Frequency dependent impedimetric cytotoxic evaluation of anticancerdrug on breast cancer cell

Rangadhar Pradhan n, Shashi Rajput, Mahitosh Mandal, Analava Mitra, Soumen DasSchool of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

a r t i c l e i n f o

Article history:Received 21 August 2013Received in revised form8 November 2013Accepted 20 November 2013Available online 3 December 2013

Keywords:MicrofabricationMCF-7ZD6474BioimpedanceCytotoxicity

a b s t r a c t

The present work reports the impedance characteristics of MCF-7 cell lines treated with anticancer drugZD6474 to evaluate the cytotoxic effect on cellular electrical behaviour using miniature impedancesensors. Four types of impedance sensing devices with different electrode geometries are fabricated bymicrofabrication technology. The frequency response characteristics of drug treated cells are studied toevaluate cytotoxic effect of ZD6474 and also to assess the frequency dependent sensitivity variation withelectrode area. A significant variation in magnitude of measured impedance data is obtained for drugtreated samples above 10 mM dose indicating prominent effect of ZD6474 which results in suppression ofcell proliferation and induction of apoptosis process. The results obtained by impedimetric method arecorrelated well with conventional in vitro assays such as flow cytometry, cell viability assays andmicroscopic imaging. Finally an empirical relation between cell impedance, electrode area and drug doseis established from impedance data which exhibits a negative correlation between drug doses andimpedance of cancer cells.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Breast cancer stands second in cancer-related mortality ofwomen. It originates in the epithelial cells of the terminal ductulo-lobular system of the breast tissue and is classified pathologically onthe basis of morphological features of tumours. The majority of thebreast carcinomas (about 40–75%) cannot be classified satisfactorily.Therefore they are termed as invasive ductal carcinoma (IDC) nototherwise specified (NOS). The specialized pathological subtypes ofbreast carcinoma include invasive lobular carcinoma (5–10%), medul-lary carcinoma (1–7%), mucinous carcinoma (2%), tubular carcinoma(o2%), and metaplastic carcinoma (o1%). Besides, cribriform, papil-lary, micropapillary, apocrine, glycogen-rich clear cell, lipid-rich,inflammatory, and adenoid cystic carcinomas are the rare form ofspecialized invasive carcinomas found in breast tissue (Fabbri et al.,2008; Viale, 2012). Although researchers are working for advancedscreening programme and targeted drug delivery for combating thisdisease process, it is still a challenging issue to reduce the highmortality rate throughout the world (Nagalingam et al., 2012).Previously the conventional cell based assay was used to study thecell proliferation and viability which has been replaced by label freeand non-invasive techniques in recent time. The different label-freetechnologies available to monitor cellular processes in real-time

include electric cell–substrate impedance sensing (ECIS), quartzmicrobalance and optical light mode spectroscopy (Hong et al.,2011). Among them ECIS has been evolved as the most effectivetechnique to measure the real time impedance of cultured cells onthe surface of microelectrode which was first reported by Giaeverand Keese (1984). At present ECIS is an established non-invasiveelectrochemical technique that has been successfully used to monitoradhesion, growth and differentiation of cells in real time (Chen et al.,2012; Hong et al., 2011; Janshoff et al., 2010; Park et al., 2011; Xiaoand Luong, 2003, 2010; Yu et al., 2011), cell migration (Burns et al.,1997; Chan et al., 2010; Hsu et al., 2010; Lee et al., 2004),morphological changes during apoptosis (Arndt et al., 2004; Yinet al., 2007), taste sensor (Ceriotti et al., 1991; Hui et al., 2013),cytotoxicity of several drugs (Asphahani and Zhang, 2007; Campbellet al., 2007; Ceriotti et al., 2007; Curtis et al., 2009; Klo et al., 2008;Male et al., 2010; Muller et al., 2011; Opp et al., 2009; Sun et al., 2013;Wang et al., 2013; Xiao and Luong, 2005; Xu et al., 2012) and stemcell research (Bagnaninchi and Drummond, 2011; Maercker et al.,2008; Park et al., 2011; Reitinger et al., 2012). However, furtherresearches are required to explore the different technical aspects ofECIS methods.

In this paper, an attempt has been made to study the electricalimpedance of cells treated in various doses of anticancer drugto correlate the electrical properties with their physiologicalconditions. ZD6474 has unique potential to combat breast canceramong the recently discovered various anticancer drugs. ZD6474 isa novel heteroaromatic-substituted anilinoquinazoline (Fig. S1),

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Biosensors and Bioelectronics

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bios.2013.11.060

n Corresponding author. Tel.: þ91 322 228 1228; fax: þ91 322 228 2221.E-mail address: rangadhar@iitkgp.ac.in (R. Pradhan).

Biosensors and Bioelectronics 55 (2014) 44–50

which acts as a potent and reversible inhibitor of Adenosine TriPhosphate (ATP) (Hennequin et al., 2002). The inhibitory effect ofZD6474 on epidermal growth factor receptor (EGFR) tyrosinekinase has been reported earlier (Ciardiello and Tortora, 2001).ZD6474 inhibits two key pathways in tumour growth process. Thefirst pathway targets indirectly by reducing vascular endothelialgrowth factor (VEGF)-dependent tumour angiogenesis and VEGF-dependent endothelial cell survival (Gille et al., 1997; Goldmanet al., 1993; Perrotte et al., 1999; Salomon et al., 1995). The secondpathway targets via inhibition of EGFR-dependent tumour cellproliferation and survival (Weber et al., 2003). However theimpedimetric monitoring of cytotoxicty of ZD6474 has not beencarried out till date.

In this paper, experiment has been carried out to understandthe cellular behaviours of MCF-7 subjected to various drug dosesof ZD6474 using fabricated ECIS devices. Among several breastcancer cell lines, MCF-7 cells (malignant and invasive) are widelyused in experimental therapeutics as the cell line maintains idealcharacteristics of mammary epithelium (Levenson and Jordan,1997). The frequency response of electrical impedance of ZD6474treated MCF-7 cells at 24 h has been investigated and comparedwith the results of standard techniques like MTT assay, flowcytometry, phase contrast and fluorescence imaging. In thisexperiment, additionally MCF-10A cells are used as negativecontrol for an effective comparisoni of impedance data obtainedfor drug treated MCF-7 cells. MCF-10A cell is a non canceroushuman breast cell line frequently used in scientific research fortheir high capacity to differentiate and proliferate in vitro. Effortshave also been given for quantitative estimation of the effect ofdrug dose on the cells using impedance data.

2. Materials and methods

2.1. Materials and reagents

Pyrex glass wafers were purchased from Semiconductor waferInc., Taiwan. Polydimethylsiloxane (PDMS, Sylgard 184) was sup-plied by Dow Corning, Inc., Midland. SU8 was purchased fromMicroChem, Newton. RPMI 1640 medium, foetal bovine serum,trypsin/EDTA solution, penicillin, and streptomycin were purchasedfrom Himedia, India. Propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen, India. Allother required reagents were supplied by Sigma-Aldrich, India.

2.2. Cell culture

MCF-7 and MCF-10A were purchased from the American TypeCulture Collection (ATCC, Manassas, VA). The cells were culturedon electrode surfaces by using RPMI 1640 containing 10% foetalbovine serum (FBS), 1% penicillin, and 100 mg/mL streptomycin at37 1C in a humidified atmosphere of 5% CO2. For seeding purposes,cells were harvested by trypsinizing the cell with 0.05% trypsin/EDTA and the cell suspensions were prepared by using standardtissue culture techniques.

2.3. Design and fabrication of ECIS devices

ECIS devices were designed by using the design rule extractedfrom previous literatures. Mishra et al. (2005) designed thedevices by keeping the working electrode (WE) area smaller thanthe reference electrode (RE) to minimize the influence of theimpedance of the electrolyte in active region and to detect anysmall changes happening in the sensing electrode. Brett and Brett(1993) have shown that the surface area ratio of counter electrode(CE) and WE should be larger than 10 in order to support the

current generated at the working electrode. In the present paper,the ratio of WE/RE and WE/CE were fixed to 0.01 to restrict higherdevice area. The CE and RE were placed at a distance of 100 mmfrom WE position in all the designs to avoid cross contamination(Breckenridge et al., 1995; Lind et al., 1991). From the activeelectrode region, each electrode was connected to a large contactpad by intermediary connection (IC) having 250 mm width and25 mm in length. The coating layer was provided by using SU8polymer of thickness 50 mm to eliminate the artifacts in impe-dance measurement provided by IC (Pradhan et al., 2013). In thispaper, four different configurations of ECIS devices were designedwith varying dimensions of WE, RE, and CE as provided in Table 1.

The realization of impedance sensing device by microfabrica-tion technique was described in authors’ previous paper (Pradhanet al., 2012) and is presented with schematic of the process flow asshown in Fig. S2. The biosensor was fabricated on Pyrex wafersusing thin film deposition and photolithography techniques.Initially, the wafers were cleaned and thin layers of chromium(Cr) and gold (Au) were deposited by thermal evaporation tech-nique. Subsequently, the electrode patterns and its contact padswere lithographically defined on the deposited metal film. Next,another photosensitive polymer (SU-8) layer was spin coated andlithographically patterned to obtain polymer passivation coatingover the metal electrodes. The patterning of SU-8 layer wasperformed in such a way that the active electrode areas andcontact pads were kept exposed to culture cells and to applyelectrical signals respectively. The individual device was diced andfixed on a PCB board following the attachment of a cloningcylinder around the three electrode system to serve as electrolytereservoir for tissue culture. The entire assembly of the ECIS devicesand a enlarge view of the three electrode regions of the fabricateddevice is shown in Fig. 1.

2.4. Experimental procedures

Prior to the cell seeding, the ECIS device was sterilized with 75%ethanol for 15 min, dried with nitrogen, and irradiated withultraviolet radiation for 15 min. RPMI 1640 (1 mL) was then addedto the ECIS device and incubated at 37 1C for 20 min to record thebackground impedance value (ZNo cell). Next, the cell suspension ofMCF-7 and MCF-10A (1 mL, 1�106 cells) was added into each cellculture chamber. After a 10 min equilibration, the device wasplaced into the incubator for cell culture and impedance sensing.Then different doses of ZD6474 (0, 5, 10 and 15 mM) were addedinto the well of ECIS devices after 30 min of inoculation to studythe cytotoxic effects of the drug.

2.5. Impedance measurement

The frequency responses of cytotoxic effects were studied bymeasuring the impedance of MCF-7 cells treated in different dosesof ZD6474 within frequency range from 100 Hz to 1 MHz with 51

Table 1Dimensions of different design of three electrode based ECIS devices.

Devices WE area(mm2)

RE andCE area (mm2)

IC width(mm)

IC length(mm)

WE/RE andWE/CE

SU8thickness(mm)

Design 1 0.05�0.05 0.5�0.5 0.25 25 0.01 0.05Design 2 0.1�0.1 1�1 0.25 25 0.01 0.05Design 3 0.15�0.15 1.5�1.5 0.25 25 0.01 0.05Design 4 0.2�0.2 2�2 0.25 25 0.01 0.05

R. Pradhan et al. / Biosensors and Bioelectronics 55 (2014) 44–50 45

points in a logarithmic scale after 24 h of drug treatment.In frequency response experiment, MCF-10A cells without drugtreatment were taken as negative control to compare the impe-dance data of drug treated samples after 24 h. All the measure-ments were repeated for ten times and then averaged to get theimpedance value for each frequency. The impedance spectroscopydata found from experiment were fitted by using the equivalentcircuit, extracted from the previous literature (Pradhan et al., 2013)and was described in Fig. S3. The sensitivity of the biosensor wascalculated by using the following Eq. (1), extracted from theprevious literature (Wang et al., 2008).

Sensitivityðf Þ ¼ ðjZCell ðf Þj�jZNo cellðf ÞjÞQ �1cell ð1Þ

where f is the sensing frequency and Qcell is the maximum celldensity (about 106 cells cm�2).

2.6. Morphological studies and confocal microscopy

MCF-7 cells were plated on coverslips in RPMI 1640 completemedium. The cells were treated with varying concentrations(0, 5, 10, 15 μM) of ZD6474 for 24 h. Phase contrast images of theMCF-7 cells were taken for the drug treated cells after 24 h ofincubation by using Carl Zeiss Observer Z1 attached with a CCDcamera using monochromatic phase contrast mode. For flores-cence imaging, the cells were fixed using 3.7% paraformaldehyde,permeabilized with 0.1% Triton X-100, and stained with DAPI asper manufacturer’s instructions. Cells were analysed by confocallaser scanning microscopy (Olympus FluoView FV1000, Version1.7.1.0, TYO, Japan) using the appropriate wavelength.

2.7. Flow cytometric analysis

Cells were treated with ZD6474 for 24 h at varying concentra-tions (0, 5, 10 and 15 μM) as described in the previous literature(Rajput et al., 2013). After treatment, cells were collected, washedand incubated in 70% ethanol, kept at �20 1C overnight forfixation. Cells were centrifuged, washed, and then incubated withPI solution (40 mg/mL PI, 100 mg/mL RNase A in PBS) at 37 1C for1 h. The distribution of cells in the different cell-cycle phases wasanalysed from the DNA histogram using Becton-Dickinson FACSCalibur and Cell Quest software, CA, USA.

2.8. MTT assay and cell viability count

MCF-7 cells (1�104 cells/well) were seeded in 96-well tissueculture plates and allowed to grow for 24 h. ZD6474 at varyingconcentrations (10 nM–50 μM) was added to the different wellsafter 30 min of inoculation. Cell viability was measured by MTTdye reduction assay as described previously (Sarkar et al., 2010).The viability of cells after 24 h of drug treatment was countedusing TC 20 automated cell counter (Bio-RAD). The experimentswere carried in triplicate and the corresponding data wererepresented with the standard deviations.

2.9. Correlation between drug doses and impedance

A quantitative relationship between drug doses, electrode area,and impedance was required to design complex electrode geome-tries of ECIS devices to study the cellular behaviours on electrodesurfaces. In this paper, the magnitude of impedance and phaseangle data were plotted with the independent variables such asdrug doses and working electrode area to establish an empiricalrelationship by using LAB Fit curve fitting software.

3. Results and discussions

3.1. Evaluation of cytotoxic effects of ZD6474

3.1.1. Evaluation of cytotoxicty by ECIS methodsThe different doses of ZD6474 are added with cells after 30 min

inoculation of cultures on fabricated devices. The bode plots forthe cytotoxic effects of ZD6474 on MCF-7 cells after 24 h arepresented in Fig. 2 for Design 1 and Fig. S4 for Design 2, 3, and 4.The experimental data are fitted perfectly with the used equiva-lent circuit as described in Fig. S3. The results illustrate that themagnitude of the impedance value is inversely proportional to theWE area as its magnitude is highest for all the samples measuredin ECIS device for Design 1 compared to other designs as observedfrom Figs. 2 and S4. From the figures, it is evident that magnitudeof impedance decreases gradually with increase of frequency up to1 MHz and phase angle value decreases gradually up to 1 kHz.The phase angle forms a plateau in the frequency range of 1–10 kHz and gradually increases from 10 kHz to 1 MHz. It is foundthat the impedance of normal MCF-10A cells (negative control) isgreater than cancerous MCF-7 cells (control) within the frequency

Design 3 Design 2 Electrical connectingwires

Design 1 Design 4 Pyrex wafer

WE

CE

RE

Fig. 1. Photograph of fabricated ECIS devices and an enlarge view of three electrode region.

R. Pradhan et al. / Biosensors and Bioelectronics 55 (2014) 44–5046

range of 100 Hz–1 MHz. Thus this impedimetric discriminationmay be used to differentiate normal cells from cancer cells withfurther refinement. The difference between the impedance spectraof control and 5 mM drug treated sample for MCF-7 cells isnegligible. However, major differences in impedance spectra arefound for 10 and 15 mM drug treated samples as compared withcontrol. Similar experimental observation has also been reported foraction of cisplatin on MCF-7 cells (Alborzinia et al., 2011). Initialvariation of slope observed at low frequency for higher amount ofZD6474 treated samples may be attributed for enhanced interactionof drug at cellular level followed by altered cell–substrate contactwhereas impedance saturation at high frequency is anticipated forfixed capacitance from polymer coating layer (Rahman et al., 2009).The relative standard deviations (RSD) for treated and untreatedsamples for different designs are found to be below 10% which infersthe reproducibility nature of the fabricated ECIS devices.

The frequency dependent sensitivity of ECIS devices are calcu-lated using Eq. (1) and the characteristic plot of control data isshown in Fig. S5 for various electrode dimensions. Fig. S5 depictsthat the sensitivity for Design 1 is highest followed by otherdesigns which implies that the sensitivity increases with thedecrease of working electrode area. This is due to enhanced interac-tion of cells at electrode area with reduced electrolyte–electrodeinterface (Wang et al., 2008). The detection limit of the ECIS device iscalculated by culturing different cell concentrations (ranging from101 to 106 cells/mL) and the impedance is measured at 24 h.A distinct difference in impedance spectra is noticed between 102

and 103 cells/mL cell concentrations as shown in Fig. S6. Themicroscopic observations reveal that the spreading of cells onelectrode increases with the increase of cell concentrations and theactive electrode is fully covered by cells for all the designs at 106 cells/mL. To determine the exact detection limit of ECIS devices, a specificexperiment is carried out by measuring the impedance of culturedcells by varying the cell concentration between 102 and 103 cells/mL.Finally from the experiment the exact value of detection limit of ECISdevices is determined to be 6�102 cells/mL.

3.1.2. Evaluation of cytotoxicty by conventional methodsZD6474 restrains cell proliferation of MCF-7 cells in a dose

dependent manner and the cell inhibition is observed betweendrug concentrations of 1–15 mΜ by MTT assay. The IC50 value ofZD6474 in MCF-7 obtained from MTT assay is 10.6570.6325 mΜ.The percentage of living cells present for negative control, control,5 mM, 10 mM and 15 mM treated samples are 9673, 9772, 8574,

6273, 4575 respectively as measured from cell viability count.The cell death due to drug treatment is clearly demonstrated withhelp of phase contrast images as shown in Fig. 3.

The flow cytometric analysis is carried out and the treated cellsundergoing apoptosis are observed at the subG1 phase after 24 h ofincubation. There is approximately 45% and 60% relative increase insubG1 phase in cells treated with 10 and 15 μM of ZD6474 respec-tively as compared with control value. This increase in subG1 phaseis dose dependent for 24 h as shown in Fig. 4. Interestingly, ZD6474at high concentrations induce cells accumulation at subG1 phasecontributing to apoptosis during 24 h treatment.

To define the contributory roles of apoptosis and nuclearmorphological changes, nucleus staining with fluorescent dye (DAPI)is performed. The assay confirms the increased percentage ofapoptotic cells with increasing dose of ZD6474 in comparison tocontrol as shown in Fig. 5.

3.1.3. Comparison of ECIS methods with conventional methodsIt is evident from the work that the cell death is progressively

higher with the increase of ZD6474 concentration. The differencein impedance value of control and 5 mM treated sample isnegligible due to low cytotoxic effect of drug as the IC50 value ofZD6474 for MCF-7 cell is nearly 10 mM. The cytotoxic effect is muchprominent in higher doses of drug which is attributed for the dosedependent suppression of cell proliferation and induction ofapoptosis of MCF-7 cells. Previous studies reveal that ZD6474suppresses cellular proliferation of breast cancer cells (Ciardielloet al., 2003; Ryan and Wedge, 2005). In addition, it inducesapoptosis and restrains anchorage-independent colony formationand chemoinvasion in vitro (Sarkar et al., 2010). The presentexperiment demonstrates that the cell death occurs on electrodesurfaces at higher drug doses. Microscopic inspection reveals thatthe dead cells detach from the intracellular matrix near the planarelectrode surface. However the dead cell colony detached from theelectrode surfaces remains inside the medium which incorporatesnoise in impedance data. Considering this phenomena it is expectedthat less number of cells contribute to the current transport processin the impedance measurement leading to the reduction of impe-dance with the increase of drug dose. Thus the mechanism of thesensor depends on the cells cultured on the electrode surfaces andthe detached cells from the electrode surface due to drug treatment.

3.2. Correlation between drug doses and impedance

In this paper, the correlation between impedance, drug dosesand electrode area is calculated to analyse the effect of drug doseon cellular behaviour and its impedance characteristics. Themeasured impedance and phase angle data for various drug doseand electrode area are plotted using curve fitting software to getthe dependence curve as shown in Fig. S7. The empirical relationbetween magnitude of impedance (Z), drug doses (D), and workingelectrode area (A) is expressed as

Z ¼ 0:2221� 106 exp ð�0:2658� 10�4 � A�0:6327� 10�2 � D2Þ ð2Þ

The relation between phase angle (θ), drug doses, and activeelectrode area is described in

θ¼ 0:5032� Dþ0:1108� 10�3 � Aþ0:6448� 102 ð3Þ

The fitting Eq. (2) implies that the cell impedance magnitudeexponentially decreases with the increase of electrode area anddrug dose. However, phase angle depends linearly on these twoparameters as observed from Eq. (3) and thus have less impact onimpedance characterization. Compared to the electrode area, the

Fig. 2. Bode plot for effects of ZD6474 on MCF-7 cancer cells for Design 1.

R. Pradhan et al. / Biosensors and Bioelectronics 55 (2014) 44–50 47

effect of drug dose is more prominent on impedance variationbecause it varies as power of square.

The inhibitory plot of ZD6474 for MCF-7 cells is shown in termsof magnitude and phase angle of impedance with respect to drug

dose keeping frequency and working electrode area constant usingcurve fitting software (Fig. S8). The statistically averaged lineobtained in this plot shows a negative slope in case of magnitudeof impedance and a positive slope in negative directions for phase

Fig. 4. Representative histogram plot of MCF-7 breast cancer cells treated with ZD6474 at varying concentration (0, 5, 10, 15 μM) showing distribution in the different phasesof the cell cycle after 24 h treatment, determined by flow cytometry.

Fig. 3. Photomicrograph of MCF-7 cells treated with 0, 5, 10 and 15 μM of ZD6474 for 24 h showing the apoptotic effect of ZD6474 in dose dependent manner.

R. Pradhan et al. / Biosensors and Bioelectronics 55 (2014) 44–5048

angle value which implies inhibitory action of drug dose on thecells. Thus a negative correlation is found between the drug dosesand impedance.

4. Conclusions

This paper provides results about the cytotoxic effects ofZD6474 on MCF-7 cells in microfabricated devices using ECIStechnique. The frequency dependent impedance data reveal thatthe cells treated above 10 mM drug dose show predominantreduction of its impedance magnitude as compared to the control.The decrease in magnitude of impedance value with the increaseof drug doses demonstrates the dose dependent cytotoxic effect ofZD6474. Sensitivity of the devices decreases with the increase ofelectrode dimensions as interaction of electrolyte is more in case ofhigher electrode area. The impedimetric cytotoxic effect of ZD6474correlates well with the conventional methods like MTT assay, flowcytometry data, phase contrast and florescence images. Thus it isself-evident that this ECIS technique can be applied in similarstudies to measure the impact of any drug in cell culture models.

Acknowledgement

Authors would like to thank the staff members of MEMS Lab,IIT Kharagpur for microfabrication support and Indian SpaceResearch Organisation for financial support to carry out thepresent work.

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2013.11.060.

Fig. 5. Fluorescent micrographs of DAPI stained cells. The arrow indicates the nuclear blebbing in apoptotic cells. Each individual experiment has been repeated three times.

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