1476 IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014

Assessing Cytotoxic Effect of ZD6474 onMDA-MB-468 Cells Using Cell-Based Sensor

Rangadhar Pradhan, Mahitosh Mandal, Analava Mitra, and Soumen Das

Abstract— This paper presents the application of animpedance-based measurement of cytotoxicity of ZD6474 onMDA-MB-468 breast cancer cells in culture. In the present exper-iment, four types of cell-based sensors with different electrodegeometries are fabricated by using microfabrication technology.MDA-MB-468 cells are grown on the electrode surfaces to studycell adhesion, spreading, and apoptosis. The real time impedancemonitoring data show that the cells took <5 h to complete thespreading process on electrode surfaces. The cytotoxic effectof ZD6474 is dose dependant and with the increases of drugdoses, the impedance value decreases, which correlates thatZD6474 blocks cell proliferation and induces apoptosis in breastcancer cells. A quantitative relationship, developed between theimpedance and drug doses, establishes a negative correlationbetween the drug doses and impedance. General consistency hasbeen found between the impedance response and the biochemicalassay. Thus, impedance sensing method provides a simple andcost effective method to study chemical cytotoxicity in vitro.

Index Terms— Cell-based sensor, MDA-MB-468, ZD6474,bioimpedance, cytotoxicity.

I. INTRODUCTION

IN VITRO cytotoxicity assays are used for several decadesto study the effects of certain chemicals on cells in culture.

These cell-based assays play major alternative methods toanimal testing for cytotoxicity assessment of several chemicalsand drugs and help to speed up the process of new drugdiscovery. The cell motility after exposure to a drug wasstudied by several conventional in vitro cell-based assayssuch as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide test (MTT assay), neutral red uptake, colony formingefficiency or phase contrast imaging [1]. However, most ofthese assays detect only specific cellular changes and do notgive information about cell-drug interaction. Moreover, theseassays are time and labor consuming, and require complexsteps. Therefore, at this stage of advanced cell and molecularbiology research we need label-free detection methods forcell-based assays. Monitoring the bioimpedance properties ofcells using cell based sensor is one of the non-labeling tech-niques used to understand the cell functionality on electrodesurfaces. The cell based sensor is also being explored for

Manuscript received August 6, 2013; accepted December 17, 2013. Dateof publication January 2, 2014; date of current version March 18, 2014. Theassociate editor coordinating the review of this paper and approving it forpublication was Prof. Gotan H. Jain.

The authors are with the School of Medical Science and Technol-ogy, Indian Institute of Technology Kharagpur, Kharagpur 721302, India(e-mail: rangadhar@iitkgp.ac.in; mahitosh@smst.iitkgp.ernet.in; amitra@smst.iitkgp.ernet.in; sou@smst.iitkgp.ernet.in).

Color versions of one or more of the figures in this paper are availableonline at http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/JSEN.2013.2296717

continuous, automatic, and real time monitoring of cell adhe-sion and spreading as well as cell motility induced by drug.Cell based sensors have been successfully used to study thecytotoxicity of several drugs [1]–[8]. In this paper, electricalproperties of cells are studied in the presence of anticancerdrug to understand the changes in different physiologicalconditions. ZD6474 has more potential to combat breast canceramong the recently discovered anticancer drugs. ZD6474 isa novel heteroaromatic-substituted anilinoquinazoline, whichacts as a reversible inhibitor of Adenosine tri phosphate(ATP) [9]. The inhibitory effect of ZD6474 on epidermalgrowth factor receptor (EGFR) tyrosine kinase was alreadyreported [10]. ZD6474 inhibits two key pathways in tumourgrowth by inhibiting VEGF-dependent tumour angiogenesisalong with VEGF-dependent endothelial cell survival [11]–[14], and also by inhibiting EGFR-dependent tumour cell pro-liferation [15]. However the impedimetric real time monitoringof cytotoxicty of ZD6474 on MDA-MB-468 has not beencarried out till date. The purpose of this study is to under-stand cell responses subjected to various drug doses usingminiature cell based sensors. This study provides results aboutreal time monitoring of cell adhesion, proliferation, and cellmortality of MDA-MB-468 cells cultured in microfabricatedsensors using electric cell-substrate impedance sensing (ECIS)technique. Subsequently the frequency response of electricalimpedance of ZD6474 drug treated MDA-MB-468 cells isinvestigated and the results are correlated with the results ofconventional technique like MTT assay. Further, quantitativeestimation of the effect of drug dose on the cells is alsoassessed using impedance data.

II. MATERIALS AND METHODS

A. Design Rule for Cell Based Sensors

In the present work, planar three-electrode based deviceswith reference electrode (RE), working electrode (WE) andcounter electrode (CE) are used to measure the bioimpedanceof cultured MDA-MB-468 cells. The design rule involvessmaller WE area than RE area [16] to avoid electrolyticimpedance in active region. Since the surface area ratio ofCE to WE should be larger than 10 to support the currentgenerated at the WE [17]. In the present experiment, the ratioof WE/RE and WE/CE were fixed to 0.01. The CE and REwere placed at a distance of 100 μm from WE position inall the designs [18], [19] to avoid cross contamination. Theconnectors between sensing region and bond pad were coveredby a passivated coating layer using SU8 polymer of thickness

1530-437X © 2014 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission.See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.

PRADHAN et al.: ASSESSING CYTOTOXIC EFFECT OF ZD6474 1477

Fig. 1. Photograph of cell based sensors and an enlarged view of activeregions.

50 μm to remove the noises in the measurement of impedance.The present study employs four different configurations of cellbased sensors that were designed with varying dimensions ofWE, RE, and CE as described in our earlier publication [20].

B. Fabrication of Cell Based Sensors

The cell based sensors were fabricated by microfabricationtechnique as described in our earlier work [20]. The sen-sors were fabricated on 2 inch diameter Pyrex wafers usingmicrofabrication technology. Initially, the wafers were cleanedand thin layers of chromium (Cr) and gold (Au) were depositedon wafers by thermal evaporation technique. Subsequently,the sensor regions and its bond pads were lithographicallydefined using positive photoresist and the Cr and Au layersat unwanted regions were selectively removed by wet etchingprocess to define the metal patterns on the substrate. Finally,a second lithography step was performed by applying SU8layer as passivation coating over the connectors. The waferswere then diced into single devices for subsequent packagingand measurements. The individual device was fixed on a PCBboard and electrical connections were taken from device toexternal equipment using thin metal wires. Finally cloningcylinders were aligned and attached to serve as electrolytereservoirs around the active regions by using PDMS as shownin Fig. 1.

C. Culture of MDA-MB-468 Cells

MDA-MB-468 cells were cultured on electrode surfacesby using DMEM containing 10% fetal bovine serum (FBS),1% penicillin, and 100 μg/ml streptomycin at 37° C in ahumidified atmosphere of 5% CO2. Different doses of ZD6474(0, 5, 10 and 15 μM) were added into the well of cell basedsensor after 30 min of inoculation to study the cytotoxic effectsof drug.

D. MTT Assay and Cell Viability Count

MTT assay of MDA-MB-468 cells and its drug treatedsamples was carried out in quadruplicate 96-well tissue cultureplates at an optimized concentration of 104cells/ml in themedium. After 24 h of treatment with ZD6474 of various

Fig. 2. Equivalent circuit of cell based sensors.

concentrations ranging from10 nM to 50 μM along with 0.1%DMSO as control, cell viability was measured at 540 nm usinga micro-plate spectrophotometer (Bio-RAD). The percentageof cell viability was counted using TC 20 automated cellcounter (Bio-RAD).

E. Measurement of Impedance of MDA-MB-468 Cells

The real time impedance at a particular frequency andfrequency dependent impedance of MDA-MB-468 cells andits drug treated samples were measured by using computercontrolled electrochemical work station SP 150 (Bio-Logic,France). All the measurements were performed with a constantvoltage of 10 mV. The real time monitoring of cell adhesionand growth of MDA-MB-468 without drug treatment wasperformed by measuring the impedance at a fixed frequencyof 10 kHz in a time lapse of 10 minute for 7 hours durationafter seeding the cells in the well of cell based sensors. In thiscase normalised impedance (NI) value has been calculated byusing Eq. (1) to eliminate the electrolytic effects.

N I = (ZCell − ZNo cell ) Z −1No cell (1)

Where, ZCell and ZNo cell are the impedance of the system withand without the cells in culture media of same volume.

The frequency responses of cytotoxic effects were studiedby measuring the impedance of MDA-MB-468 cells treatedin different doses of ZD6474 within frequency range from100 Hz to 1 MHz with 51 points in a logarithmic scale after24 h of drug treatment. All the measurements were repeatedfor ten times and the mean value was considered to get finalimpedance for data analysis.

F. Equivalent Circuit Fittings

The impedance spectroscopy data found from experimentwas imported to ZsimpWin (Version 3.10) to fit the impedancedata. The equivalent circuit was extracted from the previousliterature [21] and was described in Fig. 2. In the used circuitRS represents the extracellular bulk resistance offered bythe conductivity of bulk solution and the wire connection.Qdl represents the dielectric property of electrode/electrolyteinterface including the coating capacitance. RI represents theresistance due to charge transfer in medium while QC andRC are the capacitance and resistance of cultured cells onelectrodes.

G. Sensitivity of Cell Based Sensors

The sensitivity of the cell based sensor was calculated byusing the following equation [22].

Sensi tivi ty( f ) = (|ZCell ( f )| − |Z No cell ( f )|) Q−1cell (2)

1478 IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014

Fig. 3. Monitoring of adhesion and spreading of MDA-MB-468 cells byimpedimetric method.

Where f is sensing frequency and Qcell is the maximumcell density (about 106 cells cm−2).

H. Quantitative Estimation by Correlation

A correlation experiment was carried out to understand therole of ZD6474 on impedance variation. In this attempt, themagnitude of impedance and phase angle data were plottedwith the independent variables such as drug doses and workingelectrode area to establish an empirical relationship by usingLAB Fit curve fitting software.

III. RESULTS AND DISCUSSION

A. Real-Time Monitoring of the Attachment and Spreadingof MDA-MB-468 Cells

Fig. 3 represents the real time monitoring of cell adhesionand spreading of MDA-MB-468 cells.

During first one hour, the NI value increases slowly whichindicates the attachment of cells on electrode surfaces of cellbased sensor. In the next 1–5 h, the NI value increases rapidlywhich infers the spreading stage. After spreading, cells start toproliferate and the impedance value remains almost constantfor subsequent measurement up to 7 h time span for eachcase. The general trend of NI plot with time is same for allthe electrode designs; however the Design 1 having lowestworking electrode area shows highest NI value. Wang et al.shows that impedance of cell covered surface is inverselyproportional to the electrode area [22] because of blockingof current path at the electrode surface as observed in thepresent experiment. Similar experimental observation has alsobeen reported in previous literatures found [23], [24].

B. Evaluation of Cytotoxic Effects of ZD6474 on Cancer Cells

The different doses of ZD6474 are added with cells after30 min inoculation of cultures on fabricated devices. The drugsstart working on cells at 22±0.25 h and the correspondingimpedance decreases. The bode plots for the cytotoxic effectsof ZD6474 on MDA-MB-468 cells after 24 h are presented

Fig. 4. Bode plot for effects of ZD6474 on MDA-MB-468 cancer cells forDesign 1.

in Fig. 4 for Design 1 and all other designs follow the sametrend as shown in Fig. 4. The experimental data are fittedperfectly with the used equivalent circuit as described in Fig. 2.The results illustrate that the impedance value is inverselyproportional to the WE area as its magnitude is highest forall the samples measured in cell based sensor for Design 1compared to other designs. From the figures, it is evident thatmagnitude of impedance decreases gradually with increase offrequency. The phase angle value decreases up to 1 KHz, formsa plateau in the frequency range of 1 to 9 kHz, and thenincreases up to 1 MHz. There is a major difference found inthe impedance spectra of control and 5 μm drug treated samplefor MDA-MB-468 cells. This result can be compared to theprevious findings which describe the impedimetric action ofZD6474 on breast cancer cells [23]–[25]. In the low frequencyrange the variation of slop is due to the drug-cell interactionaltering cell-substrate contact whereas impedance saturationat high frequency is attributed to coating capacitance of SU8[26]. The relative standard deviations (RSD) for treated anduntreated samples for different designs are varied below 10%which infers the reproducibility nature of the fabricated cellbased sensors.

ZD6474 inhibits cell proliferation of MDA-MB-468 cells ina dose dependent manner and the cell inhibition is observedbetween drug concentrations of 2–15 μM by MTT assay. TheIC50 value of ZD6474 in MDA-MB-468 obtained from MTTassay is 7.75 ± 0.457 μM. The percentage of living cellspresent for control, 5 μM, 10 μM and 15 μM treated drugare 96±3, 67±4, 47±3, 31±2 respectively as measured fromcell viability count.

It is evident from the figure that the cell death isprogressively higher with the increase of ZD6474 drug doses.As the IC50 value of ZD6474 for MDA-MB-468 cell is nearly8 μM, the effect of higher doses is much prominent asobserved in impedance measurement. This is due to the dosedependant suppression of in vitro cell proliferation, migration,and induction of apoptosis in MDA-MB-468 cells [27]. Thepresent study demonstrates that the apoptotic effects of drugZD6474 may be estimated by monitoring the impedance ofthe cells using ECIS technique.

PRADHAN et al.: ASSESSING CYTOTOXIC EFFECT OF ZD6474 1479

Fig. 5. The sensitivity of the cell based sensors for different designs.The RSDs for different designs remain below 10%.

The frequency dependent sensitivity of sensors is calculatedusing equation 2 and a characteristic plot is shown in Fig. 5for various electrode dimensions. From the figure it is evidentthat the calculated sensitivity for Design 1 is highest followedby other designs which implies that sensitivity is inverselyproportional to the area of electrode which may be associatedwith electrolytic solution, coating capacitance, etc. as reportedin the previous literature [22]. However, mid frequency rangeis suitable for cell substrate impedance sensing to achievehighest sensitivity and can be useful for monitoring the variouscell apoptosis process.

C. Correlation Between Drug Doses and Impedance

The correlation between impedance variation with drug doseand electrode area is established to analyze the effect of drugdose on cellular behavior and its impedance characteristics.The measured impedance and phase angle data for variousdrug dose and electrode area are plotted using curve fittingsoftware to get the dependence curve as shown in Fig. 6.The empirical relation between magnitude of impedance (Z),drug doses (D), and working electrode area (A) as obtained isexpressed in Eq. (3).

Z = 0.7705 × 105 × exp(−0.2857 × 10−4

× A − 0.8383 × 10−2 × D2) (3)

Similarly the relation between phase angle (θ), drug doses,and active electrode area is described in Eq. (4).

θ = (−0.3950 × 106 + A)/(0.6403 × 104

+ 0.7232 × 102 × D). (4)

The fitting Eq. (3) implies that the cell impedancemagnitude exponentially varies as negative of both the func-tions electrode area and square of drug dose. However, phaseangle depends linearly on these two parameters as observedfrom Eq. (4) and thus have less impact on impedance char-acterization. From the equation it is evident that the effect of

Fig. 6. Dependence plot for drug doses in relation to (A) magnitude and(B) phase angle.

drug dose compared to the electrode area is more prominenton impedance variation.

The inhibitory plot of ZD6474 for MDA-MB-468 cellshas been established in the terms of magnitude and phaseangle of impedance with respect to drug dose keeping fre-quency and working electrode area constant using curve fittingsoftware as shown in Fig. 7. The mathematical relationbetween impedance and drug doses is expressed in Eq. (5)and Eq. (6).

Z = −0.2789 × 104 × D + 0.5033 × 105 (5)

θ = 0.5661 × D − 0.5860 × 102. (6)

The trend line obtained in this plot shows a negative slopein case of magnitude of impedance and a positive slopein negative directions for phase angle value which impliesinhibitory action of drug dose on the cells.

1480 IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014

Fig. 7. Inhibitory plot of ZD6474 in relation to (A) magnitude and (B) phaseangle.

IV. CONCLUSION

We have used different cell based sensors to follow theactivities of MDA-MB-468 cells in response to differentconcentrations of ZD6474. It is observed from the presentpaper that the peak magnitude and the position of the nor-malized impedance changes can be correlated to cell adhesionand spreading. The impedance data shows that the cytotoxiceffect of ZD6474 is dose dependant and in higher doses theimpedance values are drastically decreases as compared to thecontrol cells. The sensitivity of the devices decreases with theincrease of electrode dimensions; however it is in the optimumrange. Quantitative evaluation of cytotoxic effect throughcorrelation experiment shows a negative correlation betweendrug doses and impedance values. The results obtained byimpedance sensing methods prove that these sensors can beused to investigate various aspects of cellular responses totoxins in general.

ACKNOWLEDGMENT

The authors would like to thank the staff members of theMEMS Lab, IIT Kharagpur for their support towards thiswork. They also thank the Indian Space Research Organization(ISRO) for financial support to carry out the present work.

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Rangadhar Pradhan received the Ph.D. andM.Tech. degrees from IIT Kharagpur, in 2013 and2006, respectively. Presently, he is a Post-DoctoralFellow with IIT Kharagpur. He received the MHRDscholarship for pursuing the M.Tech. degree as wellas the Ph.D. degree. His research area includesdevelopment of biosensors and ECIS system.

Mahitosh Mandal received the Ph.D. degree fromJadavpur University, in 1994. He is presently serv-ing as an Associate Professor with the School ofMedical Science and Technology, IIT Kharagpur.His research interest includes cancer biology, signaltransduction, apoptosis, cell cycle, angiogenesis, anddrug delivery.

Analava Mitra received the M.B.B.S. and Ph.D.degrees, in 1980 and 2003, respectively, from IITKharagpur. He is presently serving as an AssociateProfessor with the School of Medical Science andTechnology, IIT Kharagpur. His research interestincludes herbal medicine, nutraceuticals, and drugdelivery.

Soumen Das received the Ph.D. degree in 1996from the Indian Institute of Technology Kharagpur.He joined IIT Khargpur, in 1996 and is an Asso-ciate Professor with the School of Medical Scienceand Technology, IIT Kharagpur. His research areasinclude biomedical and inertial MEMS transducers,BioMEMS, and microfluidic biochips for clinicaldiagnostics and medical electronics.