results
indicated that the interior cavity of HMS could encapsulate,
transport, and then release various guest species, and the highly
permeable mesoporous shell could be easily functionalized for
bio-labeling. These properties make HMS a promising candidate as
a multifunctional delivery vehicle. However, most of recent
researches are focused on the application in drug release, and
little research in biosensor has been reported. In this regard, we
expect HMS can play a fascinating role in encapsulating electroactive probes for electrochemical immunoassay of a target virus.

Corresponding author. Tel.: 86 538 8249248; fax: 86 538 8242251.


E-mail address: ashy@sdau.edu.cn (S. Ai).

Nevertheless, numerous difculties hindered the development of


HMS in electrochemical immunosensors.
It is well recognized that an efcient delivery system should
have the capability to transport the desired guest molecules
without any loss before reaching the targeted location. Upon
reaching the destination, the system needs to release the cargo
in a controlled manner. Any premature release of guest molecules
poses a challenging problem (Slowing et al., 2007). Apparently,
individual HMS cannot meet the standard of an efcient delivery
system. Thus, it is crucial for HMS to have the capability of
controlled release of the loaded guest. In some related work, e.g.,
Zhu et al. introduced a polyelectrolyte pair, which comprised
sodium polystyrene sulfonate and the polycation poly(allylamine
hydrochloride) onto HMS spheres, and conrmed that the polyelectrolyte pair coated HMS spheres could achieve the stimuli
responsive controlled drug release. He et al. (2010) designed an
anticancer drug delivery system based on surfactant-templated
mesoporous silica nanoparticles, subsequently, his group once
again excogitated a mesoporous silica nanoparticle-based pHresponsive nanodrug delivery system by a one-pot self-assembly
strategy. All the work showed a potential encouraging result
after the unilaminar or multilayer coating, HMS could be functionalized as a stimuliresponsive controlled delivery system. Among
these researches, the aim was to prolong the release time to some
extent. But on the contrary, in the eld of electrochemical
immunosensor, a rapid release time was more appropriate.
Unlike all the past modications of HMS, we focused our
attention on the use of apoferritin. Apoferritin consists of a
spherical protein shell (around 12.5 nm in diameter) composed
of 24 subunits surrounding an aqueous cavity with a diameter of

0956-5663/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bios.2013.10.051

86

X. Wang et al. / Biosensors and Bioelectronics 54 (2014) 8590

about 8 nm that is capable of accommodating around 4500 iron


atoms. The favorable accommodating performance makes it an
ideal nanocontainer for encapsulating electroactive probes. The
other signicant feature of apoferritin is that the protein cage of it
can be disassociated into 24 subunits at low pH (2.0) and
reconstituted at a high pH (8.5) environment (Liu et al., 2007).
This unique property provides the possibility of introducing
apoferritin into the interior cavity of HMS through the mesoporous
channels. Although apoferritin can accommodate a mass of electroactive probes and realize the rapid release in electrochemical
immunoassay, it still needs a large surface area matrix for the
immobilization to increase the sensitivity in the electrochemical
immunoassay. Just recently, we attempted to solve the problem by
designing an electrochemical immunoassay of a target virus based
on graphene quantum dots and apoferritin-encapsulated Cu
nanoparticles (Wang et al., 2013). In this work, electroactive
probes-encapsulated apoferritin was coated on the graphene
quantum dots modied iron core, and the label design increased
the loading amount of electroactive probes signicantly. Accordingly, the rapid release property of apoferritin, which was also
used to encapsulate the electroactive probes, and the admirable
capacity of HMS were expected to be complementary and combined in the application in electrochemical immunoassay.
Herein, we designed a novel pH-controlled immunosensor,
combined with HMS and apoferritin, for the detection of avian
leukosis virus subgroup J (ALV-J), which has caused enormous
economic losses in chickens worldwide since 1990s (Lai et al.,
2011). The HMSapoferritin combined system was designed and
reported for the rst time, and the schematic diagram is shown in
Scheme 1. Firstly, HMS was synthesized by a template method and
functionalized to combine apoferritin. Besides the large surface
area provided by the outside surface of HMS, the available interior
cavity can also supply abundant room for apoferritin binding.
Then apoferritin was linked onto the outside and internal surfaces
of functional-group-modied HMS via a disassociation and reconstitution process based on the adjustment of pH. To be specic,
HMS was modied with amino-groups and activated by glutaraldehyde in order to combine apoferritin. And then apoferritin
was disassociated into several subunits at low pH (2.0) and
some subunits were diffused into the cavity of HMS through the
mesoporous channels. When pH of the solution was adjusted to
8, the reconstitution of these subunits was executed. Thereby,

apoferritin could be linked onto the outside and internal surfaces of


the functional-group-modied HMS. At last, a considerable number of electroactive probes (Cu) was introduced into the combined
system by an in situ reduction method (Glvez et al., 2005). By
combining the fascinating features of apoferritin and HMS, the
organicinorganic combined system can introduce a host of
electroactive probes and further increase the sensitivity of electrochemical immunosensors. And a detailed description of the
assay strategy is shown in Scheme S1. Based on the design, a Cu
labeled, secondary antibody (Ab2) functionalized, and bovine
serum albumin (BSA) blocked HMSapoferritin combined system
(HMSapoferritin-Cu/Ab2BSA) was assembled. A bare glass carbon electrode (GCE) was coated with -cyclodextrin functional
graphene sheets (CD-GS) nanocomposites for the primary antibody (Ab1) binding due to the huge surface area and superior
electrical conductivity, then after a sandwich-type assembly,
Cu was released from the combined system, and then detected
by differential pulse voltammetry (DPV), a frequently-used electrochemical detection method. Accordingly, quantication of ALV-J
was accomplished based on the condition that the concentrations
of ALV-J were proportional to the oxidation peak currents of Cu.

2. Materials and methods


2.1. Materials
Tetraethoxysilane (TEOS) and 3-ammonium propyl triethoxy
silane (APTS) were purchased from Aladdin (Shanghai, China).
Cetyl trimethyl ammonium bromide (CTAB) was from Shanghai
Bio Science & Technology Co., Ltd. Apoferritin and bovine serum
albumin (BSA) were obtained from Sigma (USA). A serum specimen with ALV-J and ALV-J antibodies (Ab) were provided and
puried from the College of Animal Science and Technology,
Shandong Agricultural University (Taian, China). A 0.1 M phosphate buffered saline (PBS) (pH 7.4) was prepared by mixing the
stock solutions of 0.1 M Na2HPO4 and 0.1 M NaH2PO4 and adjusting the pH with 0.1 M H3PO4 or 0.1 M NaOH. A 0.1 M acetate buffer
(pH 4.5) was prepared by mixing the stock solutions of 0.1 M
CH3COOH and 0.1 M CH3COONa and adjusting the pH with 0.1 M
CH3COOH or 0.1 M NaOH. Other chemicals were analytical reagent
grade and all solutions were prepared using double distilled

Scheme 1. Schematic illustration of the HMSapoferritin combined system.

X. Wang et al. / Biosensors and Bioelectronics 54 (2014) 8590

deionized water from Quartz. All the reagents were used without
further purication.
2.2. Apparatus
Electrochemical experiments were performed with a CHI660C
electrochemical workstation (Shanghai Chenhua Co., China) with a
conventional three-electrode cell. A bare or modied glassy carbon
electrode (GCE) (CHI104, d 3 mm) was used as working electrode. A saturated calomel electrode (SCE) and a platinum wire
were employed as reference electrode and auxiliary electrode,
respectively. Fourier transform infrared (FT-IR) spectra were
recorded on a Nicolet 380 FT-IR spectrometer (Thermo electron
Co., USA). The transmission electron microscope (TEM) images
were obtained at a 100 CX II transmission electron microscope
(JEM, Japan). N2 absorptiondesorption isotherm and pore size
distribution were taken with an NOVOE 4000/TriStar II 3020. All
the measurements were carried out at room temperature
(25.0 70.5 1C).
2.3. Preparation of -cyclodextrin functional graphene sheets (CD-GS)
CD-GS composites were synthesized via a simple wet-chemical
strategy (Guo et al., 2010). Briey, a 100 mL portion of the
homogeneous graphene oxide dispersion (0.5 mg/mL) was mixed
with 100 mL of 80 mg/mL -cyclodextrin (CD) aqueous solution
and 0.7 mL of ammonia solution (28 wt%), followed by the addition of 0.1 mL of hydrazine solution (35 wt%). After being vigorously stirred for a few minutes, the vial was put in an oil bath
(180 1C) for 3 h. The stable black dispersion was ltered to obtain
CD-GS composites that could be redispersed readily in water by
ultrasonication. The morphology of CD-GS was illustrated by TEM
(see Fig. S1).
2.4. Preparation of hollow mesoporous silica (HMS)
As shown in Scheme S1A, at rst, Fe3O4 nanospheres were
synthesized according to the method (Deng et al., 2005).
FeCl3  6H2O (1.35 g, 5 mmol) was dissolved in ethylene glycol
(40 mL) to form a clear solution, followed by the addition of NaAc
(3.6 g) and polyethylene glycol (1.0 g). The mixture was stirred
vigorously for 30 min and then sealed in a teonlined stainlesssteel autoclave (50 mL capacity). The autoclave was heated to and
maintained at 200 1C for 10 h, and allowed to cool to room
temperature. The black products were washed several times with
ethanol and dried at 60 1C for 6 h.
Subsequently, the coreshell Fe3O4@SiO2 microspheres were
prepared by a modied solgel method (Ji et al., 2010). Briey,
0.50 g Fe3O4 was dispersed into 50 mL 0.1 M HCl aqueous solution,
ultrasonicated for 10 min, separated with a magnet and washed
with double distilled deionized water for three times, and then
homogeneously dispersed in a mixture of 80 mL ethanol, 20 mL
double distilled deionized water and 2.0 mL 28 wt% ammonia
aqueous solution, followed by the addition of 0.20 mL TEOS.
Stirring at room temperature for 6 h, the product was separated
with a magnet and washed with double distilled deionized water
three times, dispersed in a mixed solution containing 80 mL
ethanol, 60 mL deionized water, 2.0 mL 28 wt% ammonia aqueous
solutions and 0.500 g CTAB, stirred for 1 h, and then 1.0 mL TEOS
was added. After stirring for 6 h, the product was separated with
magnet and washed with double distilled deionized water and
ethanol, and dried at 50 1C for 12 h.
Next, the synthesized Fe3O4@SiO2 microspheres were
immersed into concentrated HCl solution (36 wt%) and ultrasonicated for 30 min. Then the product was separated with centrifugation and washed with double distilled deionized water several

87

times. The obtained product was again immersed into concentrated HCl solution and ultrasonicated for 10 min. After washing
with double distilled deionized water several times, the obtained
HMS was collected by centrifugation and dried at 60 1C for 12 h.
TEM was used to study the morphology of HMS nanoparticles.
2.5. Fabrication of HMSapoferritin-Cu
Firstly, HMS was functionalized with APTS and glutaraldehyde.
20 mg HMS was dispersed in 20 mL ethanol containing 100 mL
APTS, ultrasonically for 1 h and stirred for 12 h. Then the product
was separated by centrifugation and washed with ethanol and
double distilled deionized water three times. FT-IR was used to
conrm the connection of amino-group onto HMS. As shown in
Fig. S2, the peak at 1633 cm  1 was caused by the bending
vibration of NH, and the peaks at 2926 and 2855 cm  1 were
attributed to the stretching vibration of NH, which indicated the
unity of amino-group.
Next, the product was dispersed in 20 mL 0.075 wt% glutaraldehyde solution, stirred for 4 h, and then washed with double
distilled deionized water for three times.
Then, the functional HMS was dispersed in 5 mL apoferritin
solution (pH2, 22 mg NaCl, 20 mg apoferritin), ultrasonically for
10 min. Then, pH was turned to 8 and incubated for 4 h. Aliquots of
CuSO4  5H2O (0.9 g) were added over a 2 h 30 min period with
30 min intervals between additions. The pH was continuously
adjusted to 8 by using 0.01 M NaOH. The solution was left standing
overnight, and then thoroughly dialyzed against a pH 8 buffer
solution (0.1 M tris/0.15 M NaCl) for 2 days. At last, 10 mg NaBH4
was added. The nal product was separated by centrifugation
and washed with double distilled deionized water. TEM was
used to characterize the sizes and features of HMSapoferritinCu nanoparticles.
2.6. Preparation of HMSapoferritin-Cu/Ab2BSA bioconjugates
A certain amount of Ab2 solution was added into the obtained
HMSapoferritin-Cu nanoparticles suspension for the attachment.
The mixtures were centrifuged at 9000 rpm for 15 min at 4 1C
after incubation under gently stirring for 24 h at 4 1C. Later, 0.5 mL
of BSA (2 mg/mL) was added into the residual complexes under
gently stirring for about 6 h at 4 1C to block the unspecied sites
and prevent non-specic adsorption between ALV-J and the
bioconjugates. At last, the obtained HMSapoferritin-Cu/Ab2BSA
bioconjugates were collected by centrifugation and redispersed in
1 mL PBS, and then stored at 4 1C when not used.
2.7. Fabrication of the immunosensor
The schematic graph of the fabrication process is shown in
Scheme S1B, a bare GCE was polished to a mirror-like surface with
0.3 and 0.05 mm alumina slurry on micro-cloth pads and then
thoroughly cleaned before use. Then 10 mL homogeneous suspension of CD-GS (2 mg/mL) was added onto the bare GCE surface to
load Ab1. To block the non-specic binding sites, 6 L of 1.0 wt%
BSA (PBS buffered) was placed onto the electrode for 1 h at 37 1C.
Then, various concentrations of ALV-J were incubated with the
immobilized Ab1 for 1 h at 37 1C. Thereafter, the prepared HMS
apoferritin-Cu/Ab2BSA buffer solution was dropped onto the
electrode surface and incubated for 1 h. After each step, the
modied electrode was thoroughly cleaned with PBS. Electrochemical behaviors of the step by step surface modication of the
immunosensor were investigated by cyclic voltammetry (CV)
(shown in Fig. 3). The nished immunosensor was stored at 4 1C
when not in use.

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X. Wang et al. / Biosensors and Bioelectronics 54 (2014) 8590

Before electrochemical detection, the fabricated electrode was


immersed into 5 mL HCl solution (pH 2) for 20 min to release Cu
from the cavity of apoferritin. The HCl solution treated HMS
apoferritin-Cu nanoparticles are shown in Fig. S3.
2.8. Electrochemical detection
In order to get well-dened, sharp and highly reproducible
stripping peaks for low concentrations of Cu over a low background current. A bismuth-coated carbon electrode was applied.
Briey, a polished bare GCE was immersed into a 15-mL electrochemical cell, containing 0.1 M acetate buffer (pH 4.5) and 400 g/L
bismuth. The electrodeposition of bismuth was performed with the
potential of  1.2 V, while the solution was stirred.
The measurement solution was 5 mL HCl (pH 2) solution with
released Cu nanoparticles from apoferritin. The dissolved Cu
nanoparticles were measured with differential pulse voltammetry
(DPV) using the bismuth-lm electrode. The DPV measurements
were performed by a 200 s accumulation at  1.2 V and subsequent stripping from  0.4 V to 0.2 V with a step potential of
4 mV, amplitude of 50 mV, and pulse width, pulse period and
quiet time were 0.05 s, 0.2 s and 2 s, respectively.

3. Results and discussions


3.1. TEM and N2 absorptiondesorption characteristics
HMS with a large cavity was rst synthesized by a hardtemplating method, in which Fe3O4 acted as a functionalized core.
The morphology of the synthesized HMS is shown in Fig. 1A. From
Fig. 1A, a strong contrast difference between the edges (dark) and
center (bright) implies a hollow structure. It can be observed
clearly that the TEM image demonstrates a thin wall and the

average diameter size is around 160190 nm. The N2 adsorption


desorption isotherm for HMS is shown in Fig. 2A. The type IV
isotherm curve between 0.15 and 0.3 of P/P0 indicates the presence
of mesoporous. Correspondingly, the pore size distribution of HMS
(shown in Fig. 2B) shows a narrow pore distribution with a mean
value of 2.42 nm. In addition, the sample has a high specic surface
area of 651.79 m2/g and a large pore volume of 0.39 cm3/g using the
BrunauerEmmettTeller (BET) and BarrettJoynerHalenda (BJH)
methods, respectively. At this point, the hollow and mesoporous
structures are successfully characterized.
Fig. 1B and C displays TEM micrographs of the synthesized
HMSapoferritin-Cu. As evidenced in Fig. 1B, the dense and brunet
apoferritin-encapsulated Cu (apoferritin-Cu) nanoparticles are
visible, and the interior cavities of HMS are lled with apoferritin-Cu, besides, the surfaces are also coated by apoferritin-Cu.
By referring back to Fig. 1B, the image of Fig. 1C shows some HMS
particles aggregation, and apoferritin-Cu nanoparticles are also
encapsulated by and coated on the agminated HMS. All the results
reveal the formation of HMSapoferritin-Cu. After the sandwichtype fabrication, the immunosensor was treated by HCl solution to
release the electroactive probes-Cu. TEM image of the HCl solution
treated HMSapoferritin-Cu nanoparticles is presented in Fig. S3.
It can be seen that HMS nanoparticles are unabridged, substantially all the apoferritin-Cu nanoparticles are broken and Cu
nanoparticles were released. The released Cu nanoparticles are
13 nm in diameter.

3.2. Electrochemical characterization of proposed immunosensor


As presented in Scheme S1B, fabrication of the immunosensor
was accomplished. Cyclic voltammetry (CV) was a useful technology
to probe the fabrication process of the proposed immunosensor,
which is operated in 5 mM Fe(CN)63  /4  (1:1) solution containing

Fig. 1. TEM images of HMS (A) and HMSapoferritin-Cu ((B) and (C)).

Fig. 2. (A) N2 absorptiondesorption isotherm for HMS. (B) Pore size distribution of HMS obtained by the BJH method.

X. Wang et al. / Biosensors and Bioelectronics 54 (2014) 8590

0.1 M KCl in the potential range of  0.2 to 0.7 V with the scan rate
of 100 mV/s. As shown in Fig. 3, the bare GCE (curve a) displays a
pair of well-dened voltammetric peaks. After the modication
with CD-GS (curve b), the current is much higher than that of a
bare GCE, which is ascribed to the highly effective surface area and
excellent electronic transmission ability of CD-GS. As the Ab1 is
adsorbed (curve c), Ab1/CD-GS modied GCE shows a decrease of
peak current, the result is owing to the obstruction of the electron
transfer by the covered Ab1. Then after blocked with BSA and
incubated with ALV-J, the decrease in redox peak currents (curves d
and e) suggests the presence of BSA and ALV-J, which act as an inert
layer and inhibit the electron transfer.

3.3. Optimization of detection conditions


To acquire an optimal electrochemical response, accumulation
potential is an important issue to be considered. The effect of
various accumulation potentials on the currents of the immunosensor using 104.00 TCID50/mL (TCID50: 50% tissue culture infective
dose) ALV-J as an example was investigated. As given in Fig. S4A,
the current response of the immunosensor increased with increasing the accumulation potential from  1.6 V to  1.2 V, and then
decreased with increasing the accumulation potential from  1.2 V
to  0.2 V, the optimal oxidation peak current was achieved at
1.2 V. Thus,  1.2 V was chosen for subsequent experiments. The
accumulation time is also an important parameter affecting the
analytical performance. With the accumulation potential of
1.2 V, the effect of accumulation times on the current responses

89

of the immunosensor using 104.00 TCID50/mL ALV-J as an example


was inquiried. Fig. S4B shows that the current response of the
immunosensor increased with increasing the accumulation time
and reached a plateau at 200 s. As a result, the optimum accumulation time was set at 200 s.
3.4. Analytical performance of the immunosensor
Quantitative analysis of ALV-J was accomplished based on the
concentration dependence of the oxidation peak currents of Cu.
Under optimal immunoassay conditions, the proposed immunosensor was examined with different concentrations of ALV-J.
As shown in Fig. 4A, the DPV peak currents increased with increasing
concentration of ALV-J, and the calibration plot (shown in Fig. 4B)
showed a good linear relationship between the peak currents and
the logarithm values of the ALV-J concentrations ranging from
102.02 to 104.50 TCID50/mL, with a detection limit of 102 TCID50/mL
(S/N 3). The results indicated enough sensitivity for monitoring
of ALV-J.
3.5. Selectivity, reproducibility and stability of the immunosensor
To further investigate the specicity of the proposed immunosensor for ALV-J detection (see Fig. S5A), some potential interferents, such as subgroup A of avian leukosis virus (ALV-A) and
avian reticuloendotheliosis virus (AREV) were used to evaluate
selectivity of the immunosensor. The proposed immunosensors
were separately exposed to ALV-J, ALV-A and AREV with the
concentrations of 103.35, 103.70 and 104.00 TCID50/mL, respectively.
As shown in Fig. S5A, only the immunosensor incubated in ALV-J
had obvious oxidation peak currents, which indicated the high
selectivity of the prepared immunosensor. The reproducibility of
the immunosensor was evaluated by determining 103.70 TCID50/mL
ALV-J with six immunosensors (see Fig. S5B). The responses of
these immunosensors showed the RSD of 2.84%, indicating acceptable reproducibility. Additionally, the long-term storage stability
was investigated over a period of 21 days of storage at 4 1C.
The response currents decreased by about 8.9%, which suggested
the acceptable stability of the immunosensors.

4. Conclusions

Fig. 3. CVs for bare GCE (a), CD-GS (b), Ab1/CD-GS (c), BSA/Ab1/CD-GS (d), and ALVJ/BSA/Ab1/ CD-GS (e) modied GCE in 5 mM Fe(CN)63  /4  (1:1) solution containing
0.1 M KCl in potential range of  0.2 to 0.7 V with the scan rate of 100 mV/s.

In summary, ultrasensitive electrochemical immunodetection


of ALV-J is achieved by combining HMS and apoferritin-Cu nanoparticles. The objective of this study was to explore the relevant
properties of HMS and apoferritin. The results demonstrate that

Fig. 4. (A) DPV responses of the immunosensor with increasing ALV-J concentrations under the optimal conditions (102.02, 102.25, 102.55, 102.75, 103.00, 103.35, 103.70, 104.00,
and 104.50 TCID50/mL). (B) Calibration curve.

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X. Wang et al. / Biosensors and Bioelectronics 54 (2014) 8590

the HMSapoferritin combined system is successfully assembled


and able to introduce a considerable number of electroactive
probes, which are crucial in the electrochemical detection. Moreover, the proposed immunosensor shows excellent analytical
performance for the measurement of ALV-J with low detection
limit, high sensitivity, good reproducibility and stability. We also
expect this work would supply an entirely different point of view
in the design of HMS-based controlled release materials.

Acknowledgments
This work was supported by National Natural Science Foundation of China (Nos. 21375079 and 21105056) and Natural Science
Foundation of Shandong province, China (Nos. ZR2010BM005 and
ZR2011BQ001).

Appendix. Supplementary material


Supplementary material associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.bios.2013.10.051.

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