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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
108
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
109
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
110
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].
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. 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.
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
111
112
Table 1
The recovery of the proposed immunosensor in human serum.
Serum samples
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.
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