Hermann Scharfetter, Robert Merva (Eds.): ICEBI 2007, IFMBE Proceedings 17, pp. 94–97, 2007 www.springerlink.com © Springer-Verlag Berlin Heidelberg 2007

Measurements on cultured cells using screen printed sensors M. Brischwein1, B. Gleich1, T. Weyh1, P. Los2

1 Technische Universität München/Heinz Nixdorf-Lehrstuhl für Medizinische Elektronik, München, Deutschland 2 IDMoS plc, Dundee, UK

Abstract— Impedance sensors in thick film technology have been designed as a tool for electric cell impedance sensing. The screen printed gold electrodes and the flexible PET polymeric chip substrate are homogeneously grown with Hela cells as documented by fluorescence microscopy. In contrast to previ-ously published cell impedance measurements, we discuss the use of revised experimental conditions to improve the accuracy of the measurements. Responses of confluent cell monolayers to the treatment with two bioactive compounds are tested: (1) Histamine, triggering a cell morphological reaction and (2) the non-ionic detergent Triton X-100, leading to a complete dis-ruption of cell membranes. The impedance is measured and compared with a standard, planar electrode arrangement for statistical evaluation.

Keywords— bioimpedance, screen printing, active shielding, cell culture

I. INTRODUCTION

The measurement of electric impedance of planar elec-trode structures grown with living cells as a parameter to detect cell morphological changes was first described by Giaever and Keese [1]. Since then, numerous applications of “ECIS” (i.e. Electric Cell-substrate Impedance Sensor) techniques for cell-analytical purposes have been described. Among these are studies of cell growth and proliferation, spreading and adhesion, migration and motility, invasive activities, cell receptor activation and the analysis of cell transduction pathways, cell metabolism, cell volume changes, infection with pathogenic species, cytotoxicity/cell death and aggregation of platelets to test blood coagulation. The method of extracellular sensing avoids the use of labels, does not interfere with normal cellular behaviour in vitro and allows a real-time monitoring of cells. The physical background of the technique is the electrically insulating effect of cell membranes at low frequencies. The impedance of sensor electrodes is sensitive to a covering with such membranes and to any morphological change of the adher-ent cell layer.

So far, thin film technologies on glass and silicon sub-strates have been used to construct “cell on a chip” systems. Screen printed electrodes on ceramic substrates are already used as the basis of commercially available enzyme elec-trodes, as they can be produced as disposable sensors in

medium and large scales at rather low prices, even on flexi-ble, transparent polymers. Despite of this fact, to the best knowledge of the authors screen printed electrodes have only previously been tested for ECIS applications [2].

This study focuses on impedance sensors for the on-line detection of changes in cell adhesion/cell morphology. The approach involves a long-term culturing of cells on the chip. First results with such a sensor configuration fabricated with screen printing are described.

In addition to using screen printed electrodes, a sandwich setup is used employing two chips carrying aligned face-by-face with a distance of approximately 2.5 mm. Only one of these chips is grown with cells. Along with active guard electrodes on each of the chips this configuration in a tetrapolar measurement should lead to an optimised geome-try of the electric field.

II. EXPERIMENTAL

A. Materials and Methods

Electrodes: A low-temperature process was used to print gold electrodes on PET (poly-terephtalate) sheets (size: 24,0 x 33,8 cm2). The layout of the structure consisting of a cen-tral, circular electrode and two U-shaped surrounding elec-trodes is shown in Fig. 1. The conducting pathways are insulated. The sheets were obtained from Dinamic Biotech-nology Innovation Center (Tarragona, Spain).

Mechanical setup: To handle the sandwich-like configu-ration of electrode sheets a mechanical setup consisting of

Fig. 1: Chip layout with two active electrodes and the outer, U-shaped electrode for active shielding. The cell culture area is within the circle.

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anodically coated aluminium plates was constructed (Fig. 2). It accommodates two “sandwiches”, with the two sheets of each sandwich aligned “face by face” and sepa-rated by a 2,5 mm silicon rubber ring. Compounds for cell stimulation are added by syringes/needles through the sili-con ring. Apertures in the aluminium plates allow a visual control of cell growth by microscopy before and after the data recordings. The electrodes are contacted via push pin contacts (not shown in Fig. 2).

During measurements, the whole setup is placed in an in-cubator serving both as an effective Faraday cage and ther-mostatting device at the temperature of 37 oC.

Cell culturing and stimulation: Hela cells (human cervix carcinoma) tested for mycoplasma were used. One ml of cell suspension (5 x 105 cells) was added into the volume of app. 440 µl between the sandwiched sheets. The medium was Dulbeccos Modified Eagles (DME) medium, without bicarbonate, supplemented with 20 mM HEPES and 5% FCS. Cells were allowed to settle down, to adhere and to spread on the bottom sheet to form a confluent monolayer. A monolayer was usually formed during incubation over night. The setup was connected to the measurement instru-ment ≈24 hours after cell seeding on the chips. In order to stimulate the cellular test responses with the expected effect on impedance spectra, a protocol with addition of 25 µM Histamine (acting on the histamine H1-receptors) was ap-plied. From previous studies, this receptor stimulation is known to cause characteristic changes of cell morphology detectable with impedance sensing technology [3]. Subse-quently, the non-ionic detergent Triton X-100 (solubilizing completely cellular membranes) was added. For the repro-ducible addition of these compounds, a peristaltic pump was used (flow rate 8 µl s-1, stopped flow during acquisition of spectra).

Fluorescence microscopy: The presence of adherent cells on the non-transparent electrodes was tested with fluores-

cence microscopy after staining with bisbenzimide dye (Fig. 3), labelling the nuclei of cells.

Impedance measurements: A solartron SI 1260 instru-ment (Solartron Analytical, Farnborough, UK) was used to record impedance spetra in the range of 10 Hz – 20 MHz. The amplitude was 10 mVpp, DC potential 0 mV. Elec-trodes were connected in a 4-electrode setup to the instru-ment according to the configuration shown in Fig. 5. As shown in this figure, the outer U-shaped electrodes are addi-tionally connected to voltage followers (amplifiers), allow-ing to operate these electrodes as actively shielding ele-ments (active guard ring).

Table 1 Measurement protocol

time (min) action

0 starting the experiment 30, 35, 40, 45 acquisition of spectra 50 addition of 25 µM histamine 75, 80, 85, 90 acquisition of spectra 95 addition of 0.2% Triton X-100 120, 125, 130, 135 acquisition of spectra

Fig. 2: Mechanical setup for holding two “sandwiches” of electrodes in a

face-by-face position, separated by a distance of 2,5 mm.

Fig. 5: Electrode configuration and connections to the solartron instrument.The “Gen Out” and “Input” ports are used for AC supply while the “HI

and “LO” ports are for signal detection.

Fig. 3: Hela cells growing on screen printed electrodes. The optical focus

is on the surface plane of an electrode.

96 M. Brischwein, B. Gleich, T. Weyh, P. Los

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B. Impedance Data

Impedance spectra: A series of 100 impedance spectra will be recorded to obtain a significant database showing the effectiveness of the used method. Since this series of measurements is still ongoing at the time of writing this contribution, only exemplary results without statistical evaluation can be shown. However, it appears that both stimuli (receptor-mediated effect of histamine on cell mor-phology, detergent-mediated dissolution of cell membranes) can be detected (Figs. 6-8). It emerges that the spectral differences between the various conditions of cell cultures are most pronounced in the range <1 kHz, which is consis-tent with previous observations. [2]

The effect of active shielding on the sensitivity of detec-tion is another issue to be analysed. In Fig. 8, spectra with

activated and deactivated guard electrode are shown. Al-though it is too early for general conclusions, there are evi-dent spectral differences between these two modes of re-cording. However, since at the moment there is no consistent relation identifiable between the exact nature of such differences and cellular/experimental conditions, we would like to avoid any interpretation of the presented re-sults.

III. CONCLUSIONS

The aim of this study is to investigate screen printed elec-trode structures as a substrate for the analysis of cellular behavior, using the effects of living cells on the electro-chemical interface. In addition to using screen printed elec-trodes, new strategies to increase the sensitivity of meas-urement are employed: These include a 3-dimensional geometry of the electrode setup and the implementation of an actively shielding element directly at the site of meas-urement to obtain a more homogeneous distribution of the electric field. The available intermediary results of the study show the suitability of the electrodes and the experimental setup for a proper detection of cell-mediated effects on the electrochemical interface. The results show clear effects of active shielding on the obtained spectra. However, the exist-ing database is too small at the time of writing this manu-script for an interpretation of these differences. Further-

Fig. 6: Bode plot of a single experiment (treatment of a Hela cell culture with histamine and Triton X-100).

Fig. 7: Nyquist plot corresponding to Fig. 6

Fig. 8: Nyquist plot corresponding to the experiment shown in Fig. 6 and 7, showing exemplary the effect of active shielding. The graphs representing

the initial state of the cell culture (before histamine addition) have been omitted for better clearness. In this example, there is a particularly strong

difference in the spectra (with/without active shielding) after the addition of histamine.

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more, the exact benefit of using the sandwich geometry remains to be analysed experimentally.

Due to the low amplitude of the current and the large sur-face area of the screen printed electrode material (leading to a low current density) electrode polarisation is not assumed to contribute significantly to the measured impedance. Thus, the experimental setup should be suited to reveal small changes of the impedance caused by the biological material between the electrodes.

ACKNOWLEDGMENT

The authors are indebted to Dr. Ioanis Katakis and Diego Bejarano for providing the electrode sheets and Wolfgang Ruppert, Till Federsel, Julia Kohler and Lin Koh for their help in preparing the experiments.

REFERENCES

1. Giaever I., Keese C R (1984) Monitoring fibroblast behavior in tissue culture with a applied electric field. Proc Natl Acad Sci 81: 3761-3764

2. Brischwein M, Herrmann S et al (2006) Electric cell-substrate imped-ance sensing with screen printed electrode structures. Lab on a Chip 6: 819-822 DOI: 10.1039/B602987F

3. Motrescu E M, Brischwein M et al (2001) Comparison of Ca2+ sensitive dye probes and microsensor-based monitoring in the as-sessment of cellular signaling. Biol Cell 93: 225

Address of the corresponding author:

Author: Martin Brischwein Institute: Heinz Nixdorf-Lehrstuhl für Medizinische Elektronik, Tech-

nische Universität München Street: Theresienstrasse 90/N3 City: München Country: Germany Email: brischwein@tum.de