Analytica Chimica Acta 746 (2012) 107113

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Analytica Chimica Acta

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The sandwich-type electrochemiluminescence immunosensor for -fetoprotein

based on enrichment by Fe3 O4 -Au magnetic nano probes and signal amplication
by CdS-Au composite nanoparticles labeled anti-AFP
Hankun Zhou a , Ning Gan a, , Tianhua Li a , Yuting Cao a , Saolin Zeng a , Lei Zheng b, , Zhiyong Guo a
a
The State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Material Science and Chemical Engineering of Ningbo University, Ningbo 315211,
China
b
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

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

 Sandwich immunoreaction, testing a

large number of samples simultaneously.
 The magnetic separation and enrichment by Fe3 O4 -Au magnetic nano
probes.
 The amplication of detection signal
by CdS-Au composite nanoparticles
labeled anti-AFP.
 Almost no background signal, which
greatly improve the sensitivity of
detection.

a r t i c l e

i n f o

Article history:
Received 18 May 2012
Received in revised form 9 August 2012
Accepted 17 August 2012
Available online 28 August 2012
Keywords:
Electrochemiluminescence
Sandwich immunoreaction
Magnetic capture probes
CdS-Au signal tag

a b s t r a c t
A novel and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor was fabricated on a
glassy carbon electrode (GCE) for ultra trace levels of -fetoprotein (AFP) based on sandwich immunoreaction strategy by enrichment using magnetic capture probes and quantum dots coated with Au shell
(CdS-Au) as the signal tag. The capture probe was prepared by immobilizing the primary antibody of AFP
(Ab1) on the core/shell Fe3 O4 -Au nanoparticles, which was rst employed to capture AFP antigens to
form Fe3 O4 -Au/Ab1/AFP complex from the serum after incubation. The product can be separated from
the background solution through the magnetic separation. Then the CdS-Au labeled secondary antibody
(Ab2) as signal tag (CdS-Au/Ab2) was conjugated successfully with Fe3 O4 -Au/Ab1/AFP complex to form
a sandwich-type immunocomplex (Fe3 O4 -Au/Ab1/AFP/Ab2/CdS-Au), which can be further separated by
an external magnetic eld and produce ECL signals at a xed voltage. The signal was proportional to a
certain concentration range of AFP for quantication. Thus, an easy-to-use immunosensor with magnetic
probes and a quantum dots signal tag was obtained. The immunosensor performed at a level of high sensitivity and a broad concentration range for AFP between 0.0005 and 5.0 ng mL1 with a detection limit of
0.2 pg mL1 . The use of magnetic probes was combined with pre-concentration and separation for trace
levels of tumor markers in the serum. Due to the amplication of the signal tag, the immunosensor is
highly sensitive, which can offer great promise for rapid, simple, selective and cost-effective detection of
effective biomonitoring for clinical application.
2012 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +86 574 87609987; fax: +86 574 87609987.
Corresponding author. Tel.: +86 20 61642147; fax: +86 20 62787681.
E-mail addresses: ganning@nbu.edu.cn, ssrs20060621@163.com (N. Gan), nfyyzl@163.com (L. Zheng).
0003-2670/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aca.2012.08.036

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H. Zhou et al. / Analytica Chimica Acta 746 (2012) 107113

1. Introduction
It is well known that the protein -fetoprotein (AFP) in human
serum is clinically measured as a biomarker for hepatocellular cancer. The average concentration of AFP is approximately 25 ng mL1
in healthy human serum but rises greatly in patients with liver
cancer [1]. Thus, sensitive detection of AFP levels of cancer biomarkers plays an important role in the early detection of disease and
highly reliable predictions. Until now, conventional immunoassay
methods include the enzyme-linked immunosorbent assay (ELISA)
[2,3], uoroimmunoassay [4], ow injection chemiluminescence
[5], chemiluminescence enzyme immunoassay [6]. However, the
challenge remains for obtaining a rapid sample, sensitive detection and low-cast for early and ultrasensitive screening of cancer
biomarkers [7]. The conventional sandwich-type immunoassay is
one of the major analytical techniques for sensitive and selective
detection of protein and has wide application in clinical diagnosis
and biomedical research [8]. It is employed as a sandwich format
in which analytes are captured and detected by an excess immobilized primary antibody (Ab1) and labeled secondary antibody
(Ab2). The general sandwich-type immunoassay protocol requires
the primary antibodys (Ab1) immobilization of the bare electrode
or modied electrode, then its conjugation with the antigen by
the immunoreaction. Then, a uorescence-labeled antibody was
assembled to construct an immunosensor for protein detection.
Unfortunately, long incubation time and washing steps for separation of bound free antibodies and antigens are the two main
drawbacks [9]. It is easy to change the uorescence intensity during
the elution condition. Now, electro-immunosensors are important
analytical tools designed to tumorous processes and tumor markers
in human serum can be used in screening for a disease [10,11]. As
a valuable detection method, electrochemiluminescence (ECL) has
received considerable attention due to its versatility, low-cost, lowbackground, easy operation procedure and high sensitivity [1214].
Electrochemiluminescence immunoassay has been applied in biological detection and quantifying it combines the high sensitivity
of ECL detection and the specicity of immunosensors [15]. Some
kinds of ECL reagents such as, Tris (2,2 -bipyridyl), ruthenium (II)
(Ru(bpy)3 2+ ) [1618], luminol and its derivatives [19], quantum
dots or semiconductor nanocrystals (NCs) [2022], have been used
to construct ECL immunosensors. However, the bioanalysis based
on these conventional luminescent reagents possesses some limitation. A high degree of ruthenium labeling at multiple sites may
result in the loss of biological activity of the biomolecules [23] and
the luminol ECL system is weak in the neutral solution [14,24].
Recently, quantum dots (QDs), as a new kind of ECL luminophore,
with size-tunable, optical, narrow emission spectra and broad excitation spectra [2528], have been widely used in fabricating all
kinds of photoluminescence probes in biological analysis. Zhu and
co-workers used semiconductor nanoparticles to develop a series of
electrochemiluminescence biosensors for various biosystem assays
where the immunosensor provides a convenient specic method
for protein detection [15,20,29]. Lius group presents a versatile
immunosensor using a quantum dot coated silica nanosphere as
a label for IgG detection [30]. Among these strategies, the quantum dot based amplication has received special attention for its
possible outstanding optical, electronic, and biocompatible performance of fabricated electrochemiluminescence sensor for clinical
diagnosis [31].
In many applications, gold nanoparticles serve as an attractive
candidate for biosensors and modied electrodes due to their good
biocompatibility and stability. They have also facilitated the transfer of electrons between the electrode and the biomolecules [32].
There is growing interested in developing a new, advanced material for using a novel biosensor construct. Nanocomposite materials
constitute a rapidly evolving eld of science and technology. They

have attracted the interest of researchers, due to their different

nanoscale functionalities and capability of endowing the substrate
with enhanced properties [33]. Actually, the core/shell composite
nanoparticles Fe3 O4 -Au, with Au coating the magnetic nanoparticles, exhibits suitable intrinsic properties of the magnetic core and
Au shell. It is anticipated that incorporation of Au coating on a magnetic core could attain both the advantages of chemical stability,
biocompatibility of Au and magnetic separation of Fe3 O4 [3437].
Herein, in our work, the protocol for a novel sandwich
immunoassay, with quantum dots labeled and excellent magnetic/luminescent properties, is fabricated. For the primary
antibody and secondary antibody there was simultaneous incubation with the Fe3 O4 -Au and CdS-Au, respectively. A sandwich-type
immunosensor based on CdS-Au, labeled secondary antibody, was
constructed with the following process. The core/shell magnetic
materials Fe3 O4 -Au was labeled as the rst antibody (Fe3 O4 Au/Ab1) by the Au-S band of the Au-antibody and the CdS-Au
composite nanoparticles was labeled as the secondary antibody
(CdS-Au/Ab2), which carried huge amounts of ECL signal probe.
With the Fe3 O4 -Au/Ab1 capturing antigen onto the antibody site
by the specic bond of the antigenantibody, the CdS-Au, labeled
the secondary antibody, could be successively conjugated with the
antigen via the immunoreaction. Based on the ECL signal related
to the concentration of detected antigen, the immunoassay could
be accomplished successfully. Therefore, this ECL immunosensor
can effectively amplify the detected signal, avoid the inactivation
of proteins and exhibit attractive sensitivity.
2. Experimental
2.1. Materials
The AFP antibody (anti-AFP, 12 mg mL1 ) and antigen (AFP)
were bought from Biocell Company (Zhengzhou, China). Gold chloride (HAuCl4 ) and bovine serum albumin (BSA) was obtained
from Sinopharm Chemical Reagent Co. Ltd. The phosphate-buffered
saline (PBS), 0.1 M with various pH value was prepared by mixing
the stock solution of NaH2 PO4 and Na2 HPO4 and then adjusting
the pH with 0.1 M NaOH and H3 PO4 . PBS (0.1 M, pH 7.4) containing
0.1 M K2 S2 O8 and 0.1 M KCl was used as the electrolyte. All other
reagents were of analytical reagent grade and used without further
purication.
2.2. Apparatus
Electrochemiluminescence measurements were performed
using a Model MPI-B electrochemiluminescence analyzer (Xian
Remax, China) with the voltage of the photomultiplier tube (PMT)
set at 700 V in the detection process. A three-electrode conguration was employed consisting of a modied glassy carbon electrode
(4 mm diameter) as a working electrode, a platinum wire as the
counter electrode and an Ag/AgCl (sat. KCl) reference electrode. The
transmission electron microscope (TEM) image was performed on
a TEM (H-7650, Japan) operating at an acceleration voltage of 60 kV.
The XRD patterns of prepared powder samples were collected using
a Bruker AXS (D8) X-ray diffractometer with a Cu target (40 kV,
40 mA). The magnetization curve was measured with a vibrating
sample magnetometer (Lake Shore 7410) at room temperature.
All electrochemiluminescence experiments were performed in a
5.0 mL quartz cell at room temperature.
2.3. Preparation of the core/shell Fe3 O4 -Au nanoparticles and
CdS-Au composite nanospheres
The Fe3 O4 -Au nanoparticles were prepared by chemical coprecipitation methods according to the reference [33]. The Fe3 O4 -Au

H. Zhou et al. / Analytica Chimica Acta 746 (2012) 107113

109

Scheme 1. Scheme of the preparation procedures of the immunosensor.

nanoparticles were obtained and dispersed in distilled water to a

nal volume 5.0 mL.
Monodisperse CdS nanospheres were prepared according to the
literature [28] and the nal product was dried to yield a powder.
For the preparation of gold NPs (GNPs) assembler CdS nanospheres,
20 mg of CdS nanospheres were rst dispersed in 2.0 mL of ethanol
and treated with 0.2 g cysteine. After stirring for 6 h, the suspension was centrifuged and washed with ethanol repeatedly for three
times, and thiol-functionalized nanoparticles were obtained. Then
the thiol-functionalized CdS nanospheres were dispersed in a mixture of 1.0 mL of gold NPs, the mixed suspension was stirred at 4 C
for 12 h, unbound gold NPs were removed by successive centrifugation and washed with water several times.
2.4. Immobilization of anti-AFP on to the Fe3 O4 -Au and CdS-Au
nanoparticles (Fe3 O4 -Au/Ab1, CdS-Au/Ab2)
The process for the preparation of magnetic capture probes
(Fe3 O4 -Au/Ab1) and signal tag of CdS-Au/Ab2 is shown Scheme 1.
First, 20 mg of Fe3 O4 -Au nanoparticles were reached with 1.0 mL
PBS (pH 7.4), 1.0 mL of 50 g mL1 Ab1. The solution was stirred at
4 C for 24 h. Unbound Ab1 was removed by successively washing
the Fe3 O4 -Au nanoparticles with pH 7.4 PBS. After magnetic separation and washing with PBS three times, the magnetic capture
probes (Fe3 O4 -Au/Ab1) were redispersed in 1.0 mL of pH 7.4 PBS
and stored at 4 C for a later experiment. Simultaneously, (same as
above), 20 mg CdS-Au was added to 1.0 mL PBS (pH 7.4), 1.0 mL of
50 g mL1 Ab2 solution and stirred at 4 C for 24 h. Subsequently,
the mixture was centrifuged and washed with water several times
to obtain the signal tag (CdS-Au/Ab2). Finally, the CdS-Au/Ab2 were
dispersed with 0.1 M of pH 7.4 PBS to a nal volume of 1 mL and
stored at 4 C for later usage.
2.5. The sandwich immunoreaction
As shown in Scheme 1, 1.0 mL Fe3 O4 -Au immobilized Ab1 (A)
and CdS-Au immobilized Ab2 (B) were rst prepared and incubated in 1.0 mL of 1% BSA at room temperature (20 C) for 1 h to
block nonspecic binding sites. A concentration of AFP was rst
added in the Fe3 O4 -Au immobilized Ab1 and incubated at room
temperature for 1 h to obtain the Fe3 O4 -Au/Ab1/AFP (C). The prepared Fe3 O4 -Au/Ab1/AFP and CdS-Au/Ab2 was mixed in a pipe
and incubated at room temperature for 1 h to form the sandwich
products, which were magnetically separated from the background

solution. The product was washed with PBS (pH 7.4) for three
times, and then re-dispersed in 50 L of PBS (pH 7.4). As a result,
the magnetic separation and quantum dots labeled sandwich-type
(Fe3 O4 -Au/Ab1/AFP/CdS-Au/Ab2) construction was obtained (D).
2.6. Preparation of ECL immunosensor
A glassy carbon electrode (GCE) with 4 mm diameter was rst
polished carefully to a mirrorlike surface with 0.30.05 m alumina slurry, then rinsed and ultrasonically in ethanol and distilled
water. Before modication, the bare electrode was cyclic-potential
scanned in the potential range of 0.2 to 0.6 V in 5.0 103 M
K3 [Fe(CN)6 ] solution containing 0.1 M KCl supporting electrolyte
until a pair of well-dened redox peaks was obtained. After the
electrode was dried under nitrogen at room temperature, 10 L
of solution (which was prepared) dropped onto the surfaced of
the clean glassy carbon electrode, and allowed to air-dry at room
temperature, to obtain the ECL sensor.
2.7. ECL detection
The ECL measurements of the modied electrodes above were
performed in 5 mL of 0.1 M PBS (pH 8.0) containing 0.1 M K2 S2 O4
and 0.1 M KCl and the potential scanned from 1.4 to 0.2 V with
scan rate of 100 mV s1 . The ECL emission intensity was recorded
by a MPI-B multifunctional chemiluminescence analyzer, with the
PMT set at 700 V, and the AFP concentrations were measured
related to the ECL signals.
3. Results and discussion
3.1. Characterization of core/shell magnetic NPs
Fig. 1 shows the property characterization of the Fe3 O4 -Au
MNPs. Fig. 1A shows the typical image of Fe3 O4 -Au MNPs prepared
using about 40 nm. Fig. 1B shows the vibration sample magnetometer (VSM) magnetization curves of Fe3 O4 and Fe3 O4 -Au MNPs at
room temperature. It is evident that the sample presented a saturation magnetization (Ms) of 23.89 emu g1 and 15.84 emu g1 for
Fe3 O4 and Fe3 O4 -Au MNPs. The saturation magnetization of the
nanoparticles reduced after coating with a layer of gold. This maybe
ascribed to the nonmagnetic gold layer.
The coated nano Au shell of Fe3 O4 -Au magnetic nano-probes
was conrmed by the X-ray diffraction (XRD) pattern. Fig. 1C shows

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H. Zhou et al. / Analytica Chimica Acta 746 (2012) 107113

Fig. 2. ECL potential curves of (a) the Fe3 O4 /GCE, (b) the Fe3 O4 -Au/GCE, the
CdS/GCE, and the CdS-Au/GCE in 0.1 M PBS (pH 8.0) containing 0.1 M KCl and
0.1 M K2 S2 O8 . Scan rate: 100 mV s1 , the voltage of the PMT was 700 V.

the XRD spectra of synthesized Fe3 O4 (a) and core/shell Fe3 O4 Au (b) MNPs. It reveals the face-centered-cubic magnetite (Fe3 O4 )
structure (JCPDS Card No. 19-06290), and Fe3 O4 -Au MNPs exhibited diffraction peaks (at 2 = 38.25, 44.46, 64.69 and 77.72), which
were indexed to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of gold cubic
phase, respectively. Compared with the XRD spectra of two samples, the Fe3 O4 -Au MNPs displayed all the diffraction peaks of pure
Fe3 O4 MNPs; it can be infered that a thin gold was successfully
coated on the Fe3 O4 core [37].

3.2. Electrochemical and ECL behaviors of CdS-Au nanospheres

Fig. 1. TEM image of the Fe3 O4 -Au (A) MNPs; (B) magnetization measurements of
applied eld for pure Fe3 O4 (a) and core/shell Fe3 O4 -Au MNPs (b); (C) XRD patterns
of Fe3 O4 (a) and Fe3 O4 -Au MNPs (b).

Fig. S1, Supporting information shows the TEM image of the

CdS (A) and CdS-Au (B) nanoparticles. According to the TEM observation, the average size of the CdS nanoparticles is 50 nm and
their size distribution is relatively uniform, and the gold NPs with
the diameter of 10 nm was successfully assembled on the surface
of CdS. As shown in the insert of Fig. S1(B), displayed the XRD
spectra of prepared CdS-Au assembler nanoparticles. It reveals the
typical diffraction peaks of CdS (at 2 = 28.20, 36.65, 43.73, 50.93
and 58.34) and which also include the peaks of gold nanoparticles.
Fig. 2 shows the ECL intensity of the Fe3 O4 /GCE (curve a),
Fe3 O4 -Au/GCE (curve b), CdS/GCE (curve c), and CdS-Au/GCE (curve
d) electrode in 0.1 M PBS (pH 7.4) containing 0.1 M KCl and
0.05 M K2 S2 O8 , respectively. It can be seen that the Fe3 O4 MNPs
(a) modied the electrode in the solution almost had no ECL signal, and the ECL signal was slightly enhanced with the Fe3 O4 -Au
modied electrode, but still weak. Curve c and d shows the ECLpotential curves of the pure CdS NCs and CdS-Au composite lm,
respectively and the strong ECL signal obtained. The results indicate
the ECL signal from the reaction between CdS and S2 O8 2 . Compared with curve c and d, it should be noted that the ECL intensity
from the CdS-Au composite lm is about 2.5-fold higher than that
observed from the pure CdS NCs lm. The reason may be that the
gold particles exhibit great catalytic activity and enhanced electrical conductivity, which results in the occurrence of enhanced ECL
signal.
Furthermore, as shown in the Fig. 2 (curve a and b), the ECL signal
was very low, in the absence of the CdS NCs. The results indicate
that the Fe3 O4 and Au could not generate the ECL signal, and thus
was generated from the CdS NCs.

H. Zhou et al. / Analytica Chimica Acta 746 (2012) 107113

Fig. 3. ECL potential curves of (a) the bare GCE, (b) Fe3 O4 -Au/Ab1/AFP, (c) Fe3 O4 Au/Ab1/AFP/Ab2/CdS, and (d) Fe3 O4 -Au/Ab1/AFP/Ab2/CdS-Au modied glassy
carbon electrodes in 0.1 M PBS (pH 8.0) containing 0.1 M KCl and 0.1 M K2 S2 O8 . Scan
rate: 100 mV s1 , the voltage of the PMT was 700 V.

3.3. ECL behavior of sandwich immunoassay using CdS-Au/Ab2 as

the label
The process of sandwich immunoassay used to determine AFP
was shown in Scheme 1. Specically, the Ab1 was rst anchored
on the surface of the Fe3 O4 -Au nanospheres by the AuS bound.
The products with an Ab1 immobilized on the surface of the
nanosphere to capture AFP (antigen) from a solution, containing a concentration of AFP. Finally, the protein-labeled CdS-Au
nanoparticles were introduced to the immunoreaction with the
exposed part of AFP by an incubation period. This produced a
sandwich-type complex, with the magnetic separation for performing the following electrochemiluminescence analyses. In this
work, Fe3 O4 -Au/Ab1/AFP, Fe3 O4 -Au/Ab1/AFP/Ab2/CdS and Fe3 O4 Au/Ab1/AFP/Ab2/CdS-Au sandwich-type complex were used to
construct an ECL immunosensor. Before using CdS and CdS-Au as
the label for preparation of immunosensor, the performance of
the bare electrode and Fe3 O4 -Au/Ab1/AFP modied electrode in
detecting of ECL signal was investigated and compared with the ECL
response of bare electrode (Fig. 3a) and Fe3 O4 -Au/Ab1/AFP modied electrode (Fig. 3b) toward the same condition as detected in the
PBS solution. As shown in Fig. 3, they were the ECL proles of the
bare electrode and Fe3 O4 -Au/Ab1/AFP modied electrode, respectively. It could be concluded, that both electrodes of the ECL signal
were very low, without CdS or CdS-Au as the label. However, the
ECL intensity was increased obviously after the CdS or CdS-Au prepared immunocomplex Fe3 O4 -Au/Ab1/AFP/Ab2/CdS (Fig. 3c) and
Fe3 O4 -Au/Ab1/AFP/Ab2/CdS-Au (Fig. 3d) sandwich-type complex
modied on the electrode. The results show that the ECL response
of CdS-Au as the label is much higher then the pure CdS labeled
immunocomplex. It can also be concluded that the CdS-Au labeled
antibody was successfully conjugated with the Fe3 O4 -Au/Ab1/AFP
by the specic bond of the antigenantibody.

Fig. 4. Effects of incubation time on the response of ECL emission.

time and reached the maximum when incubation time was 60 min.
Afterwards, the emission intensity was slightly decreased and variation was mild. The results indicated a saturated binding of the
immobilized CdS-Au/Ab2. The optimal incubation time was 60 min
in this experiment. The pH was another important issue for the
formation of immunocomplex, as shown in Fig. 5, the ECL intensity
increased with an increase in pH values from 6.0 to 8.0, and then
decreased with pH higher than 8.0, indicating that pH played an
important role in building the immunocomplex process. Thus, the
ECL measurements were performed in pH 8.0 PBS solution.
3.5. Performance of the sandwich-type ECL immunosensor for
AFP detection
Under optimal conditions, Fig. 6 displays the ECL response of the
immunosensor before (a) and after (bn) reacting with different
concentrations of AFP. It is found that the ECL intensity enhanced
linearly with the concentration of AFP. As shown in the insert, a
linear relationship between the logarithm of the I (I = Is I)
and the logarithm of concentration of AFP wherein Is and I represent the ECL intensity of the immunosensor in the absence and
presence of AFP, respectively. The standard calibration curve was
found to be log(I) = 2.7638 + 0.2906 log C, with a correlation coefcient of 0.9939, covering the AFP concentration range from 0.0005

3.4. Optimization of major parameters for immunoreaction

One of the most important issues in the development of an efciency ECL sensor is the dependence of ECL signals on the CdS-Au
signal tag. The detection sensitivity of the immunosensor depended
on the immunocomplex of the CdS-Au/Ab2 suspension. Therefore,
the effect of incubation time and pH were studied in this study.
As seen in Fig. 4, at room temperature, electrochemiluminescence
emission intensity increased along with an increase in incubation

111

Fig. 5. Effect of pH value of substrate solution on the response signals.

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H. Zhou et al. / Analytica Chimica Acta 746 (2012) 107113

Table 1
The recovery of the proposed immunosensor in human serum.
Serum samples

Proposed method (pg mL1 )

Reference method (ng mL1 )
Relative error (%)

0.0552a
6.02
8.6

5.39b
6.02
10.5

52.9c
6.02
12.2

7.80b
7.12
9.5

8.22b
7.24
13.5

ac

The serum samples were diluted at 1.0 104 , 1000 and 100 times, respectively.

be seen from Fig. 7, the ECL response of the proposed immunosensor showed no remarkable change in the AFP mixed with interfering
agents compared to AFP only. On the contrary, many weak ECL
responses were exhibited by replacing AFP with PSA, CEA, BSA and
HIgG in the solution. The results showed a good selection of the
proposed immunosensor for AFP detection.
3.6. Application

Fig. 6. ECL proles of the immunosensor in the absence (a) and increasing of AFP
concentration (bn) in 0.1 M PBS (pH 8.0) containing 0.1 M KCl and 0.1 M K2 S2 O8 .
The inset is the calibration curve, AFP concentration (ng mL1 ): (a) 0, (b) 0.0005, (c)
0.001, (d) 0.002, (e) 0.005, (f) 0.01, (g) 0.02, (h) 0.05, (i) 0.1, (j) 0.2, (k) 0.5, (l) 1.0, (m)
2.0, (n) 5.0. Scan rate: 100 mV s1 , the voltage of the PMT was 700 V.

to 5.0 ng mL1 . The detection limit is calculated to be 0.2 pg mL1

based on a signal-to-noise of 3. The results indicate an acceptable quantitative behavior of the proposed method used for the
AFP detected. Moreover, the ultrasensitive detection at this level of
concentration was much lower than the previously reported ECL
detection of AFP [13,18,32].
The selectivity of the proposed immunosensor for AFP detection,
the as-prepared capture probe (Fe3 O4 -Au/Ab1), was incubated in
the AFP solution containing different interfering agents, such as
PSA, CEA, BSA and HIgG, and the results are shown in Fig. 7. As can

To determine the feasibility of the immunoassay system for

clinical applications, the proposed method was evaluated by comparing the assay results of real serum samples using the present ECL
immunosensor with reference values obtained by the commercial
ELISA method. The serum samples were appropriately diluted with
0.05 M pH 7.4 PBS solutions, a level at which the level of the serum
tumor marker was over the calibration range. Table 1 describes the
correlation between the partial results obtained by the proposed
method and the ELISA method, indicating an acceptable agreement,
with relative errors less than 13.5%. The proposed method could be
used for satisfactory clinical determination of AFP levels in human
serum.
4. Conclusions
In this work, we demonstrated a sensitive ECL-sensing protocol for detecting AFP using a sandwich immunoreaction strategy
for magnetic separation and quantum dots labeled sandwich-type
immunocomplex. Since the Fe3 O4 -Au and CdS-Au NPs displayed
good biocompatibility and strong ECL intensity, a simple and
sensitive sandwiched ECL immunosensor was fabricated successfully. Without almost any background intensity, the developed
immunosensor highly enhanced sensitivity compared to previous
reports. In particular, this novel strategy could open a new and
appealing approach for bioassays.
Acknowledgements
This work was supported by the Natural Science Foundation of
Zhejiang (Y3110479), the Natural Science Foundation of Ningbo
(2011A610018 and 2011A610006), the Social Development Project
of Ningbo (2011C50037, 2011C50038 and 2011B82014), and the
KC Wong Magna Fund in Ningbo University, Science and Technology Planning Project of Guangdong Province (2010A030300006
and 2008A050200006).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.aca.2012.08.036.
References

Fig. 7. Selectivity of the ECL immunosensor to AFP (0.5 ng mL1 ) by comparing

it to the interfering protein: prostate protein antigen (PSA, 10 ng mL1 ), carcinoembryonic antigen (CEA, 10 ng mL1 ), bovine serum albumin (BSA, 1.0 g mL1 ),
human IgG (HIgG, 10 ng mL1 ) and the mixed sample containing 0.5 ng mL1 AFP,
10 ng mL1 PSA, 10 ng mL1 CEA, 1.0 g mL1 BSA and 10 ng mL1 HIgG. Scan rate:
100 mV s1 , the voltage of the PMT was 700 V.

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