Talanta 77 (2009) 10091014

Contents lists available at ScienceDirect

Talanta
journal homepage: www.elsevier.com/locate/talanta

Rapid identication and high sensitive detection of cancer cells

on the gold nanoparticle interface by combined contact angle
and electrochemical measurements
Fang He a , Qin Shen a , Hui Jiang a , Jian Zhou a , Jian Cheng b , Dadong Guo a ,
Qingning Li a , Xuemei Wang a, , Degang Fu a , Baoan Chen b
a
State Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory), School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
b
Department of Hematology and Oncology, Afliated Zhongda Hospital, Southeast University, DingJiaQiao 87, Nanjing 210009, China

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

Article history: In this study, we have proposed a novel strategy for the rapid identication and high sensitive detection
Received 19 May 2008 of different kinds of cancer cells by means of electrochemical and contact angle measurements. A simple,
Received in revised form 24 July 2008 unlabeled method based on the functionalized Au nanoparticles (GNPs) modied interface has been uti-
Accepted 30 July 2008
lized to distinguish the different cancer cells, including lung cancer cells, liver cancer cells, drug sensitive
Available online 14 August 2008
leukemia K562/B.W cells and drug resistant leukemia K562/ADM cells. The relevant results indicate that
under optimal conditions, this method can provide the quantitative determination of cancer cells, with
Keywords:
a detection limit of 103 cells mL1 . Our observations demonstrate that the difference in the hydrophilic
Gold nanoparticles
Cancer cell identication
properties for target cellular surfaces and in the uptake efciency of the anticancer drug daunorubicin
Electrochemical detection for different cancer cells could be readily chosen as the elements of cancer identication and sensitive
Contact angle measurement detection. This raises the possibility to advance the promising clinic diagnosis and monitoring of tumors
with the aim of successful chemotherapy of human cancers.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Cancer is one of the most serious and lethal diseases around the
world. The research of fast identication and high-sensitive detec-
tion of cancer cells is extremely important for cancer diagnosis and
therapy, which can be utilized to explore and monitor the rele-
vant biological process of cancers and is critical for the early cancer
diagnosis and clinical treatments.
The intensive interests and efforts in the early cancer diag-
nosis reect the increasing demand of relevant biosensors with
good sensitivity and selectivity, rapidness, and easy operation
[1,2]. Several protocols have been developed to detect and identify
cancerous cells, including those based on polymerase chain reac-
tion (PCR) [3], quartz crystal microbalance (QCM) measurement
[4], microarrays [5,6], monoclonal antibody-coupled ferromagnetic
nanoparticles [7], aptamer-modied uorescent nanoparticles [8],
carbohydrate-mediated cell recognition using gold glyconanopar-
ticles [9], immunophenotyping by means of ow cytometry
[10], amperometric detection of enzymatic reaction products [11]
chemiluminescence (CL) [12] or diffraction-based cell detection
Corresponding author. Tel.: +86 25 83792177; fax: +86 25 83792177.
E-mail address: xuewang@seu.edu.cn (X. Wang).
[13], etc. Many of the currently available detection methods require
enrichment of the target cells in the sample or expression of u-
orescent protein markers or antibodies in the cells, and tend to
include additional steps and time-consuming assay procedures. For
example, PCR-based methods are indirect ways of detecting cells
and require prolonged isolation steps before analysis. Besides, the
variable sensitivity of PCR can limit its effectiveness as a diagnos-
tic technique and lead to false-negative results [3]. Moreover, the
common immunophenotypic analyses on basis of ow cytometry,
which require multiple antibody probes for accurate cell detection,
are usually time-consuming, complex and costly. In view of these
challenges, a simple detection system that can achieve high selec-
tivity and sensitivity without the need for target amplication and
labeling is highly desirable.
Recently, nanotechnology has been located in a unique position
to transform cancer diagnostics and to produce a new generation
of biosensors and medical imaging techniques for highly sensitive
and precise recognition. Large quantities of biocompatible nano-
materials have attracted much interest of numerous scientists in
various biomedical research areas due to their unique properties.
Among them, gold nanoparticles (GNPs), the known biocompatible
nanomaterials, have been widely concerned in the elds of biomed-
ical imaging and biosensing applications during the last ten years
[1417]. Our recent study indicates that the relevant GNPs have

0039-9140/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2008.07.063
1010 F. He et al. / Talanta 77 (2009) 10091014
negligible effect on cellular drug uptake of daunorubicin (DNR) of
normal cells but have signicant effect on the intracellular drug
accumulation of DNR on leukemia cancer cells [17]. Besides, the
functional GNPs-based materials have been well adopted for the
biosensing of biomacromolecules based on their unique mechani-
cal and electronic properties [1822].
Considering the plentiful merits of GNPs, in this study we have
utilized them for highly sensitive detection of cancer cells. The
strategy of a simple identication and sensitive detection of the
drug sensitive and drug resistant leukemia K562 cells, lung can-
cer cells and liver cancer cells on the functionalized GNPs modied
interface has been developed by using anticancer drug daunoru-
bicin as the electrochemical probe. Meanwhile, according to the
different hydrophilic characteristics of the relevant cancer cells on
the GNPs modied interface, we could utilize the contact angle
measurement to identify above different kinds of cancer cells. The
researches have realized the cancerous cell detection by electro-
chemical and contact angle measurements with high sensitivity,
selectivity and simplicity.
2. Experimental
2.1. Reagents
Daunorubicin, mercaptopropionic acid (MPA) and hydrox-
yethylpiperazine ethanesulfonic acid (HEPES) were purchased
from Aldrich. 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide
hydrochloride (EDC) was purchased from Sinopharm. Co. Ltd.,
China. Daunorubicin stock solutions were freshly prepared and
stored in the dark at 4 C. Phosphate buffer solution (PBS) and
HEPES buffer was prepared with double distilled water. All other
reagents were of analytical grade. The functionalized GNPs were
synthesized according to the literature [23] by the ligand exchange
reaction between triphenyl phosphine (PPh3 )-stabilized precursor
nanoparticles and MPA. The concentration ratio between original
Au (III) and MPA was adjusted at 1:100.
2.2. Cells and cell culture
Lung cancer cells, liver cancer cells, leukemia K562/ADM and
K562/B.W cell lines were purchased from Institute of Hematology
of Tianjin, Chinese Academy of Medical Sciences and cultured in a
ask in RPMI 1640 medium (GIBCO) supplemented with 10% fetal
calf serum (FCS, Sigma), penicillin (100 U mL1 ), and streptomycin
(100 g mL1 ) at 37 C in a humidied atmosphere containing 5%
CO2 . The drug resistant leukemia K562 cells (K562/ADM cells) were
maintained with 1 g mL1 Adriamycin (Sigma).
The real leukemia cell lines were collected and separated from
the mixture of peripheral blood of leukemic patients, PBS (0.1 M, pH
7.2) and lymphocytes separation medium with the ration of 1:1:2
(v/v/v) by centrifugation 2500 rpm for 30 min.
2.3. Electrode preparation
The glassy carbon electrode (GCE) was polished to a mirror
using 0.3 and 0.05 m alumina slurry (Beuhler) followed by rinsing
thoroughly with double distilled water. The electrodes were then
pretreated electrochemically by applying a potential of +1.75 V in
PBS (0.1 M, pH 5.0) for 300 s, and scanned between +0.3 and +1.25 V
and then +0.3 and 1.3 V until a steady-state currentvoltage curve
was observed [24]. This process led to the formation of hydroxyl
groups on the GCE surface and increased surface hydrophilicity of
the GCE. Then the pretreated electrode was immersed thoroughly
into the blending solution with 100 L GNPs and 100 L HEPES
buffer solution (0.1 M, pH 7.2) containing 0.5 mmol EDC for about
10 h. The hydroxyl groups on GCE were linked to the MPA stabilized
GNPs with the assistance of the EDC linker. The GNPs modied GCE
was then rinsed thoroughly with double distilled water to remove
the non-covalent nanoparticles before use.
2.4. Atomic force microscopy (AFM) measurements
The AFM measurements were performed through a simulta-
neous monitoring of both the amplitude and the phase of the
oscillating cantilever in tapping mode. All AFM data were collected
on a Digital Instruments using a Nanoscope III controller (Veeco
Instruments, New York) and an E-scanner operating in tapping
mode in air. Imaging was accomplished using diving board TESP
tips with integral square pyramidal tips. All data were plane t and
attened prior to analysis using the Nanoscope software.
2.5. Electrochemical investigation
The leukemia K562 cells at a concentration of 6 105 mL1 with
anticancer drug daunorubicin at a concentration of 5.3 105 M
were rstly incubated at room temperature. After the targeted
cells with DNR were pretreated for a series of incubated time,
the suspensions were centrifuged for 10 min. Then all medium
samples outside leukemia K562/ADM cells were collected and
diluted with sterilized PBS (0.1 M, pH 7.2). The electrochemical
signal of DNR residue in these samples was determined with
differential pulse voltammetry (DPV) assay for each sample by
CHI660C electrochemical analyzer. All measurements were per-
formed at ambient temperature (22 2 C) in a three-component
electrochemical cell consisting of the GNPs modied glassy carbon
electrode (GNPsGCE) as the working electrode, a Pt wire as the
counter electrode and a saturated calomel electrode (SCE) as the
reference electrode.
2.6. Contact angle detection
Contact angle detection was performed with a CAM2000 opti-
cal contact angle analyzer (KSV Instruments, Finland) using a CCD
video camera and a horizontal light source to illuminate the liquid
droplet. The droplets of solution were placed on the surface of the
GNPsGCE and the contact angle measurements were carried out
at ambient temperature (22 2 C). To extract the precise contact
angle values, the drop images were tted using the YoungLaplace
equation [25]. The contact angle values were determined with the
precision of 0.5 .
3. Results and discussion
3.1. Characterization of the GNPsGCE
After the preparation of the GNPsGCE according to the pro-
cedure described above, the GNPsGCE was rst characterized by
AFM, which is an effective way to provide nanoscale surface topog-
raphy and phase images. The AFM studies can afford direct evidence
for the morphologies of the functionalized GNPs lm covered on the
GCE. As shown in Fig. 1, the typical topographic images of the bare
GCE and the GNPsGCE have been explored and characterized by
AFM studies. The pretreated bare GCE exhibits a relatively planar
interface (Part A, Fig. 1), while the GNPs lm appears more rough
and highlighted in the AFM graphs (Part B, Fig. 1). The average sizes
of GNPs were about 15 nm and in coincidence with the sizes of dis-
persed GNPs obtained by TEM (not shown), indicating the assembly
of GNPs on the surface produces little aggregation.
In addition, the electrochemical performance of the GNPsGCE
has also been investigated by cyclic voltammetry. As shown in
 F. He et al. / Talanta 77 (2009) 10091014 1011

Fig. 1. Topological AFM images of the GCE surface before (A) and after (B) functionalized GNPs modication.

combination of the respective cancer cells with the GNPsGCE, our

Fig. 2. Cyclic voltammograms of 1 mM K3 [Fe(CN)6 ] (containing 0.1 M KCl elec-
trolyte) at (a) the functionalized GNPsGCE, and (b) the bare GCE. Scan rate: 0.1 V/s.
Fig. 2, compared with the bare GCE, the electrochemical current
of [Fe(CN)6 ]3 at the GNPsGCE enhances ca. 45%, indicating a
considerable improvement in the electron transfer. Besides, a neg-
ative shift of ca. 70 mV in the [Fe(CN)6 ]3 redox peak potentials
is observed for the GNPsGCE in comparison with that of the bare
GCE.
3.2. Identication of cancer cells by contact angle detection
Based on these studies, the multi-signal responsive biosens-
ing interface was further explored by tailoring the relevant cell
(i.e., lung cancer cells, liver cancer cells, leukemia drug sensitive
K562/B.W cells, and leukemia drug resistant K562/ADM cells) cov-
ering GNPsGCE, where a 10 L droplet of target cell solution was
pipetted onto the modied surface and dried with nitrogen. By
observations indicate that contact angle technique could offer a
new strategy for the rapid identication and detection of different
cancer cells. Fig. 3 shows the pixel images of the initial contact angle
values of PBS (pH 7.2, 0.1 M) droplet on the respective cell covered
GNPsGCE. The contact angle values of relevant droplet on lung
cancer cells, liver cancer cells, drug sensitive leukemia K562/B.W
cells, and drug resistant leukemia K562/ADM cells covered mod-
ied electrodes were ca. 77 , 70 , 64 , and 53 , respectively. It is
evident that the different cancer cells can be distinguished from
each other by using the contact angle detection. The rational behind
this may be due to the difference in the expression of the mem-
brane proteins (such as P-glycoprotein (P-gp) and others) on the
cellular surface, which greatly inuences the hydrophilicity of the
cellular surface. Thus, the different cells could be identied based
on the hydrophilic characteristics of them, using the contact angle
measurements.
The stability of the contact angle measurements of PBS on these
cancer cells covered GNPs surface have also been investigated (as
shown in Fig. S1 in Supporting Information). It is observed that the
contact angle values of the droplets on the relevant surfaces almost
kept constant in 30 s. The stable and convincing results guarantee
the simple and fast identication of target cancer. So, according to
the distinct contact angles of PBS on target cells covered GNPsGCE,
we can fast and simply identify the drug sensitive and drug resistant
leukemia K562 cells, lung cancer cells and liver cancer cells.
3.3. Identication of cancer cells based on electrochemical
detection
In view of the above studies, the more sensitive DPV technique
has been explored for the identication and detection of the can-
cer cells (i.e., lung cancer cells, liver cancer cells, leukemia drug
sensitive K562/B.W cells, and leukemia drug resistant K562/ADM

Fig. 3. Typical initial contact angle images of 7 L PBS droplets on (a) lung cancer cells, (b) liver cancer cells, (c) leukemia K562/B.W cells, and (d) leukemia K562/ADM cells
covered GNPsGCE. During the preparation of the cells covered GNPsGCE, a 10 L droplet of cell solution (6 107 cells mL1 ) was pipetted onto the modied electrodes
surface and dried with nitrogen. The contact angle values were determined with the precision of 0.5 .
1012 F. He et al. / Talanta 77 (2009) 10091014
Fig. 4. Differential pulse voltammetry of DNR residue after the leukemia K562/ADM
cells (a and c) or leukemia K562/B.W cells (b and d) were incubated with DNR
(5.3 105 M) for 2 h at bare GCE (a and b) and GNPsGCE (c and d). Pulse amplitude:
0.05 V. Pulse width: 0.05 s. Pulse period: 0.1 s. The signicant enhancement on the
GNPsGCE can be observed.
cells). As well known, multidrug resistance (MDR) is a dominating
limit in the current chemotherapy of cancers. It is mainly associ-
ated with the overexpression of several proteins on the cellular
surface, the most important being P-gp. Failure of chemotherapy
to the malignant tumor is usually attributed to the overexpression
of P-gp, which is considered to act as an energy-dependent drug
efux pump that causes a decrease in cytotoxic drug accumulation
[26]. Another failing factor of chemotherapy is that anticancer drugs
not only kill cancer cells but also poison healthy cells. Therefore, it
is much important to distinguish the drug sensitive and drug resis-
tant cancer cells to monitor the cancer therapy. Meanwhile, with
the anticancer drug DNR as the electrochemical probe, the DNR
uptake efciency for different cancer cells could be probed by the
DPV technique [27].
On the basis of these considerations, in this contribution, the
DNR uptake efciency by the different cancer cells has been investi-
gated. As we know, when different cells were treated with DNR, the
unadsorbed DNR molecules are still in the environmental solution,
and the electrochemical response of this part of the molecules can
be readily detected. Thus, the DNR residue (unadsorbed DNR) can
be adopted as the referential value of the cellular uptake efciency.
As shown in Fig. 4, our study indicates that the DNR detection sen-
sitivity could be remarkably enhanced by using the functionalized
GNPsGCE than that of the bare GCE. It is proved that the GNPsGCE
could remarkably enhance the DNR detection sensitivity and selec-
tivity for identication of the cancer cells.
It is evident that by combining with anticancer drug DNR as an
electrochemical probe, the residual amount of the DNR outside the
target cancer cells can be adopted as the referential value of the ef-
ciency. So, we have explored the possibility to identify and detect
the four kinds of cancer cells mentioned above by electrochem-
ical means. Fig. 5 illustrates the DPV study of DNR residue on the
GNPsGCE after the cells have been incubated with DNR for 2 h. The
distinct electrochemical responses of the DNR residue in the buffer
for the four different cancer cells appear at ca. 750 mV (vs SCE) on
the modied electrodes. Notably, the DPV peak current of the DNR
residue for the drug resistant leukemia K562/ADM cells is much
greater than that of the drug sensitive leukemia K562/B.W cells,
suggesting that much more DNR molecules have been absorbed
into drug sensitive cancer cells than that of the drug resistant can-
cer cells. This is consistent with the cellular surface properties of
the leukemia K562/ADM cells, i.e., the overexpression of P-gp on
the leukemia K562/ADM cells will extrude the positively charged
DNR out of the cells and lead to the less DNR accumulation in the
leukemia K562/ADM cells than that in the leukemia K562/B.W cells.
Fig. 5. Differential pulse voltammetry study on the GNPsGCE for DNR residue out-
side (a) lung cancer cells, (b) liver cancer cells, (c) leukemia K562/B.W cells, (d)
leukemia K562/ADM cells after the cells were incubated with DNR (5.3 105 M)
for 2 h. DPV of DNR (5.3 105 M) itself was also shown as the reference (e). All cell
concentrations are 6 105 cells mL1 . Pulse amplitude: 0.05 V; Pulse width: 0.05 s;
Pulse period: 0.1 s.
With a same incubation period, the relative decrease percentage in
the peak current of DNR in the buffer is 17% for leukemia K562/ADM
cells, 53% for leukemia K562/B.W cells, 74% for lung cancer cells and
89% for liver cancer cells, respectively, indicating that different can-
cer cells have different uptake facility of DNR. Scheme 1 illustrates
the relevant strategy for the identication of cancer cells by contact
angle and electrochemical study. Thus, by checking the correspond-
ing electrochemical behavior of the DNR residue in the buffer for
different cancer cells, we can quickly and sensitively identify these
different kinds of cancer cells based on the respective GNPsGCE.
The time dependence of the drug uptake efciency has also been
researched (as shown in Fig. S2 in Supporting Information), since
the interaction time of DNR with target cancer cells is important
for the fast and high-sensitive identication of the four different
cancer cells mentioned above. The corresponding electrochemical
response of DNR residue outside the lung cancer cells, liver cancer
cells, and leukemia K562/B.W cells are found to decrease with an
increasing incubation time, while only the current of the leukemia
K562/ADM cells keep stable during the period. In all cases, the
peak currents of DNR residue can reach to a constant value after
incubation for about 2 h. Therefore, to distinctly identify the target
cancer cells with rapidness and accuracy, the optimal incubation
time should be 2 h.
Furthermore, we have applied this procedure to real world
sample analyses. We analyzed the peripheral blood of leukemic
patients before and after injected with anticancer drug. As shown
in Fig. S3 in Supporting Information, the result was consistent with
the foregoing experiments. After injected with anticancer drug for
a period of time, the cancer cells in the body of patient became
leukemia drug resistant cells which less anticancer drug could be
absorbed into. So, its electrochemical responses of the DNR residue
in the buffer was stronger than that of patient without injection,
and both of them had some positive shift of the peak potential,
suggesting its potential application as the new strategy for the
development of the clinic diagnosis and monitoring of tumors.
3.4. Electrochemical method for cell quantication
To quantitatively describe the performance of the multi-signal
responsive biosensing interface, leukemia K562/B.W cell line was
chosen as the paradigm. When incubated with the same concen-
tration of DNR, the DPV peak current of DNR residue in the buffer
decreased with the increasing concentration of K562/B.W cells. The
peak current was proportional to the concentration of leukemia
 F. He et al. / Talanta 77 (2009) 10091014
Scheme 1. Scheme for the illustration of electrochemical and contact angle detection of cancer cells based on functionalized GNPsGCE interface.
K562/B.W cells ranging from 2.0 104 to 8.0 105 cells mL1 , with
a correlation coefcient of 0.99 (as shown in Fig. S4 in Support-
ing Information). The detection limit for cell number was calculated
to be approximate 1000 cells mL1 (based on the signal of DNR
with S/N = 3 criterion) and much lower than those of several meth-
ods developed by other researchers, such as 1.0 105 cells mL1 by
immunomagnetic separation/ow injection systems coupled with
mediated amperometric detection [28], and 1.0 104 cells mL1 by
immunosensor for detection of Salmonella species on a QCM [29].
This indicates that the presented strategy could provide a simple
and applicable way for cancer cell quantication with high sensi-
tivity and ne accuracy.
4. Conclusions
In summary, we have demonstrated a novel strategy for the
rapid identication and high sensitive detection of target cancer
cells by electrochemical and contact angle measurements based on
the functionalized GNPsGCE. On the basis of these observations,
the approaches described here may be readily adopted as a signif-
icant way to detect and identify various cancer cells and advance
clinic diagnosis and monitoring of tumors with the aim of success-
ful chemotherapy of human cancers. This could provide a promising
powerful tool for cancer early diagnosis and thus guide how to treat
it, a challenge faced by the relevant cancer therapy. Further, in vivo
applications of this new strategy to detect and distinguish different
kinds of cancerous cells by using electrochemical and contact angle
detection simultaneously are ongoing in process.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (90713023, 20675014, 20535010 and 60121101),
Ministry of Science & Technology of China (2007AA022007),
the State Key Laboratory of Electroanalytical Chemistry in the
1013
Changchun Institute of Applied Chemistry of the CAS, and the Pro-
gram (050462) for New Century Excellent Talents in University, the
Chinese Ministry of Education.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.talanta.2008.07.063.
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