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Monitoring cellular activities of cancer cells using impedance sensing devices
Sensors and Actuators B 193 (2014) 478–483
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Monitoring cellular activities of cancer cells using impedance sensing devices Rangadhar Pradhan ∗ , Mahitosh Mandal, Analava Mitra, Soumen Das School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, India
a r t i c l e
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Article history: Received 3 September 2013 Received in revised form 30 November 2013 Accepted 3 December 2013 Available online 9 December 2013 Keywords: Microfabrication T47D ZD6474 Bioimpedance Cytotoxicity
a b s t r a c t The present work reports the impedimetric characterization of cellular activities of T47D cells treated with anticancer drug ZD6474 using impedance sensing devices. Four types of devices with different dimensions are fabricated by micromachining technology. Real time impedance monitoring data reveals spreading stage completes within 5 h. The frequency response characteristics of drug treated cells are studied to evaluate cytotoxic effect of ZD6474 along with the sensitivity variation for different designs. Compared to control data, a significant decrease in impedance data is observed for drug treated samples above 10 M dose due to predominant cell death and detachment from the electrode surfaces. The quantitative relation developed between cell impedance and drug dose indicates that the magnitude of cell impedance varies inversely with the drug dose. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Recent advancement in biomedical screening technologies with the help of sophisticated microfabrication and signal processing techniques has resulted in the development of several novel sensor products and new applications with more accurate, cost effective and reliable approach as compared to their conventional counterparts. Until recently, most biosensors studied for biomolecular detection are label dependant sensor that detects biochemical processes as optical or electrochemical signals. It is also anticipated through intensive research that mechanical, optical, and electrical properties of the cell are also modified during their biochemical changes at various stages of diseases progression [1,2]. Thus there is a possibility to open an alternative path for label free detection/monitoring of disease progression process by identifying and quantifying the non-biological parameters of cells which may carry the signature of the disease. Among various techniques, electric cell-substrate impedance sensing (ECIS) technique is a rapid and inexpensive technique for determining electro-physiological characterization of cells [3,4]. The changes due to the alteration of cell properties during disease growth process can be detected well before by using electrical impedance technique. At present ECIS is an established, non-invasive electrochemical technique that has been successfully used to monitor cell adhesion, growth and
differentiation of cells [5–13], cell migration [14–17], morphological changes during apoptosis [18,19], and toxic effects of drugs on cellular activities [10,11,20–27]. In this paper, the electrical impedance of cells treated in various doses of anticancer drug ZD6474 is studied to correlate the electrical properties with their physiological conditions. ZD6474 is a heteroaromatic-substituted anilinoquinazoline (Fig. S1), which reduces tumour angiogenesis and endothelial cell survival [28–31]. It also inhibits tumour cell proliferation and survival [32]. However, the impedimetric monitoring of cytotoxicity of ZD6474 on T47D cells has not been carried out till date. The purpose of the present study is to understand the cell responses subjected to various drug doses using ECIS devices. This study is designed to provide results about real time monitoring of cell adhesion and proliferation of T47D cells in microfabricated devices using ECIS technique. Subsequently, the frequency response of electrical impedance of ZD6474 drug treated T47D cells is investigated and correlated with the standard technique like 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Also a correlation experiment is carried out to estimate the quantitative effect of drug doses on cellular activities of cancer cells. 2. Materials and methods 2.1. Materials and reagents
∗ Corresponding author. Tel.: +91 3222281228; fax: +91 3222282221. E-mail address: [email protected] (R. Pradhan). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.003
Pyrex glass wafers used as substrates in the present experiment were purchased from Semiconductor wafer Inc., Taiwan.
R. Pradhan et al. / Sensors and Actuators B 193 (2014) 478–483
Polydimethylsiloxane (PDMS, Sylgard 184) was supplied by Dow Corning, Inc., Midland. SU8 was procured from MicroChem, Newton. RPMI 1640 medium, foetal bovine serum, trypsin/EDTA solution, penicillin, and streptomycin were purchased from Himedia, India. All other required reagents were supplied by Sigma–Aldrich, India. 2.2. Culture of T47D cells T47D was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured on electrode surfaces by using RPMI 1640 containing 10% foetal bovine serum (FBS), 1% penicillin, and 100 g/mL streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 . The cells were harvested by trypsinizing the cell with 0.05% trypsin/EDTA and the cell suspensions were prepared by using standard tissue culture techniques. 2.3. Impedance measurement of T47D cells The electrical impedances of normal and drug treated T47D cells were carried out using electrochemical work station SP 150 (BioLogic, France). All the measurements were performed with applied voltage of 10 mV. The real time monitoring of cell adhesion and growth of T47D without drug treatment was performed by measuring the impedance at a fixed frequency of 10 kHz in a time lapse of 10 min for 7 h duration after seeding the cells in ECIS devices. In this case normalized impedance (NI) value has been calculated by using Eq. (1) to eliminate the effect of medium. NI =
−1 (ZCell − ZNo cell )ZNo cell
(1)
where ZCell and ZNo cell are the impedances of the system with and without the cells in culture media of same volume. The impedance of T47D cells treated in different doses of ZD6474 was measured from 100 Hz to 1 MHz with 51 points in a logarithmic scale after 24 h of drug treatment. The final impedance value for each frequency was the mean value of 10 subsequent measurements. 2.4. Equivalent circuit fittings The impedance data found from experiment was fitted by using ZsimpWin (Version 3.10). The equivalent circuit used for fitting purposes was extracted from the previous literature [33] and described in Fig. 1. 2.5. Sensitivity of impedance sensing devices The sensitivity of the biosensor was calculated by using the following Eq. (2) which is extracted from the previous literature [34].
−1 Sensitivity(f ) = (ZCell (f ) − ZNo cell (f ))Qcell
(2)
where f is sensing frequency and Qcell is the maximum cell density (about 106 cells cm−2 ).
RS
Qdl
QC
RI
RC
Fig. 1. Equivalent circuit of impedance sensing devices (RS represents the extracellular bulk resistance, Qdl represents the dielectric property of electrode/electrolyte interface including the coating capacitance, RI represents charge transfer resistance while QC and RC are the capacitance and resistance of cancer cells).
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2.6. Experimental protocol The impedance sensing device was sterilized with 75% ethanol for 15 min, dried with nitrogen, and irradiated with ultraviolet radiation for 15 min. RPMI 1640 (1 mL) was then added to the device and incubated at 37 ◦ C for 20 min to record the background impedance value (ZNo cell ). Next, the cell suspension of T47D (1 mL, 1 × 106 cells) was added into each cloning cylinder and the device was placed into the incubator after 10 min for cell culture and impedance sensing. Then different doses of ZD6474 (0, 5, 10 and 15 M) were added into the well of impedance sensing devices after 30 min of cell inoculation to study the cytotoxic effects of the drug. 2.7. Comparison of impedance data with MTT assay Cell viability of T47D breast cancer cells were determined by MTT-dye reduction assay. The cell suspensions were dispensed in quadruplicate into 96-well tissue culture plates at an optimized concentration of 104 cells/well in complete medium. After 24 h of treatment with ZD6474 of various concentrations ranging from 1 M to 50 M along with 0.1% DMSO as control, cell viability was measured at 540 nm using a micro-plate spectrophotometer (Bio-RAD Benchmark Plus). The viability of cells after 24 h of drug treatment was counted using TC 20 automated cell counter (BioRAD). 2.8. Statistical data analysis All the experiments including impedimetric analysis and biochemical assay were carried in triplicate and the data were represented with their corresponding relative standard deviations (RSD). 2.9. Correlation experiment A quantitative relationship was developed to understand the effects of ZD6474 on cellular activities of T47D cells. The magnitude of impedance and phase angle data were correlated with drug doses and working electrode area by using LAB Fit curve fitting software. 3. Results and discussions 3.1. Design of impedance sensing devices In the present work, three-electrode based impedance sensing devices are used to measure the cellular activities on gold electrode surfaces. The design rules for electrode fabrications are extracted from previous literatures. Mishra et al. [35] has used smaller working electrode (WE) than reference electrode (RE) while Brett and Brett [36] have used smaller WE than counter electrode (CE). Considering the limitations of authors’ microfabrication facilities and also to restrict higher device area, the ratio of WE/RE and WE/CE is fixed to 0.01. The cross contamination is avoided by placing the CE and RE at a distance of 100 m from WE position in all the designs [37,38]. The sensor areas are connected to large contact pads by interconnections and the impedances produced by those interconnections are eliminated by providing a SU8 coating of thickness 50 m [33]. In the present study, four different configurations of impedance sensing devices are designed with varying dimensions of WE, RE, and CE as given in Table 1. 3.2. Fabrication of impedance sensing devices The realization of impedance sensing device by microfabrication technique is described in authors’ previous paper [39]. The
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Table 1 Dimensions of different design of impedance sensing devices. Devices
WE area (mm2 )
RE and CE area (mm2 )
Lead width (mm)
Lead length (mm)
WE/RE and WE/CE
SU8 thickness (mm)
Design 1 Design 2 Design 3 Design 4
0.05 × 0.05 0.1 × 0.1 0.15 × 0.15 0.2 × 0.2
0.5 × 0.5 1×1 1.5 × 1.5 2×2
0.25 0.25 0.25 0.25
25 25 25 25
0.01 0.01 0.01 0.01
0.05 0.05 0.05 0.05
Fig. 2. Photograph of impedance sensing devices and an enlarge view of sensor area.
devices are fabricated on Pyrex wafers using photolithography techniques. Initially, the wafers are cleaned and thin layers of chromium (Cr) and gold (Au) are deposited by thermal evaporation technique. Subsequently, the electrode patterns and its contact pads are lithographically defined on the deposited metal film. Next, another photosensitive polymer (SU-8) layer is spin coated and lithographically patterned to obtain SU8 coating over the metal interconnections. The devices are fixed on a PCB board and the cloning cylinders are attached around the sensors by using PDMS to serve as electrolyte reservoir for cell culture as shown in Fig. 2. 3.3. Real-time monitoring of the attachment and spreading of T47D cells Fig. 3 represents the real time monitoring of cell adhesion and spreading of T47D cells. From the figure, it is inferred that the NI value increases slowly during first one h and then increases rapidly from 2–5 h. the first phase represents the cell adhesion stage while the second phase signifies the spreading stage. After spreading, cells enter in to proliferation stage and the impedance value shows a
Fig. 3. Monitoring of adhesion and spreading of T47D cells by impedimetric method.
Fig. 4. Bode plot for effects of ZD6474 on T47D cancer cells for Design 1.
little fluctuation for subsequent measurement up to 7 h time span for each case. The electrode area has a little influence on NI variation during cell adhesion time as the current path is passed through the electrolyte solution. Then the NI value increases gradually due to attachment of cells on the electrode surface. However, the NI variation is mostly controlled by the cells covering the electrode surface region during spreading stage [40,41]. Previous study predicts that current path is blocked at the electrode surface due to the presence of cancer cells. Thus the impedance of cell covered surface is inversely proportional to the electrode area [34] and NI variation is more prominent in smaller electrode device. Similar experimental observation is also reported for HeLa and HaCaT cells [33]. 3.4. Evaluation of cytotoxic effects of ZD6474 on T47D cells The different doses of ZD6474 are added with cells after 30 min of cell inoculation in the fabricated devices. The impedance decreases at 19 ± 0.24 h due to cell death and detachment from the electrode surfaces. The bode plots for the cytotoxic effects of ZD6474 on T47D cells after 24 h are presented in Fig. 4 for Design 1 and Fig. S2 for Design 2, 3 and 4. The experimental data are fitted reasonably well with the used equivalent circuit as described in Fig. 1. The results illustrate that the magnitude of impedance is highest for Design 1 and gradually decreases with the increase of electrode dimensions. Thus the magnitude of impedance value is inversely proportional to the WE area as observed from Fig. 4 and Fig. S2 for devices with different electrode area. From the figures, it is also evident that magnitude of impedance decreases gradually with increase of frequency. However, phase angle value decreases up to 1 kHz and then forms a plateau in the frequency range of 1–10 kHz and then increases up to 1 MHz. This initial variation of slop at low frequency occurs due to increased drug-cell interactions resulting cell death and detachment from the electrode surfaces. The relative standard deviations (RSD) for treated and untreated samples for different designs are found to be below 10% which showed the reproducibility nature of the fabricated impedance sensing devices. Also the role of drugs on medium in impedance values is estimated by measuring the impedance of cell free medium
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comparable to previous studies on sensitivity analysis of the ECIS sensor in which the sensitivity decreases with the increase of electrode dimensions [34, 42]. 3.5. Correlation between drug doses and impedance Numerical relations between impedance, drug dose and, electrode area are established to analyze the effect of drug doses on cellular behaviour and its impedance characteristics. The measured impedance and phase angle data for various drug doses and electrode area are plotted using curve fitting software to get the dependence curve as shown in Fig. 7. The empirical relation between magnitude of impedance (Z), drug doses (D), and working electrode area (A) as obtained is expressed in Eq. (4). Z = 0.1479 × 107 × −0.9124D × A−0.3097 Fig. 5. Impedimetric cell viability calibration curve of ZD6474 for different designs.
with different doses of drugs and the plot is shown in Fig. S3. From the figure, it is evident that the impedance values for different drug doses vary negligibly within 0.005% error. Thus it is assumed that the impedance due to drug doses in solution has no role in decrease of cell impedance. The IC50 value of ZD6474 in T47D obtained from MTT assay is 8.65 ± 0.5246 M. The percentage of living cells present in control, 5 M, 10 M and 15 M treated drug are 96 ± 3, 75 ± 4, 53 ± 3, and 34 ± 5, respectively as measured from cell viability count. The cell viability (%) is calculated from the impedance data by using the following Eq. (3) and is represented in Fig. 5. Cell viability(%) =
ZT × 100 ZC
(4)
Similarly the relation between phase angle (), drug doses, and active electrode area is described in Eq. (5). =
(−0.4073×106 +A) (0.6589×104 +0.7175×102 ×D)
(5)
The inhibitory plot of ZD6474 for T47D cells is plotted in Fig. 8 in terms of magnitude and phase angle of impedance with respect to drug dose keeping frequency and working electrode area constant.
(3)
where ZC and ZT represent the mean value of impedance for control and treated sample at 24 h respectively. The IC50 value of ZD6474, calculated from the impedance data of Fig. 5 is 8.74 ± 0.3137. Thus it is evident that the IC50 value obtained by impedimetric method correlates well with the value obtained by MTT assay method. The frequency dependent sensitivity of ECIS devices are calculated using Eq. (2) and a characteristic plot using control data is shown in Fig. 6 for various electrode dimensions. The sensitivity for Design 1 is highest followed by other designs which implies that the sensitivity is inversely proportional to the area of electrode. This may be due to enhanced interaction of cells at electrode area reducing electrolyte contact with electrode surfaces. This study is
Fig. 6. The sensitivity of the impedance sensing devices for different designs.
Fig. 7. Dependence plot for drug doses in relation to (A) magnitude and (B) phase angle.
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Acknowledgements Authors would like to thank the staff members of MEMS Lab, IIT Kharagpur for microfabrication support and Indian Space Research Organization for financial support to carry out the present work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.12.003. References
Fig. 8. Inhibitory plot of ZD6474 in relation to (A) magnitude and (B) phase angle.
The mathematical relation between impedance and drug doses is expressed in Eqs. (6) and (7). Z = 0.8433 × 105 × 0.9124D
(6)
= 0.5503 × D − 0.5882 × 102
(7)
The fittings obtained in these plots show a negative slope in case of magnitude of impedance and a positive slope in negative directions for phase angle value which implies inhibitory action of drug dose on the cells. 4. Conclusions This study provides results about impedimetric monitoring of cellular behaviours of T47D cells by using impedance sensing devices of different electrode geometries. Experimental observations reveal that the peak variation in normalized impedance is related to cell adhesion and spreading. The frequency dependent impedance data reveal that the cells treated above 10 M drug dose shows prominent reduction of its impedance magnitude as compared to the control cells. This effect is due to dose dependant cell death and detachment of the cells from electrode surfaces. Sensitivity of the devices decreases with the increase of electrode dimensions but remains within the working range. Quantitative evaluation of cytotoxic effect of drug dose on cell behaviour is established through an empirical relation indicating cell impedance magnitude varies inversely with drug dose. Thus these devices may be used to study the cellular activities of different kind of cells on electrode surfaces.
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Biographies Rangadhar Pradhan obtained his M.Sc. degree in Zoology from Utkal University, Bhubaneswar in 2003 and, M.Tech. and Ph.D. degrees from IIT Kharagpur in 2006 and 2013, respectively. Presently, he is a postdoctoral fellow at IIT Kharagpur. Dr. Pradhan got MHRD scholarship for pursuing M.Tech as well as Ph.D. His research area includes development of Biosensors and ECIS system. Mahitosh Mandal received his M.Sc. degree in 1984 and Ph.D. degree in 1990 from University of Burdwan. He is presently serving as Associate professor at School of Medical Science and Technology, IIT Kharagpur. His research interest includes cancer diagnosis, development of diagnostic and prognostic cancer markers, and targeted drug delivery for cancer therapy. He has authored more than 74 research papers in international journals. Analava Mitra received his M.B.B.S. degree in 1980 and Ph.D. degree in 2003 from IIT Kharagpur. He is presently serving as Associate professor at School of Medical Science and Technology, IIT Kharagpur. His research interest includes herbal medicine, nutraceuticals and drug delivery. He has authored more than 80 research papers in international journals and conference proceedings. Soumen Das received his M.Sc. degree in 1988 and Ph.D. degree in 1996 from Indian Institute of Technology Kharagpur. He joined as Scientific Officer in 1996 at Microelectronics Laboratory and at present serving as Associate professor at School of Medical Science and Technology, IIT Kharagpur. His research areas include biomedical and inertial MEMS transducers, BioMEMS and microfluidic biochips for clinical diagnostics, medical electronics and VLSI unit processing. He has authored more than 40 research papers in reputed international journals and conference proceedings.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Monitoring cellular activities of cancer cells using impedance sensing devices Rangadhar Pradhan ∗ , Mahitosh Mandal, Analava Mitra, Soumen Das School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, India
a r t i c l e
i n f o
Article history: Received 3 September 2013 Received in revised form 30 November 2013 Accepted 3 December 2013 Available online 9 December 2013 Keywords: Microfabrication T47D ZD6474 Bioimpedance Cytotoxicity
a b s t r a c t The present work reports the impedimetric characterization of cellular activities of T47D cells treated with anticancer drug ZD6474 using impedance sensing devices. Four types of devices with different dimensions are fabricated by micromachining technology. Real time impedance monitoring data reveals spreading stage completes within 5 h. The frequency response characteristics of drug treated cells are studied to evaluate cytotoxic effect of ZD6474 along with the sensitivity variation for different designs. Compared to control data, a significant decrease in impedance data is observed for drug treated samples above 10 M dose due to predominant cell death and detachment from the electrode surfaces. The quantitative relation developed between cell impedance and drug dose indicates that the magnitude of cell impedance varies inversely with the drug dose. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Recent advancement in biomedical screening technologies with the help of sophisticated microfabrication and signal processing techniques has resulted in the development of several novel sensor products and new applications with more accurate, cost effective and reliable approach as compared to their conventional counterparts. Until recently, most biosensors studied for biomolecular detection are label dependant sensor that detects biochemical processes as optical or electrochemical signals. It is also anticipated through intensive research that mechanical, optical, and electrical properties of the cell are also modified during their biochemical changes at various stages of diseases progression [1,2]. Thus there is a possibility to open an alternative path for label free detection/monitoring of disease progression process by identifying and quantifying the non-biological parameters of cells which may carry the signature of the disease. Among various techniques, electric cell-substrate impedance sensing (ECIS) technique is a rapid and inexpensive technique for determining electro-physiological characterization of cells [3,4]. The changes due to the alteration of cell properties during disease growth process can be detected well before by using electrical impedance technique. At present ECIS is an established, non-invasive electrochemical technique that has been successfully used to monitor cell adhesion, growth and
differentiation of cells [5–13], cell migration [14–17], morphological changes during apoptosis [18,19], and toxic effects of drugs on cellular activities [10,11,20–27]. In this paper, the electrical impedance of cells treated in various doses of anticancer drug ZD6474 is studied to correlate the electrical properties with their physiological conditions. ZD6474 is a heteroaromatic-substituted anilinoquinazoline (Fig. S1), which reduces tumour angiogenesis and endothelial cell survival [28–31]. It also inhibits tumour cell proliferation and survival [32]. However, the impedimetric monitoring of cytotoxicity of ZD6474 on T47D cells has not been carried out till date. The purpose of the present study is to understand the cell responses subjected to various drug doses using ECIS devices. This study is designed to provide results about real time monitoring of cell adhesion and proliferation of T47D cells in microfabricated devices using ECIS technique. Subsequently, the frequency response of electrical impedance of ZD6474 drug treated T47D cells is investigated and correlated with the standard technique like 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Also a correlation experiment is carried out to estimate the quantitative effect of drug doses on cellular activities of cancer cells. 2. Materials and methods 2.1. Materials and reagents
∗ Corresponding author. Tel.: +91 3222281228; fax: +91 3222282221. E-mail address: [email protected] (R. Pradhan). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.003
Pyrex glass wafers used as substrates in the present experiment were purchased from Semiconductor wafer Inc., Taiwan.
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Polydimethylsiloxane (PDMS, Sylgard 184) was supplied by Dow Corning, Inc., Midland. SU8 was procured from MicroChem, Newton. RPMI 1640 medium, foetal bovine serum, trypsin/EDTA solution, penicillin, and streptomycin were purchased from Himedia, India. All other required reagents were supplied by Sigma–Aldrich, India. 2.2. Culture of T47D cells T47D was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured on electrode surfaces by using RPMI 1640 containing 10% foetal bovine serum (FBS), 1% penicillin, and 100 g/mL streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 . The cells were harvested by trypsinizing the cell with 0.05% trypsin/EDTA and the cell suspensions were prepared by using standard tissue culture techniques. 2.3. Impedance measurement of T47D cells The electrical impedances of normal and drug treated T47D cells were carried out using electrochemical work station SP 150 (BioLogic, France). All the measurements were performed with applied voltage of 10 mV. The real time monitoring of cell adhesion and growth of T47D without drug treatment was performed by measuring the impedance at a fixed frequency of 10 kHz in a time lapse of 10 min for 7 h duration after seeding the cells in ECIS devices. In this case normalized impedance (NI) value has been calculated by using Eq. (1) to eliminate the effect of medium. NI =
−1 (ZCell − ZNo cell )ZNo cell
(1)
where ZCell and ZNo cell are the impedances of the system with and without the cells in culture media of same volume. The impedance of T47D cells treated in different doses of ZD6474 was measured from 100 Hz to 1 MHz with 51 points in a logarithmic scale after 24 h of drug treatment. The final impedance value for each frequency was the mean value of 10 subsequent measurements. 2.4. Equivalent circuit fittings The impedance data found from experiment was fitted by using ZsimpWin (Version 3.10). The equivalent circuit used for fitting purposes was extracted from the previous literature [33] and described in Fig. 1. 2.5. Sensitivity of impedance sensing devices The sensitivity of the biosensor was calculated by using the following Eq. (2) which is extracted from the previous literature [34].
−1 Sensitivity(f ) = (ZCell (f ) − ZNo cell (f ))Qcell
(2)
where f is sensing frequency and Qcell is the maximum cell density (about 106 cells cm−2 ).
RS
Qdl
QC
RI
RC
Fig. 1. Equivalent circuit of impedance sensing devices (RS represents the extracellular bulk resistance, Qdl represents the dielectric property of electrode/electrolyte interface including the coating capacitance, RI represents charge transfer resistance while QC and RC are the capacitance and resistance of cancer cells).
479
2.6. Experimental protocol The impedance sensing device was sterilized with 75% ethanol for 15 min, dried with nitrogen, and irradiated with ultraviolet radiation for 15 min. RPMI 1640 (1 mL) was then added to the device and incubated at 37 ◦ C for 20 min to record the background impedance value (ZNo cell ). Next, the cell suspension of T47D (1 mL, 1 × 106 cells) was added into each cloning cylinder and the device was placed into the incubator after 10 min for cell culture and impedance sensing. Then different doses of ZD6474 (0, 5, 10 and 15 M) were added into the well of impedance sensing devices after 30 min of cell inoculation to study the cytotoxic effects of the drug. 2.7. Comparison of impedance data with MTT assay Cell viability of T47D breast cancer cells were determined by MTT-dye reduction assay. The cell suspensions were dispensed in quadruplicate into 96-well tissue culture plates at an optimized concentration of 104 cells/well in complete medium. After 24 h of treatment with ZD6474 of various concentrations ranging from 1 M to 50 M along with 0.1% DMSO as control, cell viability was measured at 540 nm using a micro-plate spectrophotometer (Bio-RAD Benchmark Plus). The viability of cells after 24 h of drug treatment was counted using TC 20 automated cell counter (BioRAD). 2.8. Statistical data analysis All the experiments including impedimetric analysis and biochemical assay were carried in triplicate and the data were represented with their corresponding relative standard deviations (RSD). 2.9. Correlation experiment A quantitative relationship was developed to understand the effects of ZD6474 on cellular activities of T47D cells. The magnitude of impedance and phase angle data were correlated with drug doses and working electrode area by using LAB Fit curve fitting software. 3. Results and discussions 3.1. Design of impedance sensing devices In the present work, three-electrode based impedance sensing devices are used to measure the cellular activities on gold electrode surfaces. The design rules for electrode fabrications are extracted from previous literatures. Mishra et al. [35] has used smaller working electrode (WE) than reference electrode (RE) while Brett and Brett [36] have used smaller WE than counter electrode (CE). Considering the limitations of authors’ microfabrication facilities and also to restrict higher device area, the ratio of WE/RE and WE/CE is fixed to 0.01. The cross contamination is avoided by placing the CE and RE at a distance of 100 m from WE position in all the designs [37,38]. The sensor areas are connected to large contact pads by interconnections and the impedances produced by those interconnections are eliminated by providing a SU8 coating of thickness 50 m [33]. In the present study, four different configurations of impedance sensing devices are designed with varying dimensions of WE, RE, and CE as given in Table 1. 3.2. Fabrication of impedance sensing devices The realization of impedance sensing device by microfabrication technique is described in authors’ previous paper [39]. The
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Table 1 Dimensions of different design of impedance sensing devices. Devices
WE area (mm2 )
RE and CE area (mm2 )
Lead width (mm)
Lead length (mm)
WE/RE and WE/CE
SU8 thickness (mm)
Design 1 Design 2 Design 3 Design 4
0.05 × 0.05 0.1 × 0.1 0.15 × 0.15 0.2 × 0.2
0.5 × 0.5 1×1 1.5 × 1.5 2×2
0.25 0.25 0.25 0.25
25 25 25 25
0.01 0.01 0.01 0.01
0.05 0.05 0.05 0.05
Fig. 2. Photograph of impedance sensing devices and an enlarge view of sensor area.
devices are fabricated on Pyrex wafers using photolithography techniques. Initially, the wafers are cleaned and thin layers of chromium (Cr) and gold (Au) are deposited by thermal evaporation technique. Subsequently, the electrode patterns and its contact pads are lithographically defined on the deposited metal film. Next, another photosensitive polymer (SU-8) layer is spin coated and lithographically patterned to obtain SU8 coating over the metal interconnections. The devices are fixed on a PCB board and the cloning cylinders are attached around the sensors by using PDMS to serve as electrolyte reservoir for cell culture as shown in Fig. 2. 3.3. Real-time monitoring of the attachment and spreading of T47D cells Fig. 3 represents the real time monitoring of cell adhesion and spreading of T47D cells. From the figure, it is inferred that the NI value increases slowly during first one h and then increases rapidly from 2–5 h. the first phase represents the cell adhesion stage while the second phase signifies the spreading stage. After spreading, cells enter in to proliferation stage and the impedance value shows a
Fig. 3. Monitoring of adhesion and spreading of T47D cells by impedimetric method.
Fig. 4. Bode plot for effects of ZD6474 on T47D cancer cells for Design 1.
little fluctuation for subsequent measurement up to 7 h time span for each case. The electrode area has a little influence on NI variation during cell adhesion time as the current path is passed through the electrolyte solution. Then the NI value increases gradually due to attachment of cells on the electrode surface. However, the NI variation is mostly controlled by the cells covering the electrode surface region during spreading stage [40,41]. Previous study predicts that current path is blocked at the electrode surface due to the presence of cancer cells. Thus the impedance of cell covered surface is inversely proportional to the electrode area [34] and NI variation is more prominent in smaller electrode device. Similar experimental observation is also reported for HeLa and HaCaT cells [33]. 3.4. Evaluation of cytotoxic effects of ZD6474 on T47D cells The different doses of ZD6474 are added with cells after 30 min of cell inoculation in the fabricated devices. The impedance decreases at 19 ± 0.24 h due to cell death and detachment from the electrode surfaces. The bode plots for the cytotoxic effects of ZD6474 on T47D cells after 24 h are presented in Fig. 4 for Design 1 and Fig. S2 for Design 2, 3 and 4. The experimental data are fitted reasonably well with the used equivalent circuit as described in Fig. 1. The results illustrate that the magnitude of impedance is highest for Design 1 and gradually decreases with the increase of electrode dimensions. Thus the magnitude of impedance value is inversely proportional to the WE area as observed from Fig. 4 and Fig. S2 for devices with different electrode area. From the figures, it is also evident that magnitude of impedance decreases gradually with increase of frequency. However, phase angle value decreases up to 1 kHz and then forms a plateau in the frequency range of 1–10 kHz and then increases up to 1 MHz. This initial variation of slop at low frequency occurs due to increased drug-cell interactions resulting cell death and detachment from the electrode surfaces. The relative standard deviations (RSD) for treated and untreated samples for different designs are found to be below 10% which showed the reproducibility nature of the fabricated impedance sensing devices. Also the role of drugs on medium in impedance values is estimated by measuring the impedance of cell free medium
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comparable to previous studies on sensitivity analysis of the ECIS sensor in which the sensitivity decreases with the increase of electrode dimensions [34, 42]. 3.5. Correlation between drug doses and impedance Numerical relations between impedance, drug dose and, electrode area are established to analyze the effect of drug doses on cellular behaviour and its impedance characteristics. The measured impedance and phase angle data for various drug doses and electrode area are plotted using curve fitting software to get the dependence curve as shown in Fig. 7. The empirical relation between magnitude of impedance (Z), drug doses (D), and working electrode area (A) as obtained is expressed in Eq. (4). Z = 0.1479 × 107 × −0.9124D × A−0.3097 Fig. 5. Impedimetric cell viability calibration curve of ZD6474 for different designs.
with different doses of drugs and the plot is shown in Fig. S3. From the figure, it is evident that the impedance values for different drug doses vary negligibly within 0.005% error. Thus it is assumed that the impedance due to drug doses in solution has no role in decrease of cell impedance. The IC50 value of ZD6474 in T47D obtained from MTT assay is 8.65 ± 0.5246 M. The percentage of living cells present in control, 5 M, 10 M and 15 M treated drug are 96 ± 3, 75 ± 4, 53 ± 3, and 34 ± 5, respectively as measured from cell viability count. The cell viability (%) is calculated from the impedance data by using the following Eq. (3) and is represented in Fig. 5. Cell viability(%) =
ZT × 100 ZC
(4)
Similarly the relation between phase angle (), drug doses, and active electrode area is described in Eq. (5). =
(−0.4073×106 +A) (0.6589×104 +0.7175×102 ×D)
(5)
The inhibitory plot of ZD6474 for T47D cells is plotted in Fig. 8 in terms of magnitude and phase angle of impedance with respect to drug dose keeping frequency and working electrode area constant.
(3)
where ZC and ZT represent the mean value of impedance for control and treated sample at 24 h respectively. The IC50 value of ZD6474, calculated from the impedance data of Fig. 5 is 8.74 ± 0.3137. Thus it is evident that the IC50 value obtained by impedimetric method correlates well with the value obtained by MTT assay method. The frequency dependent sensitivity of ECIS devices are calculated using Eq. (2) and a characteristic plot using control data is shown in Fig. 6 for various electrode dimensions. The sensitivity for Design 1 is highest followed by other designs which implies that the sensitivity is inversely proportional to the area of electrode. This may be due to enhanced interaction of cells at electrode area reducing electrolyte contact with electrode surfaces. This study is
Fig. 6. The sensitivity of the impedance sensing devices for different designs.
Fig. 7. Dependence plot for drug doses in relation to (A) magnitude and (B) phase angle.
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Acknowledgements Authors would like to thank the staff members of MEMS Lab, IIT Kharagpur for microfabrication support and Indian Space Research Organization for financial support to carry out the present work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.12.003. References
Fig. 8. Inhibitory plot of ZD6474 in relation to (A) magnitude and (B) phase angle.
The mathematical relation between impedance and drug doses is expressed in Eqs. (6) and (7). Z = 0.8433 × 105 × 0.9124D
(6)
= 0.5503 × D − 0.5882 × 102
(7)
The fittings obtained in these plots show a negative slope in case of magnitude of impedance and a positive slope in negative directions for phase angle value which implies inhibitory action of drug dose on the cells. 4. Conclusions This study provides results about impedimetric monitoring of cellular behaviours of T47D cells by using impedance sensing devices of different electrode geometries. Experimental observations reveal that the peak variation in normalized impedance is related to cell adhesion and spreading. The frequency dependent impedance data reveal that the cells treated above 10 M drug dose shows prominent reduction of its impedance magnitude as compared to the control cells. This effect is due to dose dependant cell death and detachment of the cells from electrode surfaces. Sensitivity of the devices decreases with the increase of electrode dimensions but remains within the working range. Quantitative evaluation of cytotoxic effect of drug dose on cell behaviour is established through an empirical relation indicating cell impedance magnitude varies inversely with drug dose. Thus these devices may be used to study the cellular activities of different kind of cells on electrode surfaces.
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Biographies Rangadhar Pradhan obtained his M.Sc. degree in Zoology from Utkal University, Bhubaneswar in 2003 and, M.Tech. and Ph.D. degrees from IIT Kharagpur in 2006 and 2013, respectively. Presently, he is a postdoctoral fellow at IIT Kharagpur. Dr. Pradhan got MHRD scholarship for pursuing M.Tech as well as Ph.D. His research area includes development of Biosensors and ECIS system. Mahitosh Mandal received his M.Sc. degree in 1984 and Ph.D. degree in 1990 from University of Burdwan. He is presently serving as Associate professor at School of Medical Science and Technology, IIT Kharagpur. His research interest includes cancer diagnosis, development of diagnostic and prognostic cancer markers, and targeted drug delivery for cancer therapy. He has authored more than 74 research papers in international journals. Analava Mitra received his M.B.B.S. degree in 1980 and Ph.D. degree in 2003 from IIT Kharagpur. He is presently serving as Associate professor at School of Medical Science and Technology, IIT Kharagpur. His research interest includes herbal medicine, nutraceuticals and drug delivery. He has authored more than 80 research papers in international journals and conference proceedings. Soumen Das received his M.Sc. degree in 1988 and Ph.D. degree in 1996 from Indian Institute of Technology Kharagpur. He joined as Scientific Officer in 1996 at Microelectronics Laboratory and at present serving as Associate professor at School of Medical Science and Technology, IIT Kharagpur. His research areas include biomedical and inertial MEMS transducers, BioMEMS and microfluidic biochips for clinical diagnostics, medical electronics and VLSI unit processing. He has authored more than 40 research papers in reputed international journals and conference proceedings.