Andresen, Thomas Lars

Carminati, M.

Caviglia, Claudia

Emnéus, Jenny

Ferrari, G.

Heiskanen, Arto

Sampietro, M.

Vergani, M.

A theoretical and experimental comparison between vertical and coplanar interdigitated sensing configurations for impedimetric cell growth tracking is presented. For the first time, these widely-adopted approaches are quantitatively compared on the same cell populations and on the same 10μm interdigitated microelectrodes using a versatile custom-made monitoringplatform including a 24-channel miniaturized potentiostat. As expected, characterization of bare microelectrodes in buffer and tracking experiments with HeLa cells over 16 hours demonstrate that the coplanar configuration provides a higher sensitivity to cell adhesion and spreading (Cell Index = 1.6 vs. 0.4) albeit at a higher frequency of maximum sensitivity (100kHz vs. 24 kHz)

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Downloaded from orbit.dtu.dk on: Dec 17, 2017Comparison of microelectrode sensing configurations for impedimetric cell monitoringCaviglia, Claudia; Heiskanen, Arto; Andresen, Thomas Lars; Emnéus, Jenny; Carminati, M.; Vergani, M.;Ferrari, G.; Sampietro, M.Published in:Proceedings of the International Workshop on Impedance SpectroscopyPublication date:2012Link back to DTU OrbitCitation (APA):Caviglia, C., Heiskanen, A., Andresen, T. L., Emnéus, J., Carminati, M., Vergani, M., ... Sampietro, M. (2012).Comparison of microelectrode sensing configurations for impedimetric cell monitoring. In Proceedings of theInternational Workshop on Impedance SpectroscopyComparison of Microelectrode Sensing Configurations for Impedimetric Cell Monitoring C. Caviglia, A. Heiskanen, T. L. Andresen, J. Emnéus Department of Micro- and Nanotechnology Technical University of Denmark Lyngby, Denmark Claudia.Caviglia@nanotech.dtu.dk M. Carminati, M. Vergani, G. Ferrari, M. Sampietro Dipartimento di Elettronica e Informazione Politecnico di Milano Milano, Italy carminati@elet.polimi.it Abstract—A theoretical and experimental comparison between vertical and coplanar interdigitated sensing configurations for impedimetric cell growth tracking is presented. For the first time, these widely-adopted approaches are quantitatively compared on the same cell populations and on the same 10μm interdigitated microelectrodes using a versatile custom-made monitoring platform including a 24-channel miniaturized potentiostat. As expected, characterization of bare microelectrodes in buffer and tracking experiments with HeLa cells over 16 hours demonstrate that the coplanar configuration provides a higher sensitivity to cell adhesion and spreading (Cell Index = 1.6 vs. 0.4) albeit at a higher frequency of maximum sensitivity (100kHz vs. 24 kHz). Index Terms— impedance spectroscopy; interdigitated electrodes; cell tracking. I. INTRODUCTION The use of impedance spectroscopy, consolidated in other electrochemical applications such as corrosion studies or characterization of batteries, is becoming a routine tool also in the biomedical field, in particular for monitoring the growth and response to chemical stimulation of cell populations cultivated on planar microelectrodes. In fact, since the pioneering work by Giaever and Keese [1], a huge volume of research has flourished and various custom-made and commercial systems are available for biological investigation [2]. The success of this technique, based on the increase of impedance due to the insulating barrier represented by the cells filling the volume at a height of 10-20nm above the electrodes, relies on its simplicity enabling label-free and automatic analysis. However, as highlighted by Orazem and Tribollet [3], as impedance is not specific by itself and is affected by any variation of the interface properties, the interpretation of impedance data is particularly delicate. Elaborate equivalent models, though providing good fitting, can lead to the loss of the physical meaning of their parameters. In this work we present a theoretical and experimental comparison of two alternative sensing configurations, i) the standard “vertical” configuration (a single working electrode (WE) versus a large, distant counter electrode (CE), and ii) the interdigitated configuration (WEa comb versus WEb comb). The latter was introduced for cellular monitoring 15 years ago [4] and is now becoming commonly adopted due to its good performance [5] even in commercial systems, such as exCELLigence by Roche. The major novelty of our investigation is that for the first time a quantitative comparison is performed exactly on the same cells, alternating the two configurations when measuring impedance spectra on the same couples of electrodes, as enabled by an original and versatile monitoring platform we have recently designed [6]. II. THEORY AND MODELING The theoretical spectral characteristics of the two alternative configurations, illustrated in Fig. 1 along with their simplified lumped-parameter equivalent impedance models, will be discussed. Design rules, issues on the instrumentation side and expressions to estimate the equivalent parameters will be illustrated. It will be highlighted how the coplanar configuration results in a higher corner frequency. This frequency roughly corresponds to the optimal operating frequency, where the spectral sensitivity to cell adhesion is maximum.  For small initial cell densities (i.e. small fractions of covered electrode area, far from confluence) it is expected that the coplanar detection is more sensitive to the presence of cells in the volume between the fingers, with respect to the vertical condition, in which the solution resistance is set by the radius of the microelectrode (i.e. by the electrode border in a radial diffusion regime). III. EXPERIMENTAL RESULTS A. Electrodes Characterization Prior to the experiments with cell populations, the interfacial impedance of the bare electrodes in contact with PBS buffer has been characterized. The Au microelectrodes were fabricated with a standard lift-off process and passivated by Si3N4 layer, which was reactive ion etched to expose the active electrode areas. Each comb comprises 12 fingers with length 500μm, width 10μm and spacing 10μm. As the vertical penetration of the electric field between interdigitated electrodes is roughly equal to the gap, this size has been chosen in order to probe cells in the 5μm-20μm size range. The measured spectra (Fig. 2) excellently match the expected values, confirming the correct estimates of the double layer capacitance (CDL) and solution resistance (RS) of WEa in both cases (i.e. vs. CE and WEb respectively). The corner frequency shifts from ~30kHz (vertical) to ~300kHz (coplanar).  Figure 3.  (a) Cell Index over frequency at different time instants after cells seeding; (b) Cell Index tracking over time at 24kHz and 100kHz respectively for vertical and coplanar configuration. VAC is reduced to 200µV to limit the electromagnetic perturbation stress applied to the cell culture.100 1k 10k 100k 1M1001k10k100k1M  Measured  TheoryRS2 = 200RS1 = 1.16kCDL = 6nFVerticalImpedance Magnitude []Frequency [Hz]CoplanarFigure 2. Compared WEa spectra measured in PBS (VAC = 4mV).  CE WE (a) (b) WEa WEb MediumMediumCDL RS1 Cells Cells CDL RS2 CDLCS1CS2 ECIS Vertical Configuration Interdigitated Coplanar Configuration  Figure 1. Compared vertical (a) and coplanar (b) sensing configurations.B. Monitoring HeLa Cells Electrochemical impedance spectroscopic tracking has been used as a non-invasive biophysical approach to continuously monitor adhesion and proliferation of HeLa cells. The experiments have been performed by initially seeding about 2.5105 cells into a sterilized well containing poly-L-lysine-coated gold electrode arrays. Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin was used as culture medium. The chamber was incubated at 37° C in a humidified atmosphere having 5% CO2. Impedance spectra were acquired from each sensor element for 16 hours: during the first 4 hours after cell seeding data were recorded every 20 minutes, then every hour. A 200μV sinusoidal perturbation potential was applied (to reduce the max. current below the 1µA safety limit) and 30 points were recorded in the frequency range between 100Hz and 100kHz with an averaging time of 2s. Data recorded from the different sensor elements were averaged and processed to derive, for each frequency and for each time point, the Cell Index (CI). It is defined as CI(t,f) = (|Z(t,f)| / |Z(0,f)|) – 1, where |Z(0,f)| is the magnitude of impedance acquired when cells were not yet seeded into the well and |Z(t,f)| is the magnitude of impedance acquired after cell seeding at different time points. Fig. 3A shows examples of CI values acquired using the ECIS vertical configuration. A peak frequency, indicating the most sensitive region of the spectra, can be determined at about 24kHz. Data recorded using the interdigitated coplanar configuration showed instead a peak frequency at 100kHz. Fig. 3B shows the CI tracking, over 16 hours, acquired using the two different configuration modes and plotted at the characteristic peak frequency. The maximum value of CI, about 1.6 ± 16% and 0.45 ± 17% for coplanar and vertical configurations, respectively, was reached in both cases 10 hours after cell seeding. This clearly demonstrates that the coplanar configuration provides the highest sensitivity for monitoring cell adhesion and proliferation. REFERENCES [1] I. Giaever, C.R. Keese, “Monitoring fibroblast behavior in tissue culture with an applied electric field,” PNAS, vol. 81, pp. 3761-3764, 1984. [2] J. Hong, K. Kandasamy, M. Marimuthu. C.S. Choi, S. Kim, “Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study,” Analyst, vol. 136, pp. 237-245, 2011. [3] M.E. Orazem, B. Tribollet, Electrochemical Impedance Spectroscopy, John Wiley & Son, 2009. [4] R. Ehret, W. Baumann, M. Brischwein, A. Schwinde, K. Stegbauer, B. Wolf, “Monitoring of cellular behaviour by impedance measurements on interdigitated electrode structures,” Biosens. Bioelectron, vol. 12, no. 1, pp. 29-41, 1997. [5] J. Mamouni, L. Yang, “Interdigitated microelectrode-based microchip for electrical impedance spectroscopy study of oral cancer cells,” Biomed. Microdev., vol. 13, pp. 1075-1088, 2011. [6] M. Vergani et al., “Multichannel bipotentiostat integrated with a microfluidic platform for electrochemical real-time monitoring of cell cultures,” IEEE Trans. Biomed. Circuits Syst., DOI: 10.1109/TBCAS.2012.2187783.

Comparison of microelectrode sensing configurations for impedimetric cell monitoring

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Comparison of microelectrode sensing configurations for impedimetric cell monitoring

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