Calcium carbonate-gold nanocluster hybrid spheres: Synthesis and versatile application in immunoassays

Article abstract of DOI:10.1002/chem.201102876

Fluorescent gold nanoclusters (AuNCs) were incorporated into porous calcium carbonate spheres through electrostatic interaction. The resulting CaCO ₃/AuNCs hybrid material exhibited interesting properties, such as porous structure, excellent bi

Full text of DOI:10.1002/chem.201102876

FULL PAPER
DOI: 10.1002/chem.201102876
Calcium Carbonate–Gold Nanocluster Hybrid Spheres: Synthesis and
Versatile Application in Immunoassays
[
a]
[a]
[b]
[a]
[a]
Juan Peng, Li-Na Feng, Kui Zhang, Xing-Hua Li, Li-Ping Jiang,* and
[
a]
Jun-Jie Zhu
Abstract: Fluorescent gold nanoclus-
ters (AuNCs) were incorporated into
porous calcium carbonate spheres
through electrostatic interaction. The
ising template to assemble horseradish
peroxidase/antibody conjugates (HRP-
Ab ). By using CaCO /AuNCs/HRP-
rescent and electrochemical detection
of the cancer biomarker neuron-specif-
ic enolase (NSE). The detection limits
2
3
À1
Ab bioconjugates as probes, a versatile
of the sensor were 2.0 and 0.1 pgmL
2
resulting CaCO /AuNCs hybrid materi-
immunosensor was developed for fluo-
for fluorescent and electrochemical de-
tection, respectively. The immunosen-
sor shows high sensitivity and offers an
alternative strategy for the detection of
other proteins and DNA.
3
al exhibited interesting properties, such
as porous structure, excellent biocom-
patibility, good water solubility, and de-
gradability. These properties make the
Keywords: calcium carbonate
electrochemistry fluorescent
probes · gold · immunoassays
·
·
CaCO /AuNCs hybrid material a prom-
3
Introduction
Calcium carbonate (CaCO ) is one of the main biomineral
3
components of seashells. Polymorph control of CaCO bio-
3
Gold nanoclusters (AuNCs) have attracted great attention
in the past decade. Research interest ranges from fundamen-
mineral in seashells is achieved naturally by complex cellu-
lar cues. CaCO has three different crystalline phases: vater-
3
[1–4]
tal properties such as photoluminescence,
optical chirali-
ite, calcite, and aragonite. Porous CaCO microspheres can
3
[
5–7]
[8]
ty,
ing behavior
ferromagnetism, and quantized double-layer charg-
be used as effective hosts for the fabrication of biocompat-
ible hybrid materials due to their occurrence in nature as
biominerals and their porous structure, which makes them
[
9,10]
to potential applications in optoelectron-
[
11]
[4,12–15]
[16–18]
ics, sensing,
and bioassays.
Compared to semi-
[
20,21]
conductor quantum dots, which have larger size (3–100 nm)
and contain toxic metal species (e.g., cadmium, lead),
AuNCs are highly attractive because of their smaller size
suitable for the loading of other materials.
CaCO microspheres have wide applications in drug deliv-
Porous
3
[22]
[21,23–25]
[20]
ery,
biosensing,
and protein encapsulation.
The
[19]
and nontoxicity. Moreover, AuNCs exhibit other fascinat-
ing features, including ease of synthesis, good water solubili-
ty, and surface functionalities, which also give them great
promise in bioassays. However, direct bioconjugation of
these AuNCs with biomolecules may induce aggregation
during the cross-linking reaction, which influences the stabil-
ity, precision, and reproducibility of the assay. Thus, a prom-
ising strategy is to use a porous template to encapsulate
AuNCs.
difficulty in using this attractive material for the design of
multifunctional composites lies in its polymorphic character.
The most attractive phase of CaCO for the fabrication of
3
multifunctional materials is the metastable vaterite form
featuring spherical morphology and porous surface. Vaterite
easily undergoes phase transition to the more thermody-
namically stable phase of CaCO , calcite, which does not
3
[20]
show the spherical, porous morphology of vaterite. This
phase transformation usually occurs overnight for vaterite
[20]
samples in water, which greatly limits their further appli-
cation. CaCO also shows good degradability, because it can
3
be easily dissolved in ethylenediaminetetraacetic acid
(
EDTA) solution under mild conditions. Much research has
[
a] J. Peng, L.-N. Feng, Dr. X.-H. Li, Dr. L.-P. Jiang, Prof. J.-J. Zhu
State Key Lab of Analytical Chemistry for Life Science
School of Chemistry and Chemical Engineering
Nanjing University, Nanjing, 210093 (P. R. China)
Fax : (+86)25-583-597-204
been carried out on the fabrication and application of
CaCO microspheres with large size (larger than 3 mm).
However, the large size limits their application in bioassay.
Thus, a facile way of preparing CaCO spheres of smaller
[20,26]
3
3
E-mail: Jianglp@nju.edu.cn
size is highly required and of great challenge.
[
b] Prof. K. Zhang
Hybrid materials with specific catalytic, magnetic, or opti-
The Affiliated Drum Tower Hospital of
Nanjing University Medical School
Nanjing, 210008 (P. R. China)
[27–30]
cal properties have been fabricated.
knowledge, no report has been made on the fabrication of
To the best of our
CaCO /AuNCs hybrid spheres. Due to the good biocompati-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102876.
3
bility of both CaCO spheres and AuNCs, excellent fluores-
3
Chem. Eur. J. 2012, 18, 5261 – 5268
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5261

cent property of AuNCs, and porous structure of CaCO3,
to cerebral injury due to physical damage or ischemia
caused by infarction or cerebral hemorrhage, coupled with
increased permeability of the blood/brain barrier. The
serum concentration of NSE has also been reported to cor-
relate with the extent of damage and neurological out-
CaCO /AuNCs hybrid material could find potential applica-
3
tions in drug delivery, bioassay, and biological systems.
Immunoassays based on specific molecular recognition of
an antigen by its antibody have been widely used for quanti-
tative analysis of protein biomarkers for clinical purposes.
Numerous immunoassays have been developed for enhance-
ment of detection sensitivity by signal amplification or em-
ployment of different detection technologies. Nanoparticle-
based labeling systems have attracted special interest in im-
munoassays due to the outstanding optical, electronic, and
[33]
come. Therefore, NSE was chosen as a protein model to
evaluate the immunoassay. The coating antibody (Ab ) was
1
immobilized on polyethylenimine (PEI)-functionalized, ni-
trogen-doped multiwalled carbon nanotubes (PNCNs).
Then, NSE was bound with Ab through immunoreaction.
1
Finally, CaCO /AuNCs/HRP-Ab bioconjugates were cap-
3
2
[31,32]
biocompatible performance of nanoparticles.
Here,
tured during the specific binding event. The sensing signals
could be detected by fluorescence and electrochemical dif-
ferential pulse voltammetry (DPV).
CaCO /AuNCs hybrid spheres were employed to construct
3
a labeling system for the fabrication of dual fluorescent and
electrochemical detection platform.
In this work, a novel CaCO /AuNCs multifunctional ma-
3
terial was synthesized and further used for the encapsulation
of horseradish peroxidase/antibody conjugates (HRP-Ab2)
Results and Discussion
to
fabricate
CaCO /AuNCs/HRP-Ab
bioconjugates
Synthesis and characterization of the porous CaCO /AuNCs
3
2
3
(
Scheme 1). Fluorescent AuNCs were incorporated into
hybrid spheres: The porous CaCO spheres were synthesized
by a one-pot approach. Figure 1A shows TEM image of
3
[34]
Figure 1. TEM images of A) CaCO
3 3
spheres. B) CaCO /AuNCs hybrid
spheres. Insets: corresponding HRTEM images.
the CaCO spheres, which have diameters in the range of
3
5
(
00–700 nm. A high-resolution TEM (HRTEM) image
inset in Figure 1A) showed that the CaCO3 sphere has
a unique structure and porous surface with pore size around
0 nm. The nanosized pores and channels in the CaCO3
Scheme 1. a) Schematic illustration of fabrication process of CaCO
AuNCs/HRP-Ab bioconjugate. A) Porous CaCO sphere. B) CaCO
AuNCs hybrid sphere. C) CaCO /AuNCs/HRP-Ab
rication process of sandwich immunosensor for fluorescent and electro-
chemical detection of NSE. A) Glassy carbon electrode (GCE).
3
3
/
/
2
3
3
3
2
bioconjugate. b) Fab-
spheres provide efficient templates for encapsulation of bio-
macromolecules such as enzymes and proteins through phys-
[35]
B) PNCNs coated on GCE (GCE/PNCNs). C) Ab
PNCNs surface (GCE/PNCNs/Ab ). D) NSE targeted by immunoreac-
tion. E) CaCO /AuNCs/HRP-Ab captured by secondary immunoreac-
1
immobilized on
ical adsorption and pore diffusion. As shown in Figure S1
Supporting Information), fluorescent AuNCs around 2 nm
1
(
3
2
in size were synthesized and were well dispersed in deion-
ized water. For the adsorption of AuNCs, poly(allylamine
hydrochloride), PAH, was used to make the surfaces of the
CaCO3 spheres positively charged. Loading with AuNCs
tion. F) NSE detection by fluorescent and electrochemical DPV analysis.
CaCO porous spheres. The resulting CaCO /AuNCs hybrid
was carried out by dispersing the CaCO spheres in an aque-
3
3
3
material retained the porous spherical structure, which pro-
ous solution of AuNCs under sonication. Since the CaCO3
spheres have positive surface charge (+16.5 mV) and
AuNCs negative surface charge (À13.5 mV), the driving
force for adsorption of AuNCs into the pores was mainly at-
tributed to electrostatic interaction. Figure 1B shows TEM
vided an efficient template for the assembly of HRP-Ab to
2
construct a labeling system. The ability of CaCO /AuNCs/
3
HRP-Ab to act as probes was further examined by per-
2
forming a sandwich bioaffinity immunoassay, as shown in
Scheme 1b. Neuron specific enolase (NSE) is highly specific
for neurons. Elevated NSE levels in serum can be attributed
and HRTEM images of the CaCO /AuNCs hybrid spheres.
3
The AuNCs incorporated into the CaCO particles can be
3
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Chem. Eur. J. 2012, 18, 5261 – 5268

Calcium Carbonate–Gold Nanoclusters Hybrid Spheres
FULL PAPER
clearly observed. The spherical and channel-like interior
structure can also be observed in the HRTEM image (inset
of Figure 1B). This result confirms that the AuNCs were
successfully loaded into the CaCO spheres.
3
The fabrication process was monitored by fluorescence
spectroscopy. Figure 2A shows fluorescence spectra of the
Figure 3. Confocal laser-scanning fluorescence image of CaCO
spheres. Inset: high-magnification confocal image of a CaCO
sphere.
3
3
/AuNCs
/AuNCs
Figure 2. A) Fluorescence spectra of a) as-prepared AuNCs and b) the su-
pernatant after AuNCs adsorption. B) Fluorescence spectra of a) CaCO
particles and b) CaCO /AuNCs suspension. Inset of B) is a photograph of
CaCO /AuNCs aqueous solution under UV light.
3
3
3
CaCO /AuNCs hybrid spheres are shown in Figure 4. As can
3
be seen in Figure 4A, the surface of the CaCO particles is
3
as-prepared AuNCs solution (curve a) and the supernatant
porous and consists of numerous carbonate nanoparticles
that form the specific morphology. This unique structure
played an important role in the assembly of AuNCs. After
incorporation of AuNCs, the surface became smooth (Fig-
ure 4B). It was found that loading with AuNCs could en-
of the AuNCs solution after adsorption onto CaCO parti-
3
cles (curve b). A great loss in fluorescence intensity was ob-
served after loading of AuNCs, which indicates incorpora-
tion of the AuNCs into the CaCO spheres. No fluorescence
3
was observed for the CaCO spheres (curve a in Figure 2B).
hance the stability of CaCO spheres consisting of the vater-
3
3
In contrast, the CaCO /AuNCs hybrid spheres showed
ite polymorph. When the CaCO particles were stored in
3
3
strong fluorescence (curve b in Figure 2B), indicating effec-
tive loading of AuNCs, which are responsible for the strong-
ly fluorescent property in the hybrid spheres. The loading
water for five days (Figure 4C), calcite began to form. In
contrast, the CaCO /AuNCs hybrid particles were stable as
3
the vaterite polymorph in water for more than two months
(Figure 4D). This result is confirmed by the XRD data in
Figure S3 (Supporting Information). It is speculated that for-
amount of AuNCs in the CaCO particles could be calculat-
3
ed by the difference in peak intensity of AuNCs solution
before and after loading (Figure 2A). According to the con-
À1
centration difference before adsorption (344 mgmL ) and
À1
after adsorption (56 mgmL ), a rough estimate indicated
that 1.0 mg of CaCO particles could accommodate 288 mg
3
[36,37]
AuNCs.
Confocal laser-scanning fluorescence microsco-
py was also employed to demonstrate the distribution of the
AuNCs in the CaCO spheres (Figure 3). This showed that
3
the AuNCs could penetrate into the inner pores of CaCO3.
The porous structure and open channels in the CaCO3
spheres offer suitable microenvironments for AuNCs ad-
sorption, and resulted in a large loading amount of AuNCs.
Because of the porous structure, the CaCO spheres have
3
2
À1
a high surface area of 27.21 m g and pore volume of
.1227 cm g (curve a in Figure S2, Supporting Informa-
3
À1
0
tion). After incorporation of AuNCs, the CaCO /AuNCs
3
2
À1
hybrid spheres had a surface area of 20.60 m g and a pore
3
À1
volume of 0.0926 cm g (curve b in Figure S2, Supporting
Information), which are smaller than those of the CaCO3
spheres. In addition, a narrower pore-size distribution (inset
of Figure S2, Supporting Information) and a sharp decrease
in the number of large pores were observed, which might be
3 3
Figure 4. SEM images of A) vaterite CaCO spheres, B) CaCO /AuNCs
attributed to infiltration of the AuNCs into the CaCO parti-
3
hybrid spheres, C) calcite CaCO acquired after five days, and D) CaCO /
3
3
cles. SEM images of both the CaCO3 spheres and the
AuNCs spheres after two months.
Chem. Eur. J. 2012, 18, 5261 – 5268
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5263

L.-P. Jiang et al.
mation of thermodynamically stable compounds between
details), followed by the functionalization with polyethyleni-
mine (PNCNs). Then, by employing glutaraldehyde as cross-
the stabilizer and CaCO is the main reason for the good
3
stability of the hybrid. It is reported that a solid solution is
formed between calcite carbonate and inorganic materials
on the vaterite surface. These solid solutions show a lower
heat of formation than calcite, and thus prevent dissolution
of the vaterite polymorph. For comparison, gold nanoparti-
cles (AuNPs) with a size of 20 nm were used to fabricate
linking agent, Ab molecules could be firmly absorbed onto
1
the PNCN surface through the interaction between amino-
functionalized PNCNs and primary amino groups of the pro-
tein. As shown in Figure S5 (Supporting Information), SEM
was employed to characterize the NCNs, PNCNs, and the
[38]
Ab1 immobilized on PNCNs (PNCNs/Ab ). The NCNs
1
CaCO /AuNPs hybrid spheres. The SEM image of the
showed a well-dispersed one-dimensional structure with di-
ameter in the range of 20–40 nm. For PNCNs, no obvious
change was observed in the morphology after PEI function-
3
CaCO /AuNPs hybrid spheres (Figure S4, Supporting Infor-
3
mation) indicated that the calcite polymorph of CaCO3
could easily form after one week. AuNPs of larger size less
readily penetrate into the inner pores of CaCO3 than
AuNCs of smaller size, and hence the loading of AuNPs in
CaCO3 is greatly reduced. Thus, incorporation of AuNCs
can efficiently stabilize the vaterite polymorph of CaCO3.
alization. When Ab was immobilized on the surface, the
1
thickness of PNCNs/Ab clearly increased, which indicated
1
that Ab was effectively bound to the PNCN surface. The
1
immunoassay process is outlined in Scheme 1b. First, Ab1
was immobilized on the PNCN surface, and then NSE was
bound with Ab through the first immunoreaction. Subse-
1
Assembly of protein on CaCO /AuNCs hybrid spheres for
quently, CaCO /AuNCs/HRP-Ab bioconjugates were cap-
3
3
2
biolabeling system: The CaCO /AuNCs hybrid spheres in-
herited advantages from their parent materials, such as satis-
factory biocompatibility, good solubility in water, and
tured on the surface by the second immunoreaction. Finally,
two applications were demonstrated to detect the target
NSE concentration by using fluorescent and electrochemical
DPV analysis. The details of the procedure are described in
the Experimental section.
3
porous structure. Thus, the CaCO /AuNCs hybrid spheres
3
could be expected to be promising templates for protein
loading. Then HRP-Ab were encapsulated into the hybrid
2
spheres and the obtained bioconjugates were used as versa-
tile probes for immunoassay. Since the pI of HRP is 8.8,
Fluorescence immunoassay: To be useful as a fluorescence
biolabel, it is important that the loaded AuNCs can be re-
leased from the particles for fluorescence detection. In our
work, AuNCs can be released from the captured CaCO3/
AuNCs/HRP-Ab2 bioconjugates by dissolution of CaCO3
templates in aqueous EDTA solution. Thus, the fluorescence
of the AuNCs released from the biolabels was detected to
determine the NSE concentration. The fluorescence intensi-
ty was strongly affected by the assay conditions (Figure S6,
[39]
HRP-Ab is positively charged at pH 7.0, and thus could be
2
easily adsorbed on the hybrid spheres through electrostatic
adsorption and interactions between AuNCs and the amino
groups of the protein. Compared to CaCO /AuNCs hybrid
3
2
À1
spheres, a smaller BET surface area (13.62 m g ) and lower
3
À1
pore volume (0.0682 cm g ) for CaCO /AuNCs/HRP-Ab
3
2
indicated effective loading of HRP-Ab . Because of their
2
small size, HRP-Ab could penetrate into the pores, result-
ing in a decrease in the pore volume. Calculating the differ-
ence in enzyme concentration before and after adsorption
Supporting Information). After the Ab concentration in-
2
1
À1
creased to 50 mgmL , the fluorescence intensity increased
À1
and tended to a stable signal at 30 mgmL . Thus, an Ab
1
À1
revealed that about 180 mg of HRP-Ab was captured by
concentration of 30 mgmL was selected for the further
2
1
.0 mg of CaCO /AuNCs hybrid spheres from a protein solu-
studies. The fluorescence intensity increased with the incu-
bation time between 10 and 30 min and then leveled off
above 30 min. This result indicated that the interaction be-
tween antigen and antibody had reached equilibrium after
30 min, and hence an incubation time of 30 min was select-
ed. The fluorescence response increased with increasing con-
3
À1
tion with a concentration of 0.4 mgmL HRP-Ab . When
the hybrid material was conjugated with HRP-Ab and kept
in water for one month, vaterite was still the dominant poly-
morph. To demonstrate the potential application of CaCO3/
AuNCs/HRP-Ab bioconjugates in bioassay, a sandwich im-
2
2
2
À1
munoassay was developed.
centration and reached a platform at 50 mgmL of the bio-
conjugates. Under optimal assay conditions, the fluorescence
intensity with CaCO /AuNCs/HRP-Ab probes (curve b in
Fabrication of a sandwich immunosensor by using CaCO3/
3
2
AuNCs/HRP-Ab as probes: The utility of CaCO /AuNCs/
Figure 5A) was 8.54 times the signal when using AuNCs/
2
3
HRP-Ab bioconjugates for immunoassay was examined by
HRP-Ab probes (curve a in Figure 5A). This amplification
2
2
using a model sandwich immunoassay for detection of NSE.
Nitrogen-doped multiwalled carbon nanotubes (NCNs) have
attracted considerable interest for constructing electrochem-
ical biosensors because of their high electrical conductivity
and excellent electrocatalytic effects. To fabricate the im-
munosensing surface, NCNs were used for immobilization of
of the fluorescence signal can mainly be attributed to the
high loading amount of AuNCs on CaCO3 spheres. As
shown in Figure 5B and C, the fluorescence response of the
immunosensor with CaCO /AuNCs/HRP-Ab probes in-
3
2
[40]
creased linearly with increasing logarithm of the target NSE
À1
concentration in the range from 0.005 to 1.0 ngmL . The
Ab . To increase their solubility and biocompatibility, NCNs
were initially acid-oxidized to introduce carboxyl groups on
their surface (see Supporting Information for experimental
linear regression equation is F/a.u.=428.8+174.8lg(CNSE/
1
À1
ngmL ) with a linear regression coefficient of 0.995. The
À1
detection limit (S/N=3) is estimated to be 2.0 pgmL .
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Calcium Carbonate–Gold Nanoclusters Hybrid Spheres
FULL PAPER
2 2
Scheme 2. Oxidation of OPD by H O .
À1
Figure 5. A) Fluorescence immunoassay in the presence of 0.1 ngmL
NSE. a) With AuNC/HRP-Ab
HRP-Ab bioconjugates. B) Fluorescence immunoassay with CaCO
AuNCs/HRP-Ab bioconjugates at target NSE concentrations of 0.005,
.01, 0.02, 0.1, 0.2, and 1.0 ngmL (curves a–g, respectively). C) Linear
2 3
bioconjugates. b) With CaCO /AuNCs/
2
3
/
2
À1
0
relationship between fluorescence intensity and the logarithm of the
target NSE concentration.
DPV analysis: The target NSE concentration was also de-
tected electrochemically. It is well known that HRP can cat-
alyze the oxidation of o-phenylenediamine (OPD) by H O ,
2
2
and the mechanism of enzymatic catalysis and oxidation was
[41]
investigated previously. The DPV response can be tested
in 0.1m pH 7.0 phosphate-buffered saline (PBS) containing
OPD and H O . The mechanism of the electrochemical oxi-
2
2
À1
dation of OPD is illustrated in Scheme 2. Multiple HRP in
CaCO /AuNCs/HRP-Ab bioconjugates can catalyze the ox-
Figure 6. A) Electrochemical immunoassay in the presence of 0.1 ngmL
NSE. a) With AuNC/HRP-Ab bioconjugates. b) With CaCO /AuNCs/
2
3
3
2
HRP-Ab
2
bioconjugates. B) Typical DPV curves at target NSE concen-
idation of OPD with H O . Figure 6A shows typical DPV
À1
2
2
trations of 0.0005, 0.005, 0.01, 0.1, 1.0, and 2.0 ngmL (curves a–g, re-
spectively). C) Linear relationship between current and the logarithm of
the target NSE concentration.
curves for AuNCs-HRP-Ab and CaCO /AuNCs/HRP-Ab
2
2
3
bioconjugates. A well-defined peak was observed around
À0.521 V (vs. SCE) for the electrochemical oxidation of
[42]
OPD to 2,2’-diaminoazobenzene, the enzymatic product.
The DPV response was strongly influenced by the assay
(Figure S7, Supporting Information). An Ab1 concentration
À1
conditions. Therefore, the concentration of Ab , incubation
of 30 mgmL , an incubation time of 30 min, and a bioconju-
1
À1
time, and concentration of bioconjugates were investigated
gate concentration of 50 mgmL were selected as optimal
Chem. Eur. J. 2012, 18, 5261 – 5268
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5265

L.-P. Jiang et al.
assay conditions. The DPV signal with CaCO /AuNCs/HRP-
3
Ab probes (curve b in Figure 6A) was 4.24 times that with
2
AuNCs/HRP-Ab probes (curve a in Figure 6A). This also
2
confirms that the signal was amplified by the high loading
amount of AuNCs/HRP-Ab on porous CaCO . Figure 6B
2
3
shows typical DPV curves of the immunoassay at different
NSE concentrations under optimal conditions. The voltam-
metric peaks were well defined and the intensity was pro-
portional to the concentration of NSE in the range from
À1
0
.0005 to 2.0 ngmL . The resulting calibration plots were
linear (Figure 6C). The linear regression equation is I/mA=
À1
7
.944+2.138lg(CNSE/ngmL ) with a linear regression coeffi-
cient of 0.993. The detection limit (S/N=3) is estimated to
À1
be 0.1 pgmL .
Figure 7. Comparison of serum NSE levels determined by A) fluores-
cence assay, B) ELISA, and C) electrochemical assay.
Compared to other reported electrochemical immunosen-
[43,44]
sors,
the proposed assay showed a lower detection limit.
The high sensitivity could be attributed to the following rea-
sons: The porous CaCO sphere provided an efficient host
Conclusion
3
for multiple AuNCs/HRP-Ab2 loading, which greatly en-
hanced the detection signal. In addition, the PNCN-modi-
fied GCE was an effective matrix to immobilize biomole-
cules with high stability and bioactivity.
Porous CaCO spheres were used for loading of AuNCs to
3
fabricate CaCO /AuNCs hybrid. The vaterite polymorph of
3
CaCO can be well stabilized by AuNCs. The hybrid spheres
3
have the advantages such as porous structure, high porosity,
biocompatibility, degradability, and fluorescence property
and can be used as templates for the encapsulation of HRP-
Ab to fabricate CaCO /AuNCs/HRP-Ab bioconjugates. A
promising and versatile immunosensor was developed for
both fluorescent and electrochemical detection of NSE by
using the as-prepared bioconjugates as probes. The CaCO3/
AuNCs hybrid spheres are also expected to find other po-
tential applications such as drug delivery, diagnostics, and
optics.
Specificity and stability of the immunosensor: The selectivi-
ty of the immunoassay was evaluated by using possible in-
terfering substances such as immunoglobulin G (IgG) and
2
3
2
À1
a-fetoprotein (AFP) at a concentration of 0.1 ngmL . The
fluorescence responses for IgG and AFP were 5.8 and 7.1%
that of NSE, respectively. The DPV signals for IgG and
AFP were 6.2 and 7.4% that of NSE, respectively. This indi-
cates that the proposed sensor has sufficient selectivity for
NSE detection, and is capable of distinguishing NSE from
its analogues in complex samples. The reproducibility of the
immunoassay was estimated by assaying one NSE level for
five replicate measurements with relative standard devia-
tions (RSD) of 6.5%. The storage stability of CaCO3/
AuNCs/HRP-Ab2 was also investigated by comparing the
signals after a sandwiched immunoreaction. When the
CaCO /AuNCs/HRP-Ab bioconjugates were stored in PBS
Experimental Section
Chemicals: H
amine (OPD) were purchased from Shanghai Chemical Reagent Co.
Shanghai, China). Human neuron specific enolase (NSE) antigen, NSE
antibody coating (Ab , monoclonal), and NSE antibody (Ab , polyclone)
2 2 4 2
O (30% w/v solution), HAuCl ·4H O and o-phenylenedi-
3
2
(
at 48C, the signals remained at about 93.4% for fluores-
cence response and 94.6% for DPV signal after one month.
This indicates that CaCO /AuNCs/HRP-Ab has good stor-
1
2
labeled with HRP (HRP-Ab ) were purchased from Shanghai Linc-Bio
2
Science Co. Ltd. Poly(allylamine hydrochloride) (PAH, M=15000), poly-
ethylenimine (PEI), bovine serum albumin (BSA), glutaraldehyde
3
2
age stability at 48C.
(
GLU), and Tween-20 were obtained from Aldrich Chemical Co. All
other chemicals were of analytical grade. Doubly distilled water was used
throughout the experiments.
Application of the immunosensor in human serum: The fea-
sibility of the immunoassay in clinical applications was in-
vestigated by analyzing several real samples, in comparison
with the ELISA method. Figure 7 shows the correlation be-
tween the results obtained by fluorescence immunoassay,
electrochemical immunoassay, and the ELISA method. The
relative deviations between fluorescence immunoassay and
ELISA method were in the range from À4.4 to +6.8%, and
the relative deviation between electrochemical assay and
ELISA method was 3.2–7.2%. Since there are no significant
differences among the results of the three methods, the de-
veloped immunoassay may provide an interesting alternative
tool for detection of proteins in clinical laboratories.
Synthesis of CaCO
3
/AuNCs hybrid spheres: AuNCs were synthesized ac-
cording to the literature (see Supporting Information for experimental
[
19]
[34]
details).
For CaCO
3
CaCO spheres were synthesized by a one-pot approach.
3
/AuNCs synthesis, CaCO
3
spheres were dispersed in PAH
À1
aqueous solution (2 mgmL ) and sonicated for 10 min to make the sur-
3
face positively charged. Then, CaCO (50 mg) was dispersed in 50 mL of
AuNCs solution (pH 7.0) and sonicated for 30 min. After centrifugation,
the composites were obtained. The composites were further washed with
distilled water three times and dried.
Assembly of HRP-Ab
CaCO
2
into the CaCO
/AuNCs was dispersed in a solution of HRP-Ab
3
/AuNCs spheres: 10 mg of
À1
3
2
(0.4 mgmL
)
and shaken for 1 h at 48C for enzyme absorption. The bioconjugates
were then centrifuged and washed with distilled water three times. Then,
the CaCO /AuNCs/HRP-Ab was dispersed in PBS.
3 2
5266
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Chem. Eur. J. 2012, 18, 5261 – 5268

Calcium Carbonate–Gold Nanoclusters Hybrid Spheres
FULL PAPER
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Received: September 14, 2011
Revised: December 31, 2011
Published online: March 15, 2012
5268
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Chem. Eur. J. 2012, 18, 5261 – 5268