Fibroblast Cells: A Sensing Bioelement for Glucose Detection by

de Collongues, 69134 Ecully Cedex, France, Laboratoire U352 Biochimie et Pharmacologie de ... The linear calibration curve was established in the ...
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Anal. Chem. 2003, 75, 3340-3344

Fibroblast Cells: A Sensing Bioelement for Glucose Detection by Impedance Spectroscopy Chaker Tlili,*,†,‡ Karine Reybier,† Alain Ge´loe 1 n,§ Laurence Ponsonnet,† ,† § Claude Martelet* Hafedh Ben Ouada, Michel Lagarde,§ and Nicole Jaffrezic-Renault*,†

Laboratoire d’Inge´ nierie et de Fonctionnalisation des Surfaces, UMR-CNRS 5621, ECL-Lyon, 36 Avenue Guy de Collongues, 69134 Ecully Cedex, France, Laboratoire U352 Biochimie et Pharmacologie de la me´ diation lipidique, INSERM-CNRS, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France, and Laboratoire de Physicochimie des Interfaces, Universite´ de Monastir, Avenue de l’Environnement, 5019 Monastir, Tunisie

Modifying the electrical properties of fibroblasts against various glucose concentrations can serve as a basis for a new, original sensing device. The aim of the present study is to test a new biosensor based on impedancemetry measurement using eukaryote cells. Fibroblast cells were grown on a small optically transparent indium tin oxide semiconductor electrode. Electrochemical impedance spectroscopy (EIS) was used to measure the effect of D-glucose on the electrical properties of fibroblast cells. Further analyses of the EIS results were performed using equivalent circuits in order to model the electrical flow through the interface. The linear calibration curve was established in the range 0-14 mM. The specification of the biosensors was verified using cytochalasin B as an inhibitor agent of the glucose transporters. The nonreactivity to sugars other than glucose was demonstrated. Such a biosensor could be applied to a more fundamental study of cell metabolism. A global epidemic of diabetes is predicted for the first quarter of the 21st century. In the United States in 1998, ∼6.5% of the population (over 16 million people) were diagnosed with type 2 diabetes and a roughly equivalent number was estimated to be undiagnosed.1 Recent evidence reveals that the prevalence of diabetes in the United States increased by 33% from 1990 to 1998 with a further 6% increase over the following year. These statistics are not confined to the United States. The worldwide prevalence of diabetes in persons g20 years old was 4% in 1995 and is expected to reach 5.4% (over 300 million people) by 2025. This means that diagnosing diabetes has become a major issue. Diabetes is diagnosed exclusively by demonstrating a persistent increased glucose concentration in the blood. Continuous glucose sensing with a stable in vivo glucose sensor is expected to lead to improved regulation of the glucose concentration and thus to a reduction of the number of complications in diabetes * Corresponding authors. E-mail: [email protected]; chaker_tlili@ yahoo.fr. † UMR-CNRS 5621. ‡ INSERM-CNRS. § Universite´ de Monastir. (1) Greenberg, R. A.; Sacks, D. B. Clin. Chim. Acta 2002, 315, 61.

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mellitus patients. Since Clark and Lyons2 reported their enzyme electrode for measuring glucose in 1962, an enormous amount of literature has been published concerning enzyme-based biosensors. Amperometric biosensors based on glucose oxidase are the most widely used biosensors. This enzyme catalyses the oxidation of β-D-glucose consuming molecular oxygen, producing gluconolactone, gluconic acid, and hydrogen peroxide. Biosensor response is generally based on the electrochemical oxidation of H2O2.3,4 This kind of biosensor is often used in short-term evaluation5,6 only due to an activity loss of the enzyme and the relative toxicity of H2O2 for biological entities. Several studies on glucose sensing by numerous investigators have aimed at developing an implantable glucose sensor,7-10 but to obtain reliable glucose measurements in vivo, the sensor must have an outer coating in order to prevent contact between tissue and the enzymatic membrane,10,11 and its lifetime does not usually exceed 3 months. This is why the development of new biosensors is of considerable interest, and microorganisms are good candidates. Some authors have proposed to replace the enzyme with fragments of yeast cell wall,12 since bacteria and yeasts are recognized as organisms that metabolize all kinds of sugars very well.13,14 However, to improve the selectivity of such biosensors, there must be use a second anti-interference enzymatic-layer. The use of living cells as sensor elements provides the opportunity for high sensitivity in a broad range of biologically active substances that affect the response of the cells. It has long (2) Clark, L. C.; Lyons, C. Ann. N. Y. Acad. Sci. 1962, 102, 29. (3) Cosnier, S.; Senillou, A.; Gra¨tzel, M.; Comte, P.; Vlachopoulos, N.; JaffrezicRenault, N.; Martelet, C. J. Electroanal. Chem. 1999, 469, 176. (4) Poyard, S.; Jaffrezic-Renault, N.; Martelet, C.; Labbe, P.; Besombes, J. L.; Cosnier, S. Sens. Actuators, B 1996, 33, 44. (5) Ward, W. K.; Jansen, L. B.; Anderson, E.; Reach, G.; Klein, J. C.; Wilson, G. S. Biosens. Bioelectron. 2002, 17, 181. (6) Wilkins, P.; Atanasov, B.; Muggenburg, A. Biosens. Bioelectron. 1995, 10, 485. (7) Schmidtke, D. W.; Heller, A. Anal. Chem. 1998, 70, 2149. (8) Pickup, J. C. Diabetes Cares 1999, 16, 535. (9) Kros, A.; Gerritsen, M.; Sprakel, V. S. I.; Sommerdijk, N. A. J. M.; Jansen, J. A.; Nolte, R. J. M. Sens. Actuators, B 2001, 81, 68. (10) Galeska, I.; Chattopadhyay, D.; Moussy, F. Biomacromolecules 2000, 1, 202. (11) Quinn, C. A. P.; Connor, R. E.; Heller, A. Biomaterials 1997, 18, 1665. (12) Barlı´kova´, A.; Svorc, J.; Miertus, S. Anal. Chim. Acta 1991, 247, 83. (13) Rotariu, L.; Bala, C.; Magearu, V. Anal. Chim. Acta 2002, 458, 215-222. (14) Svitel, J.; Curilla, O.; Tka´c , J. Biotechnol. Appl. Biochem. 1998, 27, 153. 10.1021/ac0340861 CCC: $25.00

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been recognized that the membrane of biological materials that include cells exhibits dielectric properties. For example, when culturing cells over electrode contacts, any change in the effective electrode impedance permits an assay of cultured cell adhesion, spreading, and mortality.15-20 Impedance measurements rely on the observation that living cells have excellent insulating properties at low frequencies. As cells grow or migrate to increase coverage over an electrode surface, the effective electrode impedance rises. Impedance measurements have been used to monitor the behavior of an array of nonexcitable cell types including macrophages,16 endothelial cells,17 and fibroblasts.18 This paper describes a new fibroblast-based sensing device for the determination of glucose using impedance spectroscopy. Indeed, 3T3-L1 fibroblasts are able to metabolize glucose through the activation of specific glucose transporters (Glut 1 and Glut 4). EXPERIMENTAL SECTION Materials. Glass slides coated with 110-nm-thick indium tin oxide (ITO) films (R < 20 Ω/cm2) were purchased from Merck Display Technologies. Ultrapure water was used throughout this study (Millipore-Q-Systems; R > 18 MΩ/cm), and all other chemical reagent were purchased from Aldrich. ITO Electrodes. The active area of these electrodes was 0.07 cm2. Before cell culture, ITO electrodes were carefully cleaned by ultrasonication, twice in 2% Fluka cleaning (ref 61257) solution for 30 min and then twice in ultrapure water for 30 min. Each sonication step was followed by 10 rinsing cycles in ultrapure water. ITO surfaces were dried under a nitrogen flow. The ITO treated with cleaner presents sufficient hydrophilicity for cell attachment (θeau ) 60°).19 The sterilization of the probes was performed using an autoclave at 137 °C and 2.4 bar for 30 min. Fibroblast Cell Monolayer. 3T3-L1 fibroblasts were obtained from American Type Culture Collection (Manassas, VA). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum, 4 mM glutamine, 4 nM insulin (Actrapid Human, Novo), 10 mM Hepes, and with 25 µg of sodium ascorbate, 100 IU of penicillin, 100 µg of streptomycin, and 0.25 µg of amphotericin B per milliliter at 37 °C in water-saturated atmosphere at 5% CO2 in air, in a Heraeus incubator (BB16). Probes were used 2-4 days after cell addition once they had reached the confluent state. Under these conditions, cells formed a thin regular monolayer. For impedance measurements, the medium was replaced by DMEM to which increasing concentrations of glucose were added. Impedance Spectroscopy. Impedance spectroscopy was recorded using a Voltalab40 (Radiometer Analytical SA) impedance analyzer, which provides the perturbation sine wave voltage signals, records the current response of the system, and calculates the complex impedance in polar coordinates |Z(ω)| and |φ(ω)|. The experimental setup is shown in Figure 1. Because of the (15) Giaever, I.; Keese, C. R. IEEE Trans. Bio-Med. Eng. 1986, 33, 242. (16) Mitra, P.; Keese, C. R.; Lawrence, D. A.; Giaever, I. Biotechniques 1990, 11, 504. (17) Xiao, C.; Lachance, B.; Sunahara, G.; Luong, J. H. T. Anal. Chem. 2002, 74, 1333. (18) Luong, J. H. T.; Habibi-Razaei, M.; Meghrous, J.; Xiao, C.; Male, K. B.; Kamen, A. Anal. Chem. 2001, 73, 1844. (19) Ehret, R.; Baumann, W.; Brischwein, M.; Schwinde, A.; Stegbauer, K.; Wolf, B. Med. Biol. Eng. Comput. 1998, 36, 365. (20) Ehret, R.; Baumann, W.; Brischwein, M.; Schwinde, A.; Stegbauer, K.; Wolf, B. Biosens. Bioelectron. 1997, 12, 29.

Figure 1. Schematic of the experimental setup. The impedance analyzer is controlled by a computer and generates the perturbation ac voltage, which is applied between the two ITO electrodes. The current response of the system is measured, and the analyzer calculates the complex impedance Z.

polarizable properties of ITO, a reference electrode was not required for Ubias ) 0 mV, but in the case of the Mott-Schottky analysis, where different bias potentials are necessary, the threeelectrode setup is applied. We used sine waves between a small ITO electrode (0.07 cm2) and a large ITO counter electrode (0.385 cm2) with an amplitude of (5mV (peak to peak) for frequencies in the range 10-2-105 Hz at five fixed values of the frequency that were equidistant on the logarithmic scale. The impedance data are evaluated by fitting (nonlinear least squares) the parameters of appropriate equivalent circuits to the experimental data using the Equivcrt.Pas (EQU) (version 4.51) program written by Boukamp.25 To relate the measured complex impedance to the electrical behavior of the system, the semiconductor, the cells, and the electrolyte were represented by simple electrical elements in equivalent circuits. As demonstrated previously,26,27 each element contributes predominantly to different frequency regimes, which allows separation of the effects of the different components of the electrical interface. However, the impedance spectra derived from equivalent circuits composed of ideal elements, like capacitances or resistance, do not fit the measured spectra in a satisfactory way because of inhomogeneities of surface. To overcome this problem, it is possible to replace the ideal elements by constant-phase elements (CPEs) CCPE,28,29 which account for the nonlinearities and the (21) Kowolenko, M.; Keese, C. R.; Lawrence, D. A.; Giaever, I. J. Immunol. Methods 1990, 127, 71. (22) Hillebrandt, H.; Abdelghani, A.; Abdelghani-Jacquin, C.; Aepfelbacher, M.; Sackmann, E. Appl. Phys. A 2001, 73, 539. (23) Giaever, I.; Keese, C. R. Nature 1993, 366, 591. (24) Lee, J. H.; Lee, S. J.; Khang, G.; Lee, H. B. J. Biomater. Sci. Polym. Ed. 1999, 10, 283. (25) Boukamp, A. B. Equivalent circuit users manual. Report CT88/265/128. Department of Chemical Technology, University of Twente, Twente, The Netherlands, 1989. (26) Stelzle, M.; Weissmu ¨ ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974. (27) Gritsch, S.; Nollert, P.; Ja¨hnig, F.; Sackmann, E. Langmuir 1998, 14, 3118. (28) MacDonald, J. R. Impedance spectroscopy, Emphasing solid materials; John Wiley and Son: New York, 1987. (29) Omanovic, S.; Metikos-Hukovis, M. Thin Solid Films 1995, 266, 31.

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frequency dependence of the elements by expressing the electrical impedance in terms of a simple power law.

ZCPE ) Kω-R

(1)

For ideal elements, the frequency exponent is R ) 1 and K ) 1/C for a capacitance and R ) 0 and K ) R for a resistance, respectively. The deviation of the exponent R from the ideal values is attributed to the inhomogeneities of the analyzed layer. By using of the CPEs, the quality of the fitting of the experimental data by equivalent circuit models can be dramatically improved. Medium for Experiments. Serum has the disadvantage of being made up of numerous components whose composition fluctuates, which renders its use difficult for reproducible experiments. For adherent cells, the intensity of adhesion is a very important test factor, which is largely dependent upon the medium. Experiments concerning the influence of serum components have already been performed with an interdigitated electrode structure.20 In this study, we have chosen to perform impedance analysis in DMEM, to improve reproducibility of measurements. RESULTS AND DISCUSSION Effect of Biological Cells on Interface Impedance. Biological cells are very poor conductors at low frequencies (at least below 10 kHz), and therefore, force electrical currents to bypass them. As cells grow adherently on the ITO electrode, this reduces the available electrode area, and the interface impedance is increased as it is inversely proportional to the total electrode area reached by the current.30 The gap between the cell membrane and the substratum limits the influence of the cell membrane capacity on the interface impedance of the electrodes. Impedance Analysis of the Electrodes/Culture Medium System without Cells. The Bode diagram of a bare semiconductor ITO electrode, immersed in culture medium DMEM (corresponding to an electrolyte/semiconductor interface), is shown in Figure 2a. The module Z showed a plateau region at 105-103 Hz and increased linearly to 10-2 Hz. The phase shift φ decrease from 105 to101 shows a constant in the vicinity of -90° from 10 to 10-2 Hz. The linear increase of |Z(ω)| from 103 to 10-2 Hz indicates a single capacitance corresponding to the semiconductor/electrolyte interface. The impedance spectrum of this system can therefore be analyzed using an equivalent circuit that consists of a resistor and a CPE (replacing the capacitor) in series. The resistor R0 is mainly due to the conductivity of the bulk solution and the wire connection whereas the CCPE represents the dielectric properties of the electrode/electrolyte and additional qualitative information on the surface morphology of the ITO electrode. Applying a fitting procedure to the Bode diagram of Figure 1a yielded the value of R0 ) 33.8 ( 0.7 Ω cm2, CCPE ) 24.0 ( 0.3 µF cm-2, and R ) 0.929 ( 0.005. Impedance Spectra of Fibroblast Cell Monolayer Grown on ITO Electrode. The influence of cells on the impedance signal is clearly shown in Figure 2b, which presents the Bode diagram with fibroblast cell monolayer 3 days after confluence. The (30) Schwan, H. P. In Determination of biological impedance in physical techniques in research, Electrophysiological Methods, part B; Nesluk, W. L., Ed.; Academic Press: New York, 1963; Vol. 6, p 323.

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Figure 2. (a) Impedance spectrum of bare ITO electrode in contact with DMEM electrolyte. The squares represents the measured data, while the solid line is the theoretical spectrum of the equivalent circuit inside the figure. (b) Impedance spectrum of ITO electrode covered with a confluent monolayer of fibroblast cells (3T3-L1).

presentation of the data in the form of a Bode impedance plot clearly reveals the presence of three separate frequency-dependent segments. In the high-frequency region, the absolute impedance curve is almost quasi-independent of frequency, when the phase angle value approaches 0°. This is a typical response for a resistive behavior, which corresponds to a resistance of the DMEM electrolyte between the working electrode and the reference electrode and to the existence of a cell monolayer on the small electrode ITO. There is not a great difference between a bare electrode and an electrode bearing a cell layer, since the junctions between the cell are very weak.31 In the intermediate frequencies, a linear relationship can be observed between the absolute impedance and the frequency with a slope of -0.8 and a phase angle of ∼-80° with a minimum at 10 Hz. This frequency range corresponds to a capacitive behavior of the electrode/electrolyte interface. In the lower frequency region, there is a change in the slope of the absolute impedance and the phase angle presents another minimum after 4 × 10-2 Hz, indicating an increase in the resistive behavior of the electrode, relative to a capacitive behavior. The impedance spectrum is interpreted in terms of the equivalent circuit shown in the inset of Figure 2b, where R0 ) (31) Wegener, J.; Sieber, M.; Galla. H, J. J. Biochem. Biophys. Methods 1996, 32, 151.

Figure 3. Nyquist diagram of the effect of D-glucose on confluent monolayer of fibroblast cells.

34.4 ( 0.7 Ω cm2, CCPE ) 68 ( 1 µF cm-2, and R ) 0.871 ( 0.004, but R ) 6.5 ( 0.3 kΩ cm2 and C ) 45 ( 2 µF cm-2 are the resistor and the capacity of the layer of the adsorbed proteins32 on the oxide surface and Rc ) 5.425 ( 0.720 Ω cm2 and Cc ) 0.76 ( 0.24 µF cm-2 corresponds to the cell layer. Effect of Glucose. Figure 3 shows the effect of glucose addition on the electrical properties of the fibroblast cell monolayer in terms of impedance spectra. In this case, the Nyquist diagram representation was preferred since such a representation allows a more sensitive analytical treatment. Analyses have been performed in a wide range of glucose concentrations from 1 to 23 mM, which is wider than the physiological range to test the responsiveness of our biosensor. Each glucose addition induced a change of the Nyquist diagram, which comes from a change both in the resistance and in the capacity of the cell monolayer. This variation is correlated to the change of the dielectric properties σm and m of cellular membrane under the effect of D-glucose.33 As no impedance variation was recorded after glucose additions in the case of a bare ITO electrode, the variations recorded are due to an “interaction” between the cell monolayer and glucose molecules. This interaction could correspond to the glucose uptake through Glut 1 and Glut 4 transporters present on the plasma membrane of the fibroblasts. Figure 4 shows that, at glucose concentrations below 14 mM, a typical calibration graph of the absolute impedance response at 4 kHz of fibroblast/ITO electrode versus glucose concentration is produced. But when the concentration goes over 14 mM, the system reaches a saturation, as can be clearly seen from the figure. To correlate this variation of impedance to fibroblast behavior, the fitting procedure with the previous equivalent circuit, defined without glucose, is applied to impedance variation as a function of glucose concentration. Values of R0, CCPE, R, and C, remaining constant in the equivalent circuit, values of Rc and Cc components vary as a function of glucose concentration as shown in Figure 5. The error bars include the experimental error on impedance measurements and the discrepancy of experimental values related (32) Hiromoto, S.; Noda, K.; Hanawa, T. Corros. Sci. 2002, 44, 955. (33) Caduff, A.; Hayashi, Y.; Livshits, L.; Feldman, Y. 2nd International Conference on Broadband Dielectric Spectroscopy and Its Applications, Leipzig, Germany, September 2-6, 2002.

Figure 4. Calibration plot of the glucose biosensor. The signal is linear from 0 to 14 mM glucose concentration and reaches saturation for higher concentration. Error bars were determined from three experimental results on the same electrode.

Figure 5. Variation of the Rc (9) and Cc (b) versus D-glucose concentration with calculated error bars according to the equivalent circuit used to interpret the experimental variations of Z modulus versus glucose concentration.

to the model equivalent circuit. The general evolution of Rc and Cc shows that they translate the fibroblast behavior, and a saturation effect is observed for both components for concentrations higher than 14 mM, which corresponds to the saturation effect on the experimental curve (cf. Figure 4). To highlight the specificity of the cells toward glucose and to reinforce the hypothesis of an impedancemetric signal correlated to glucose uptake by fibroblasts, the same experiments have been performed in the presence of D-mannitol instead of glucose, a sweetener not metabolized by the cells. As shown in the Nyquist diagram presented in Figure 6, no variation was recorded for D-mannitol additions in the range from 1 to 15 mM. Consequently, the changes in capacity and resistance of the cell monolayer observed in the presence of glucose (Figure 3) result from the incorporation of glucose in the fibroblast monolayer itself and not from indirect changes induced by the increased concentrations of glucose such as, for example, increased osmotic pressure. Inhibition of Glucose Uptake. To strengthen our hypothesis, we inhibited the glucose transporters of the 3T3-L1 fibroblasts. This was achieved by using cytochalasin B, which inhibits Glut Analytical Chemistry, Vol. 75, No. 14, July 15, 2003

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Figure 6. Nyquist diagram of the effect of D-mannitol on confluent monolayer of fibroblast cells.

1- and Glut 4-mediated glucose uptake.34 The cell monolayer was exposed for 10 min to 10-4 M cytochalasin B. Then cytochalasin B was removed and replaced by a DMEM solution to which increasing quantities of glucose were added. Figure 7 presents the Nyquist diagram after inhibition by cytochalasin B and glucose additions (10 and 20 mM). After glucose additions, the curves are superimposed, which indicates that the resistance and the capacitance of the cell monolayer are not modified and consequently that glucose is no larger incorporated into the cells. These results demonstrate that the changes in the resistance and in the capacity of the cell monolayer recorded in Figure 2 directly result from glucose uptake and metabolization of glucose by the cells. CONCLUSIONS The present study shows for the first time the feasibility of glucose sensing by fibroblasts over the physiological range of glucose concentrations, using impedancemetry analysis. Indeed, (34) Karnieli, E.; Zaenowski, M. J.; Hissin, P. J.; Simpson, I. A.; Salans; L. B.; Cushman, S. W. J. Biol. Chem. 1981, 256, 4772.

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Figure 7. Nyquist diagram of the inhibition of glucose uptake by cytochalasin B on confluent monolayer of fibroblast cells.

the addition of a carbohydrate not metabolized by cells did not generate any change in the impedance parameters of the sensor. Furthermore, inhibiting glucose uptake by cytochalasin B, a specific inhibitor of glucose transporters, completely abolished the signals relating to the modification of glucose concentration. Further studies, over a wider frequency range, are under way in order to improve the intensity of the performance of the sensing device. Subsequently, this system could furnish fresh insights for a better understanding of glucose metabolism in cells. ACKNOWLEDGMENT The authors acknowledge financial support from MIRA Research Program and valuable discussion with Professor Belcourt Alain, Vice-President and Director Centre Europeen D’Etude du Diabete.

Received for review January 29, 2003. Accepted April 25, 2003. AC0340861