Biosensors and Bioelectronics 66 (2015) 321–326

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Biosensors and Bioelectronics

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Short communication

Sensitive detection of tumor cells by a new cytosensor with 3D-

MWCNTs array based on vicinal-dithiol-containing proteins (VDPs)
Yanan Xu, Hui Wu, Chusen Huang, Caiqin Hao, Beina Wu, Chongchong Miao, Shen Chen,
Nengqin Jia n
The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials and Shanghai Municipal
Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Department of Chemistry, Shanghai Normal University, Shanghai 200234,
PR China

art ic l e i nf o a b s t r a c t

Article history: Vicinal-dithiol-containing Proteins (VDPs) are overexpressed in cancer cells and become a potential
Received 9 September 2014 biomarker for aggressive tumors, the synthesized 2-p-aminophenyl-1, 3, 2-dithiarsenolane (VTA2) is
Received in revised form proved to be a highly selective ligand for vicinal dithiols of VDPs in cells. In this report, we developed a
21 October 2014
new cytosensor based on three-dimensional (3D)-like VTA2-conjugated multiwalled carbon nanotubes
Accepted 7 November 2014
Available online 21 November 2014
(VTA2@MWCNTs) array modified indium tin oxide (ITO) electrode for sensitive and selective detection of
VDPs-overexpression tumor cells. The layer-by-layer assembling and cellular detection performances of
Keywords: the 3D-VTA2@MWCNTs-based cytosensor were investigated by atomic force microscopy (AFM) and
Cytosensor electrochemical methods including cyclic voltammetry (CV), electrochemical impedance spectroscopy
3D-MWCNTs array
(EIS) and differential pulse voltammetry (DPV). Attributed to signal amplification and targeting re-
Electrochemical impedance spectroscopy
cognition of the 3D-structured MWCNTs@VTA2, the fabricated cytosensor showed high specificity and
VDPs
ITO sensitivity to the detection of VDPs-overexpression HL-60 cells ranging from 2.7
102 to
2.7
107 cells mL  1 with a low detection limit of ca. 90 cells mL  1. Furthermore, the captured cancer
cells on the cytosensor also could be directly visualized by optical microscopy technology. This proposed
3D-MWCNTs array-based cytosensing strategy provides a simple, sensitive approach for non-invasive cell
detection, presenting potential applications in cancer diagnosis.
& 2014 Elsevier B.V. All rights reserved.

1. Introduction cell-surface vicinal dithiols on VDPs display key roles in main-

taining extracellular redox homeostasis and cellular functions,
The homeostasis of cellular redox environment is one of the which is found to be responsible for cancer (Lee et al., 2014; Weiss
most important foundations of living systems. Among the im- et al., 2001; Yang et al., 2007), platelet function (Sugatani et al.,
portant factors associated with redox homeostasis and signaling, 1987), and human immunodeficiency virus type 1 (HIV-1) (Mat-
vicinal dithiol-containing proteins (VDPs) with space-closed thiol thias and Hogg, 2003; Ou and Silver, 2006; Sahaf et al., 2005).
groups was especially attractive for its direct involvement of many Especially, VDPs are overexpressed in aggressive tumors and the
biological processes through the interconvert between protein tumor cells become more dependent on these vicinal dithiols of
vicinal dithiols and disulfides in protein synthesis and post VDPs such as Trx and TrxR system (Dilda et al., 2008; Lu et al.,
translational modification (Maron et al., 2013; Poole and Nelson, 2007; Zhang et al., 2007). Many efforts have been devoted to
2008; Reddie and Carroll, 2008; Ying et al., 2007). Commonly, studying of the functionality of VDPs including identification of
new functional VDPs by arsenite-affinity chromatography (Huang
mammalian cell surface (exofacial membrane) is rich in disulfide
et al., 2011; Krauth-Siegel et al., 2005; McStay et al., 2002;
bonding, which can maintain the structure and functionalities of
Schuppe-Koistinen et al., 1994), development of fluorescent probes
cells in the presence of oxidizing extracellular environment (Ali
for specific detection of VDPs, and synthesis of new drug candi-
and Hynes, 1978; Hynes and Destree, 1977). However, recent stu-
dates for cancer therapy based on the active vicinal dithiols of
dies demonstrated that VDPs were also present on mammalian
VDPs. All these studies were based on the active vicinal dithiols of
cell membrane. And as the reductive form of protein thiols, these VDPs. Thus the high levels of vicinal dithiols of VDPs in cancer cells
will become a potential biomarker for aggressive tumors. Captur-
n
Corresponding author. Fax: þ 86 21 64321833. ing and detection of cancer cells can provide an easy and effective
E-mail address: [email protected] (N. Jia). way for monitoring the progression of cancer and their related

http://dx.doi.org/10.1016/j.bios.2014.11.008
0956-5663/& 2014 Elsevier B.V. All rights reserved.
322 Y. Xu et al. / Biosensors and Bioelectronics 66 (2015) 321–326

biological processes (Galanzha et al., 2009; Uhr, 2007; Xu et al., (PBS, pH 7.4) containing 14 mM KH2PO4, 87 mM Na2HPO4, 2.7 mM
2009). Therefore, development of rapid, selective and economical KCl and 137 mM NaCl. Ultrahigh purity H2O (18.2 MΩ) was used
approach for detection of cancer cells is highly desirable. Recent throughout the experiments.
years have witnessed a great development in the field of electro-
chemical biosensors for the rapid, selective, and sensitive detec- 2.2. Preparation of arylamine film on ITO electrode
tion of cancer cells with low cost. However, to best of our
knowledge, there are no electrochemical biosensors reported for Indium tin oxide (ITO) electrodes (resistivity ¼10–15 Ω) were
capturing and sensitive detection of cancer cells based on the cut into 2 cm
0.3 cm and rinsed by ultrahigh purity water
vicinal dithiols of VDPs. (18.2 MΩ), ethanol, and then ultrasonic washing in acetone and
The applications of three dimensional (3D)-nanostructured isopropyl alcohol. The electrochemical working area is controlled
electrodes are highly desirable for the improvement in the per- via use of epoxy glue with a size of 2 mm
3 mm. For the pre-
formance of electrochemical biosensors (Mendes et al., 2007; Zhu paration of arylamine film, p-phenylenediamine (7.25 mL, 10 mM)
et al., 2014). Especially, three-dimensional (3D) array or vertically hydrochloric acid solution was rapidly added to NaNO2 (2.1 mL,
aligned structure with functionalized nanohybrids, based on a 1 M) solution in an ice bath and allowed for 15 min complete re-
solid basement, has shown excellent performance in enhancement action. The arylamine film was electrografted to the ITO surface by
of surface area and low cytotoxicity (Malhotra et al., 2010; McCune cycling once from  0.6 to 0.4 V vs SCE (v ¼100 mV s  1), followed
et al., 2012). A number of 3D-structure is used as ideal support in by depositing at 0.6 V for 2 min. To block the further reduction
bio-sensing and biomedical fields (Han et al., 2014; Kim et al., of the diazonium salt, two final cyclic voltammetric scans were
2011; Sardesai et al., 2011). For instance, 3D synthetic biodegrad- performed.
able polymer scaffold promotes cell survival without affecting
cytochrome oxidase activities, allowing actual liver function more 2.3. Fabrication of the 3D-MWCNTs@VTA2 cytosensor
complete simulation (Kim et al., 1998). Other research uses
IrO2–Hemin–TiO2 nanowire arrays to detect glutathione and Hela Firstly, target ligand VTA2 was synthesized, and the detailed
cells extracts by photoelectrochemical detection (Tang et al., 2013). synthetic procedures were demonstrated in the supplementary
Due to the electrochemical property of the superior electron materials. Then, the cytosensor was fabricated as following pro-
transfer and efficient electrical conductivity, carbon nanotubes cess (Fig. 1A). Acid-treated shortened MWCNTs (5 mg, 1 mg mL  1)
(CNTs) were typically used as the ideal leader in ultrasensitive, dispersed in DMSO solution (5 mL) containing amide condensa-
nanostructured electrochemical sensing platform design (Rawson tion agent (HATU, 5 mg, 1 mg mL  1) and VTA2 (5 mg, 1 mg mL  1),
et al., 2012; Tu et al., 2009). Some study has demonstrated that and then shaked for 30 min at room temperature. Then the
well aligned multiwalled carbon nanotubes (MWCNTs) array em- VTA2@MWCNTs were vertically anchored to arylamine modified
bedded in a SiO2 matrix was significant for improving the sensi- ITO electrodes at 65 °C for 24 h. After preparation, the cytosensors,
tivity of electrochemical detection method (Li et al., 2002). Herein, VTA2@MWCNTs/ITO electrodes, were rinsed with phosphate buf-
we take MWCNTs as the nanostructured surface for constructing fer solution (PBS, 10 mM, pH 7.4) and dried for subsequent cell
the target electrochemical biosensor. In addition, some studies detections.
have suggested that trivalent arsenic based compounds could bind
to VDPs efficiently (Donoghue et al., 2000; Huang et al., 2013). Our 2.4. Apparatus
previous work is also found that 2-p-aminophenyl-1, 3, 2-di-
thiarsenolane (VTA2) is a highly selective acceptor for vicinal di- All electrochemical experiments containing electrochemical
thiols of VDPs in cells. Due to the overexpressed levels of vicinal impedance spectroscopy (EIS), cyclic voltammetry (CV) and dif-
dithiols of VDPs in tumor cells, VTA2 was introduced into the ferential pulse voltammetry (DPV) were performed with a CHI
MWCNTs as the selective ligand in construction of our target 660B electrochemical workstation (Shanghai Chenhua Co. Ltd.,
electrochemical biosensor. China). A standard three-electrode cell was used during the ex-
Up to now, no electrochemical techniques for the detection of periment. The functionalized ITO electrode as a working electrode
vicinal cancer cells based on that the higher levels of vicinal di- with a SCE reference electrode and a Pt wire counter electrode.
thiols of VDPs were present in cancer cells compared to the low The electrolyte consists of a 10 mM pH 7.4 PBS solution containing
levels of vicinal dithiols of VDPs in the normal cells. Here is an K3Fe(CN)6 (10 mM), K4Fe(CN)6 (10 mM) and KCl (0.1 M). The im-
attempt to design a cytosensor based on 3D-MWCNTs@VTA2 pedance spectra were recorded within the frequency ranging from
modified indium tin oxide (ITO) electrode for detecting VDPs 10  2 to 105 Hz with signal amplitude of 5 mV. The value of Ret (the
overexpressing tumor cells. The ITO electrode was also used as a diameters of Nyquist diagrams) was obtained by fitting with
platform for fabricating VTA2@MWCNTs structure not only due to ZsimpWin software version 3.10. The differential pulse voltam-
its conductivity for electrochemical signal collection, but also for metry (DPV) measurements were performed from  0.2 to 0.6 V
that the captured cancer cells could be tracked directly by optical with a pulse amplitude of 50 mV and width of 0.02 s.
microscope technology. This constructed electrochemical cyto- Atomic force microscopy (AFM) images were obtained on the
sensor may enable for a dual functional detection of cancer cells Bruker AXS GmbH at MultiMode Nanoscope.
based on both electrochemical and optical image technology.

3. Results and discussion

2. Experimental
3.1. Fabrication and characterization of the 3D-MWCNTs@VTA2
2.1. Materials and reagents cytosensor

MWCNTs were purchased from Shenzhen Nanotech Port Co. p- To fabricate a cytosensor with 3D array of VTA2@MWCNTs, the
phenylenediamine (AP) was purchased from Aladdin. Indium Tin ITO electrode is initially modified by arylamine layer through
Oxide (ITO, resistivity ¼10–15 Ω) was purchased from Zhuhai electrochemical reduction of the aryl diazonium cation. As sche-
kaivo company. Other Reagents were of analytical grade and used matically illustrated in Fig. 1A, electrochemical reduction
without further purification. The 10 mM phosphate buffer solution was applied to control the functionalization of ITO surface,
 Y. Xu et al. / Biosensors and Bioelectronics 66 (2015) 321–326 323

Fig. 1. (A) Schematic illustration of assembling processes of 3D-MWCNTs@VTA2 cytosensor for detection of VDPs overexpressed tumor cell. (B) The corresponding AFM
images of (a) bare ITO electrode, (b) arylamine modified ITO and (c) MWCNTs@VTA2 modified ITO electrode.

electrochemical reduction of the aryl-diazonium salt anchored to of ∼60 nm. The root-mean-squared surface roughness (rms) values
the ITO substrate by forming an arylamine layer. The mechanism of bare ITO, arylamine modified ITO surface and MWCNTs@VTA2
for formatting a multilayer film structures has been proposed anchored ITO surface were 0.818 70.09, 2.781 70.37 and
previously (Delamar et al., 1992; Doppelt et al., 2007). The in situ 4.818 70.32 nm, respectively. Obviously, the increase in roughness
generating of aryl diazonium cation grafted to ITO surface is and the results of AFM demonstrate the 3D surface structure of
shown in Fig. S1A (see the supplementary materials), it can be MWCNTs@VTA2 modified ITO electrode is built. Furthermore, the
seen that one reduction peak appeared at  0.15 V on the first scan stepwise assembly on the ITO electrode could be efficiently de-
of cyclic voltammograms (CVs) from  0.6 V to 0.4 V, indicating tected by electrochemical impedance spectroscopy (EIS) and CV.
the reduction of the aryl diazonium salt and its electrografting on EIS is regarded as a powerful tool for probing the interface features
the ITO electrode. On the consecutive scan, at the ITO electrode, no of the surface modified electrodes, and its impedance spectra in-
subsequent cathodic peak is observed for the reduction of the aryl cludes a semicircle portion and a linear line portion, which cor-
diazonium cation due to the blocking nature of the formed ar- respond to the electron transfer process and diffusion process,
ylamine film, suggesting that the arylamine film with approximate respectively. The diameter of the semicircle represents the elec-
thickness had electrodeposited on the electrode surface and thus tron-transfer resistance (Ret) at the electrode surface. Fig. S2A
hindered further reduction of aryl diazonium cation (Delamar shows Nyquist diagrams changed substantially after step-by-step
et al., 1992; Doppelt et al., 2007). Furthermore, to confirm this assembly of arylamine, VTA2@MWCNTs, HL-60 cells on the ITO
surface modification, electrochemical behavior of the probe electrodes using [Fe(CN)6]3  /4  as a redox probe. At a bare ITO
[Fe(CN)6]3  /4  was performed and the resulting CVs are shown in electrode, the redox process of the probe showed a low electron-
Fig. S1B. At the unmodified ITO electrode, a couple of well-defined transfer impedance (Ret) value (curve a). After the successive
redox peaks of the probe [Fe(CN)6]3  /4  were investigated, conjugation of arylamine and VTA2@MWCNTs, the Ret value obviously
whereas this characteristic CV peaks completely vanished at the increased (curve b, c), suggesting the VTA2@MWCNTs had successfully
arylamine film modified electrode, which could further verify that assembled on the ITO modified electrode and thereby blocked the
ITO surface was well functionalized by aryl diazonium cation and electron exchange between the redox probe. Especially, after HL-60
thereby modified by the arylamine film. cells (4.8
103 cells mL  1) were attached to the VTA2@MWCNTs
Following that, VTA2@MWCNTs structure was then prepared modified electrode, owing to the dielectric behavior of cells for inter-
by covalent interaction between amine group of VTA2 and facial electron transfer processes (Chen et al., 2013; Zhang et al., 2010),
carboxyl group of MWCNTs. The target cytosensor was finally a barrier of the cell membrane would further hamper the redox probe
synthesized by a covalent tether between VTA2@MWCNTs and close to the electrode surface and thus the Ret value increased greatly
arylamine functionalized ITO via amide bonds. Atomic force mi- (curve d), which could be applied as a rapid and label-free strategy for
croscopy (AFM) was used to characterize the surface topology of detection of tumor cells and bio-analysis. In addition, cyclic voltam-
layer-by-layer assembling on the ITO electrode (Fig. 1B), displaying metric characteristics of the redox probe at these modified electrodes
vertically aligned carbon nanotubes array with an average length (Fig. S2B) were also consistent with the EIS results.
324 Y. Xu et al. / Biosensors and Bioelectronics 66 (2015) 321–326

Fig. 2. Histogram of electrochemical impedance spectroscopy for (A) ITO, ITO-CNTs, ITO-CNTs@VTA2 coincubated with HL-60 cells (9.8
106 cells mL  1) separately, and
(B) for control experiments of Dithiothreitol (DTT) and N-ethylmaleimide (NEM)-treated MWCNTs@VTA2 modified ITO electrode co-incubated with HL-60 cells
(9.8
106 cells mL  1), respectively.

The corresponding histogram of typical electrochemical bound to vicinal dithiols of VDPs, the VTA2@MWCNTs modified
impedance spectroscopy for ITO, ITO-CNTs, and ITO-CNTs@VTA2 ITO electrode co-incubated with Dithiothreitol (DTT) or N-ethyl-
incubated with the same concentration of HL-60 cells (9.8
maleimide (NEM) in HL-60 cells (9.8
106 cells mL  1) was studied
106 cells mL  1) is illustrated in Fig. 2A. The increase in corre- by using electrochemical impedance spectroscopy (EIS) (Fig. 2B).
sponding histograms shows different anchor ability for tumor The DTT treated cells-anchored ITO electrode showed enhanced
cells, and it can be observed that ITO-CNTs@VTA2 exhibited a higher in an electron transfer resistance (Ret) response than the
highly selective for cancer cells. We can deduce that this higher cell-coated ITO electrode (without reagent treated), which was
specificity might be ascribed to the specific interaction between explained that DTT reagent was used to reduce disulfide bonds,
VTA2 anchored to ITO-CNTs@VTA2 and overexpressed vicinal di- and enable higher levels of vicinal dithiols of VDPs expressed by
thiols of VDPs in cancer cells. To further verify the process of VTA2 cells. However, a strongly decreased response was observed on the

Fig. 3. (A) Typical EIS responses of bare ITO (a) and VTA2@MWCNTs modified ITO electrode after capture of HL-60 cells with different concentrations: (b) 2.7
102, (c)
2.7
103, (d) 2.7
104, (e) 2.7
105, (f) 2.7
106, (g) 2.7
107 cells mL  1. Inset was the corresponding equivalent circuit model. (B) Calibration curve between logarithm
values of cells concentration and impedance for detecting HL-60 cells. (C) EIS responses of (a) VTA2@MWCNTs modified ITO electrode and after respective incubation with
(b) leukocytes (mouse bone marrow leukocytes), (c) NEC (human pancreatic cancer adjacent tissue cells), (d) CEC (human pancreatic cancer cells) and (e) HL-60 cells.
(D) Corresponding histogram of impedance data into Ret values. The concentration of all kinds of cells was same (1.0
105 cells mL  1).
 Y. Xu et al. / Biosensors and Bioelectronics 66 (2015) 321–326 325

NEM treated cells-anchored ITO electrode. It is attributed to NEM demonstrate that there are overexpressed VDPs on tumor cells and
that it is commonly used thiol-alkylation reagent that can in situ the levels of vicinal dithiols present on tumor cells are higher than
block vicinal dithiols of VDPs in cells, which could reduce the le- normal cells. Thus, the higher levels of vicinal dithiols (or the
vels of vicinal dithiols of VDPs in cells, and there was little efficient overexpressed VDPs) could become a new biomarker for cancer
sites for binding by VTA2 in ITO-CNTs@VTA2. These results sug- cells. It is especially interesting to note that the human pancreatic
gested that ITO-CNTs@VTA2 was a highly specific cytosensor for cancer adjacent tissue cells (NEC) gave little response compared
cancer cells through the specific interaction between VTAF2 in with the response of the Human pancreatic cancer cells (CEC) cell
ITO-CNTs@VTA2 and vicinal dithiols of VDPs in cells. lines, which further confirmed that our proposed cytosensor (ITO-
CNTs@VTA2) could selectively determine cancer cells based on the
3.2. Detection performances and selectivity of the cytosensor higher levels of vicinal dithiols of VDPs.

To attain adequate target conjugation and ideal cell capturing ca-

pacity of the constructed cytosensor, the suitable concentration of 4. Conclusions
VTA2 and incubating time of ITO-CNTs@VTA2 biosensor for cell de-
tection were firstly optimized (Fig. S3). Then the assay performances of In summary, we have designed a new cytosensor (ITO-
the proposed cytosensor in the detection of HL-60 cells were in- CNTs@VTA2) based on 3D-like VTA2@MWCNTs array for detection
vestigated. The cytosensor was based on the measurement of electron of tumor cells via specific recognition of VDPs expression patterns
transfer resistance (Ret) with [Fe(CN)6]3 /4 as a redox probe. As on the cellular surface. Three-dimensional architecture MWCNTs
electrochemical impedance spectroscopy (EIS) response immediately array equipped with excellent conductivity and enhanced surface
changed due to the electronic shielding effect from the cell membrane area performed as an appropriate sensing layer for the attachment
and the electron-transfer impedance (Ret) depended on the surface of large populations of VTA2 molecules and could be beneficial
coverage of the cells anchored onto the electrode surface, herein, EIS for the proposed cytosensor to achieve highly selective and
was applied for quantitative determination of tumor cells. Fig. 3A il- sensitive detection of VDPs overexpressed cancer cells. The
lustrates its EIS response with increasing concentration of tumor cells VTA2@MWCNTs-coated ITO modified electrode presented wide
ranging from 2.7
102 to 2.7
107 cells mL  1 measured in the pre- linear range, low detection limit and fast response as well as direct
sence of [Fe(CN)6]3 /4 as a redox probe and the equivalent circuit visualized observation in the detection of HL-60 tumor cells. An-
model inset in Fig. 3A, representing the typical electrochemical inter- other novel features of this constructed assay system is that the
face, was used to fit the impedance data into Ret values. It can be seen small-molecule ligand VTA2 conjugated with MWCNTs affords an
that electron transfer resistance, Ret, increased with the increasing cell alternative to antibody-based biosensor. A detection limit for tu-
concentration. The relationship between ΔRet (ΔRet ¼Ramp  Rblank) mor cells on the biosensor was equivalent to or better than other
and logarithm concentrations of HL-60 cells in the range from antibody-based measurement systems. Therefore, with a simple
2.7
102 to 2.7
107 cells mL  1 was fitted linearly with the equation and effective 3D nanoscale components assembling on a sensing
ΔRet (Ω)¼ 1323.18402þ514.18655
lg C[HL-60] (cells mL  1) with a surface, this electrochemical cytosensing provides a feasible and
correlation coefficient R¼0.99488 (Fig. 3B). Additionally, based on its sensitive method for the detection of cancer cells and may hold
transparent characteristics of the ITO electrode, the process for cap- great promise in clinical applications for early diagnosis of cancer.
turing different concentrations of cells could be visualized by optical
microscope technology (Fig. S4). This result from direct observation of
HL-60 cells with different concentrations on the VTA2@MWCNTs-ITO Acknowledgments
modified electrode was consistent with the response in EIS (Fig. 3A).
The detection limit of the proposed cytosensor was estimated to be This work was supported by the National Natural Science
approximately 90 cells mL  1 (signal-to-noise, S/N¼ 3) which are much Foundation of China (21373138, 21302125), National 973 Project
lower than those reported cytosensors (Table S1 in the supplementary (2010CB933901), Shanghai Sci. & Tech. Committee (12JC1407200,
materials). The relative standard deviations (RSD) of five ITO electro- 13ZR1458800), program for Changjiang Scholars and Innovative
des with the same modified procedure independently were 4.1% and Research Team in University (IRT1269), Doctoral Fund of Ministry
5.7% at the cell concentrations, 2.7
103 and 2.7
106 cells mL  1, of Education of China (Grant no. 20133127120005).
signifying an acceptable precision. All these results provide a good
performance of the VTA2@MWCNTs/ITO electrode in the quantitative
determination of HL-60 cells. Appendix A. Supplementary material
Further, the selectivity of the VTA2@MWCNTs/ITO biosensor
was evaluated by measuring its electrochemical impedance spec- Supplementary data associated with this article can be found in
troscopy (EIS) response toward tumor cells and human normal the online version at http://dx.doi.org/10.1016/j.bios.2014.11.008.
cells (Fig. 3C). Generally, there is overexpressed VDPs on tumor
cells compared to normal cells, which suggested the higher levels
of vicinal dithiols of VDPs were present in cancer cells. Here, we References
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