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64-Channel extended gate electrode arrays for extracellular signal recording
Electrochimica Acta 48 (2003) 3355 /3362 www.elsevier.com/locate/electacta
64-Channel extended gate electrode arrays for extracellular signal recording H. Ecken a, S. Ingebrandt a, M. Krause b, D. Richter b, M. Hara c, A. Offenha¨usser a,* a
Institute for Thin Films and Interfaces (ISG2), Research Center Ju ¨ elich, D-52425 Juelich, Germany b Max Planck Institute for Polymer Research, D-55128 Mainz, Germany c The Institute of Physical and Chemical Research (RIKEN), Frontier Research System, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 10 December 2002; received in revised form 24 March 2003; accepted 10 April 2003
Abstract A 64-channel amplifier system for the recording of extracellular signals with planar metal microelectrodes is presented. Gold metal microelectrodes on glass wafers were fabricated using standard photolithographic techniques. The measurement system was divided into a headstage preamplifier and a main amplifier. The inherent noise of the extracellular recording system was minimized by using an independent battery supply. The metal electrodes were directly connected to the gates of low noise junction field effect transistors (JFETs) using a specially designed electronic circuit. With this set-up, it was possible to record extracellular signals with planar metal microelectrodes without any surface modification for impedance reduction. A feedback circuit in the first amplification stage compensated slow drifts of the gold microelectrodes, which made online sampling of all 64 channels with a sampling rate of 10 kHz possible. Recordings were taken from rat cardiac myocytes cultured on fibronectin coated sensor chips. The system exhibited a good signal-to-noise ratio. It was able to detect the signal propagation within the cardiac cell layer and it could be used for pharmacological investigations involving the heart. # 2003 Elsevier Ltd. All rights reserved. Keywords: Extended gate electrodes; Extracellular recording; Sensor array; Action potential; Rat cardiac myocytes
1. Introduction Recording of extracellular signals from living cells cultured on planar devices has been the subject of intense studies in recent years [1 /5]. The concept of biosensors utilizing living cells as the sensing element coupled to planar metal microelectrodes offers the possibility of gaining information about the reaction between cells and external stimuli. The cell-integrated extracellular sensor hybrid-system can be used for many applications such as drug screening [6], neurophysiology [7], toxicology [8], and environmental measurements [9]. This system shows very high sensitivity to minute amount of chemical changes within the cellular environment. The concept of extracellular sensors has several advantages compared with traditional methods of internal recording of cells using glass capillaries, patch * Corresponding author Tel./fax: /49-2461-612-330. E-mail address: [email protected] (A. Offenha¨usser). 0013-4686/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0013-4686(03)00405-5
clamp electrodes [10] or fluorescence methods with voltage sensitive dyes [11]. The major disadvantage of optical methods is the toxicity of the dyes to the cells upon illumination, which makes it unsuitable for longterm recordings. However, with extracellular sensors, non-invasive long-term recordings of up to several weeks have been demonstrated [12]. We have shown that it was possible to observe the development of the cells in vitro with multiple recordings during the day and incubating them overnight [6]. There are already commercially available measurement systems [13]. However, all these systems need to perform a surface modification with porous platinum coating [14] or TiN micro pillars [15] of the microelectrode in order to lower the input impedence. These coatings change the electrical properties of the microelectrodes and they can degrade over time. With the 64-channel system presented in this article we were able to record extracellular signals from planar microelectrode chips. The sensor chips are reusable for up to 100 cultures and therefore very robust
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but inexpensive. With the almost dc-like amplification unit, we were able to interpret signal shapes of the recorded action potentials [16]. The system has a very good signal to noise ratio and by using an electrode array it was possible to map the propagation of electrical excitation-couplings inside the cell layer. By using a positive chronotrope, norepinephrine, which stimulates and increases the rate of the beating cardiac myocyte layer, we demonstrated the possible use of the system in pharmacological bioassays.
2. Materials and methods 2.1. Experimental set-up 2.1.1. Preamplifier headstage The extended gate electrode (EGE) preamplifier headstage was developed based on the knowledge gained from a former preamplifier unit [17]. The housing of the headstage consisted of a ground plate and a cover with an opening in the middle of the upper lid. It was designed for the use with either an inverted or an upright microscope. In the ground plate the electronic printed circuit board (PCB) with all electronic components in surface mount technique (SMD) were fixed (Fig. 1). On this board, a modified test socket (PLCC68 T/B IC51, YAMAICHI Inc.) was fixed, which provided 68 gilt spring contacts. The top cover of this test socket has been re-designed in black anodized aluminum. This cover acted as an additional shield and held an isolated Ag/AgCl ground electrode. The test socket provided a quick and reliable exchange of the EGE-chips. There were two connectors at the back of the EGE headstage. One connector (68 sub D) contained the data lines and one connector (50 sub D) was used for the voltage supply of the headstage.
Fig. 1. Preamplifier headstage with a mounted microelectrode array. The connectors of the external power supply and the signal lines of the microelectrodes are located at the backside.
2.1.2. Preamplifier circuit Essential elements of the first amplification circuit are low noise junction FETs (JFET PMBF5484 from Philips Semiconductors, USA). These transistors exhibited in the relevant frequency range (0.1 Hz /10 kHz) with a high signal strength and a very low inherent noise. The metal electrodes of the EGE array were directly connected to the gates of these JFETs [17]. The electrolyte solution was grounded with an Ag/AgCl electrode at the socket cover, which acted as ground potential when the gate-source and the drain-source voltages were applied to the JFETs. The voltage supply for the JFETs was located inside the main amplifier stage. The input at the metal microelectrode was converted into a current signal IDS by the JFET. An operational amplifier (OP-97F Analog Devices Inc., USA) was used for the I /V conversion with V /5 kOhm /I (Fig. 2). The converted current inputs were guided through the data lines as preamplified voltage signals. Therefore, the data lines did not have to be particularly shielded. In the predefined working point, a constant working current of the JFET in the 0.5 mA range superimposed the measurement signals Isignal, which were typically in the 10 nA range.
2.1.3. 64-Channel main amplifier The 64-channel main amplifier used a feedback circuitry for the compensation of this constant dc-part of the recorded signal (Fig. 2). The compensation was performed using a feedback loop, which subtracted the output signal from the baseline. The time constant of the feedback loop was in the second range and the 3 dB point of the set-up lied at 1.82 Hz. Therefore, the set-up was no longer a real-time dc measurement set-up compared to the set-up described in Ref. [17], but it left the shape of the slowest heart beat signals (up to 300 ms) unchanged. The advantages of it being that it compensated the very slow dc drifts of the FET and EGE devices caused by ionic and temperature changes. The set-up used an independent battery voltage supply for all electronic components. It is completely controlled with a standard PC (minimum requirements: PII with 400 MHz and a fast 10 GB hard disc), a PCI 6071 E data acquisition card from National Instruments, USA, and a 24-channel digital in- and output card (PIO-24 I/O card, BMC Messysteme, Germany). For data acquisition, the Panasonic MED64 Conductor 3.1 software was used. The front panel of the 64-channel main amplifier provided 64 plugs for additional control of the amplified signals with an oscilloscope. The almost dc-like measurement set-up without any active filtering elements offered the possibility of gaining deeper knowledge about signal shapes of the recorded action potentials [16 /18].
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Fig. 2. Block diagram of the extended gate amplification circuit. The 64 microelectrodes are connected to the gates of junction FETs. Current signals (modulated by the electrode potentials) are converted to voltages in the headstage, compensated and amplified in the main amplifier.
2.2. Sensor arrays The EGE arrays were manufactured using standard silicon planar technology. The electrodes were arranged in an 8 /8 matrix with a spacing of either 100 or 200 mm. Details of the sensor production process have been described [17]. In short, the gold interconnecting lanes, microelectrodes and bondpads were fabricated on 5ƒ glass wafers (¥ 125 mm, Borofloat 33, SCHOTT DESAG AG, Germany) using electron beam evaporation and a lift off process. For better adhesion of the 300nm gold on glass substrates, it was sandwiched between two 30 nm titanium layers. Three layers of 500 nm SiO2, 500 nm Si3N4 and 100 nm SiO2 were deposited using plasma enhanced chemical vapor deposition (PECVD) for stable passivation against the electrolyte. Sensor spots and outer electrode contact holes (bondpads) were opened with reactive ion technique (RIE) in CHF3 atmosphere and with wet etching of the upper titanium layer in a NH4F-mixture.
center and gold galvanized interconnecting lanes. In the first step, a conductive two-component epoxy glue (EPO-TEK H20E-PFC; Epoxy Technology Inc., USA) was printed on the contact areas with a screen printer (ESSEMTEC SP-002, ESSEMTEC AG, Switzerland). With the aid of a very precise xy -positioning system (Fineplacer 96, Finetech, Germany) the EGE arrays were glued onto the contact points of the PCB. After curing of the conductive glue, the gaps between the contacts were filled with a dielectric two component underfill (U300, Epoxy Technology Inc., USA) in order to prevent short-circuits between individual contacts. A petri-dish for cell culture was formed by gluing two glass rings with diameters of 7 mm and 16 mm and filling up the space between the rings with (poly)dimethylsiloxane (PDMS) (Sylgard 96-083, Dow Corning, Germany). The free area for cell culture is 38.5 mm2 and the sensor chip was able to hold 600 ml of culture medium. The fully encapsulated EGE array is shown in Fig. 3. 2.4. Chip cleaning and protein coating
2.3. Encapsulation of the EGE chips Compared to the sensor arrays used in previous studies [17] we tried to minimize cost and time of the encapsulation process. The single EGE chips (11 /11 mm2) were mounted with flip chip technology [19] on a 24 /24 mm2 and 1 mm thick printed circuit board (PCB). The PCB board has a 9 /9 mm2 opening in the
Before the culturing of living cells on the sensor surfaces, the EGE arrays have to be cleaned and primed with biocompatible protein layers. The encapsulated sensors are very robust and can be used several times for culturing of cells. Initially, mechanical cleaning of cell debris using cotton buds was performed. It was followed by ultrasonification for 10 min in 2 % detergent
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Fig. 3. EGE recording device. Two glass rings are forming the cell culture dish.
(Hellmanex II, Helma GmbH & Co. KG, Mu¨hlheim, Germany), a rinsing step with Milli-Q water and further ultrasonification in Milli-Q water. The main cleaning step was treating the cell culture containers with 20% sulphuric acid for 30 min at 80 8C followed by intensive rinsing with Milli-Q water. Immersing the cleaned sensor arrays in 70% ethanol solution was adequate for sterilisation. An intensive cleaning procedure for the sensors appeared to be crucial in maintaining optimal cell culture conditions. It is very important that the main cleaning materials, the detergent and the sulphuric acid, were completely washed off before the subsequent steps. The clean sensor surfaces were coated with fibronectin. The function of the fibronectin was to make the SiO2 surface biocompatible and to promote a close adherence of the cells onto the surface. All steps for coating the sensors with fibronectin were performed in a sterile clean bench. The sensors were removed from the storage ethanol and rinsed two times with autoclaved Milli-Q water followed by a third rinsing step in PBS-Buffer (Dulbeco, GibcoBRL). The sensitive areas of the chips were plated with 60 ml of 5 mg ml 1 fibronectin solution. The sensors were then incubated for 60 min at 37 8C in an atmosphere enriched with 5% CO2. After this incubation period, excess fibronectin solution was removed and the surfaces were gently rinsed three times with PBS.
matic dissociation was done with DNAse Type II solution (10 000 U ml 1). A solution containing HAMS F10 solution with 36% foetal calf serum (FCS), 0.5% insulin, transferrin, selenite, (ITS) solution, 6 mM L-glutamine and 2% penicillin/streptomycin mixture (5000 U per 5 mg ml 1) (all from Sigma, Germany) was used to stop trypsinization. The cell suspension was centrifuged five minutes at 1500 rpm to get rid of remaining blood or cell debris. The pellet was resuspended with HAMS F10 solution (10% FCS, 0.5% ITS, 6 mM glutamine and 2% antibiotic mixture) in cell culture flasks and incubated for two hours at 37 8C and 5% CO2. The incubation led to an accumulation of fibroblasts on the surface of the flasks preferentially over cardiac myocytes leaving a higher proportion of cardiac myocytes in suspension. The cells in the final suspension were adjusted to the desired cell concentration and transferred onto the fibronectin primed EGE arrays. One preparation resulted in 1/2 ml cell suspension of 1 million cells per ml. This quantity was sufficient to plate 25 /50 EGE arrays. After approximately three hours, the cell culture chambers were filled with medium (HAMS F10). Medium exchange was done every second day and recordings began after cells had formed a complete syncytium on the surface (2 /3 DIV). In Fig. 4 a cardiac myocyte layer (4 DIV) cultured on an EGE array is shown.
2.5. Cell culture
2.6. Pharmacology
The cell culture of embryonic cardiac myocytes was carried out following the protocol described in [20]. In short, hearts of about ten rat fetuses (Sprague Dawley; embryonic days 15 /18, E15 /E18) were removed under sterile conditions, minced and trypsinized. The enzy-
To validate the possible application of the EGE system in pharmacological bioassays, we used a wellestablished positive chronotrope, norepinephrine (NE), at three different concentrations (10 nM, 100 nM, 1mM). The different concentrations were made up in standard
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and the final concentrations were then prepared using the extracellular solution.
3. Results
Fig. 4. Cardiac myocytes cell-layer (4 DIV) being grown on a microelectrode chip.
extracellular solution (in mM: KCl, 5; NaCl, 150; MgCl2, 1; HEPES, 10; CaCl2, 2.5; glucose, 10; pH 7.35 at 22 8C, adjusted with 1 N NaOH). For the measurements, the culture medium was exchanged using extracellular solution and equilibrated in the incubator for 30 min before the experiments. The stock solution of NE (1 mM) was made up using 1/100 N HCl as vehicle
In Fig. 5, extracellular recorded signals from a cardiac myocyte layer (4 DIV) are shown. The signals were simultaneously measured from 64 differently localized gold microelectrodes (electrode diameter 30 mm; spacing 200 mm, sampling rate 10 kHz). Each signal represents the time course of the drain-source-current through the JFET connected to the microelectrode. In the time scale shown (10 s) three beats of the cell layer were detected. The signals were regular at approximately every four seconds. Some electrodes (e.g. the electrodes 30 and 46) did not show any signal, which was caused by an insufficient electrical contact between the microelectrode chip and the amplification unit. Some other electrodes showed only very weak signals e.g. the electrodes 41 and 48. The signal amplitude and the strength of the cell electrode contact were influenced by the position and the distance of the electrically active cell on the electrode [18]. Only an adequate seal of the microelectrode led to a good coupling. Best coupling in this experiment was
Fig. 5. Extracellular recordings of action potentials of a cardiac myocytes cell-layer (4 DIV) with a 64-channel EGE device.
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Fig. 6. Propagation of action potentials of cardiac myocytes cell-layer (4 DIV) recorded with 64-channel EGE device.
recorded with electrode 55 (amplitude 2 mV; signal-tonoise 11:1). The 64-channel recording can be used to localize the origin of the excitation (the so-called pacemaker center). From there, the electrical excitation spread out as an excitation wave within a few milliseconds over the entire cell layer. During the propagation of the excitation wave a temporal delay occurs in the measured signals at different places. This can clearly be seen in Fig. 6, which shows a single action potential of the cell layer is shown higher temporal resolution. The time window (20 ms) in Fig. 6 allows pursuing the signal propagation over the entire 8 /8 electrode matrix. The first signal was detected with electrode 8. Then the excitation wave-respectively the action potentials-propagates to the left and to the center. Subsequently, also the lower electrodes register the contraction of the cell layer. Therefore the pacemaker center must be located outside of the 8 /8 electrode matrix, but closest to electrode 8. The temporal difference of the signals between electrode 8 and electrode 57 is about 13 ms. The stimulative effect of the positive chronotrope norepinephrine in terms of changing the beating rate of the cell layer is shown in Fig. 7. The initial extracellular
solution and the drug-containing solutions were warmed to 37 8C before the experiment. Drugs were applied by completely exchanging the solution with a solution containing the next higher concentration. Traces of 10 s time period were shown for the initial state (no NE) resulting in a beating rate of 24 bpm, 10 nM NE resulting in 48 bpm, 100 nM NE resulting in 90 bpm and 1 mM NE resulting in 114 bpm. It can clearly be seen that the chronotrope stimulated the cells to higher beating rates.
4. Conclusion We developed a 64-channel amplifier system for the recording of extracellular signals with planar metal microelectrodes. The described extended gate electrode concept of having the metal microelectrodes directly connected to low noise JFETs offers the possibility to record extracellular signals using planar metal microelectrodes without modification of the electrode surface. With a special circuit for the compensation of dc offsets and baseline drifts, the system can sample all channels simultaneously at 10 kHz in real-time. We recorded
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tissue significantly. We have shown in a previous study [6] that the recordings obtained with the extracellular system can be used as reflection of the physiological responses of the whole organ in vivo. However there are still several refinements required in order to improve the sensitivity and durability of the sensor chips and of the recording system as a whole. The interpretation of the extracellular recorded signal shapes is still the subject of intense study [16,22]. The origin of the too high recorded signals amplitudes is still unclear and needs to be further investigated. Although several improvements and investigations are still required, the system at its present form is already capable of performing basic pharmacological experiments.
Acknowledgements
Fig. 7. Stimulation of a cardiac myocyte layer using a positive chronotropic agent, norepinephrine, at different concentrations.
signals from cardiac myocytes cultured on the sensor surfaces. The signal-to-noise ratios varied depending on the quality of the cell-electrode contacts. The best signalto-noise ratio was 11:1 and extracellular signal amplitudes of up to 2 mV were recorded. With this system, it is possible to pinpoint the focal point (or foci) of the cardiac myocyte layer and the development of the electrical excitation pattern can be followed over several days. In the first pharmacological bioassay experiment, we validated the system by using a positive chronotrope, norepinephrine, for the stimulation of the cell layer. We have chosen the cardiac myocyte culture for this study, because these cells provide auto-rhythmic extracellular signals to evaluate the EGE system without the use of artificial stimulation. Extracellular recording systems were used as additional tools in electrophysiology as a substitute for existing optical and intracellular methods. A review for cell-based biosensors has been published by Pancrazio et al. [21]. The concept of extracellular recording has several advantages compared to classical techniques such as patch-clamp recording or fluorescence methods. The recording is non-invasive, can be up-scaled to many different localized sensor spots and long-term measurements over several days can be performed as long as sterility of the cell culture is maintained. With a single cell culture up to 50 electrode chips can be prepared, which reduces the use of animal
We would like to acknowledge Prof. Dr. W. Knoll of the Max-Planck-Institute for Polymer Research, Mainz, with whom this project was initiated. We would like to acknowledge Dr. C.-K. Yeung of the Department of Ophthalmology and Visual Sciences, the Chinese University of Hong Kong, for establishing the experimental methods using cardiac stimulants and relaxants in this project and for the critical review of this manuscript. We thank Mr. Lacher, Dr. T. Zetterer and Mr. W. Staab from the Institute for Microtechnique in Mainz for the support during fabrication of the EGE sensor chips. We acknowledge the excellent technical support of Mr. Mu¨ller and the members of the electronic laboratory, and Mr. Gerstenberg and Mr. Christ from the mechanical workshop of the MPI for Polymer Research. We would like to thank Prof. Dr. M. J. Scho¨ning from the Research Center Ju¨elich and the Fachhochschule Ju¨elich for valuable discussions and support during this work. We would like to acknowledge Mrs. J. Hayashi from the RIKEN Institute, Frontier Research System, Wako-shi, Japan for the cell culture. All the measurements were done at the RIKEN institute. This work was partially funded by the Northrhine /Westphalian Ministry of Schools, Education, Science and Research (Project: ELMINOS).
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[6] C.K. Yeung, S. Ingebrandt, M. Krause, A. Offenha¨usser, W. Knoll, J. Pharmacol. Toxicol. Methods 45 (2001) 207. [7] S.I. Morefield, E. W: Keefer, K.D. Chapman, G.W. Gross, Biosens. Bioelectron. 15 (2000) 383. [8] M. Lehmann, W. Baumann, M. Brischwein, R. Ehret, M. Kraus, A. Schwinde, M. Bitzenhofer, I. Freund, B. Wolf, Biosens. Bioelectron. 15 (2000) 117. [9] B.D. DeBusschere, G.T.A. Kovacs, Biosens. Bioelectron. 16 (2001) 543. [10] L.A. Geddes, Principles of Applied Biomedical Instrumentation. Wiley, New York, 1992, p. 137. [11] A. Grinvald, W.N. Ross, I.C. Farber, Proc. Natl. Acad. Sci. USA 78 (1981) 3245. [12] E.W. Keefer, A. Gramowski, G.W. Gross, J. Neurophys. 86 (2001) 3030. [13] Multi Channel Systems, Germany: http://www.multichannelsystems.com, MED systemsTM, Panasonic, Japan: http:// www.med64.com, Center for Network Neuroscience, UNT, USA: http://129.120.38.103.
[14] D.A. Robinson, Proc. IEEE 56 (1968) 1065. [15] W. Nisch, J. Bock, U. Egert, H. Hammerle, A. Mohr, Biosens. Bioelectron. 9 (1994) 737. [16] S. Ingebrandt, C.K. Yeung, M. Krause, A. Offenhausser, Biosens. Bioelectron. 16 (2001) 565. [17] M. Krause, S. Ingebrandt, D. Richter, M. Denyer, M. Scholl, C. Spro¨ssler, A. Offenha¨usser, Sensors and Actuators B 70 (2000) 101. [18] C. Spro¨ssler, M. Denyer, S. Britland, W. Knoll, A. Offenha¨usser, Phys. Rev. E 60 (1999) 2171. [19] J.H. Lau, Flip Chip Technologies. McGraw-Hill, New York, 1995, p. 223. [20] M.C.T. Denyer, M. Riehle, J. Hayashi, M. Scholl, C. Sproessler, S.T. Britland, A. Offenhaeusser, W. Knoll, In Vitro Cell. Dev. Biol. Anim. 35 (1999) 352. [21] J.J. Pancrazio, J.P. Whelan, D.A. Borkholder, W.A. Ma, D.A. Stenger, Ann. Biomed. Eng. 27 (1999) 697. [22] S. Vassanelli, P. Fromherz, J. Neurosci. 19 (1999) 6767.
64-Channel extended gate electrode arrays for extracellular signal recording H. Ecken a, S. Ingebrandt a, M. Krause b, D. Richter b, M. Hara c, A. Offenha¨usser a,* a
Institute for Thin Films and Interfaces (ISG2), Research Center Ju ¨ elich, D-52425 Juelich, Germany b Max Planck Institute for Polymer Research, D-55128 Mainz, Germany c The Institute of Physical and Chemical Research (RIKEN), Frontier Research System, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 10 December 2002; received in revised form 24 March 2003; accepted 10 April 2003
Abstract A 64-channel amplifier system for the recording of extracellular signals with planar metal microelectrodes is presented. Gold metal microelectrodes on glass wafers were fabricated using standard photolithographic techniques. The measurement system was divided into a headstage preamplifier and a main amplifier. The inherent noise of the extracellular recording system was minimized by using an independent battery supply. The metal electrodes were directly connected to the gates of low noise junction field effect transistors (JFETs) using a specially designed electronic circuit. With this set-up, it was possible to record extracellular signals with planar metal microelectrodes without any surface modification for impedance reduction. A feedback circuit in the first amplification stage compensated slow drifts of the gold microelectrodes, which made online sampling of all 64 channels with a sampling rate of 10 kHz possible. Recordings were taken from rat cardiac myocytes cultured on fibronectin coated sensor chips. The system exhibited a good signal-to-noise ratio. It was able to detect the signal propagation within the cardiac cell layer and it could be used for pharmacological investigations involving the heart. # 2003 Elsevier Ltd. All rights reserved. Keywords: Extended gate electrodes; Extracellular recording; Sensor array; Action potential; Rat cardiac myocytes
1. Introduction Recording of extracellular signals from living cells cultured on planar devices has been the subject of intense studies in recent years [1 /5]. The concept of biosensors utilizing living cells as the sensing element coupled to planar metal microelectrodes offers the possibility of gaining information about the reaction between cells and external stimuli. The cell-integrated extracellular sensor hybrid-system can be used for many applications such as drug screening [6], neurophysiology [7], toxicology [8], and environmental measurements [9]. This system shows very high sensitivity to minute amount of chemical changes within the cellular environment. The concept of extracellular sensors has several advantages compared with traditional methods of internal recording of cells using glass capillaries, patch * Corresponding author Tel./fax: /49-2461-612-330. E-mail address: [email protected] (A. Offenha¨usser). 0013-4686/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0013-4686(03)00405-5
clamp electrodes [10] or fluorescence methods with voltage sensitive dyes [11]. The major disadvantage of optical methods is the toxicity of the dyes to the cells upon illumination, which makes it unsuitable for longterm recordings. However, with extracellular sensors, non-invasive long-term recordings of up to several weeks have been demonstrated [12]. We have shown that it was possible to observe the development of the cells in vitro with multiple recordings during the day and incubating them overnight [6]. There are already commercially available measurement systems [13]. However, all these systems need to perform a surface modification with porous platinum coating [14] or TiN micro pillars [15] of the microelectrode in order to lower the input impedence. These coatings change the electrical properties of the microelectrodes and they can degrade over time. With the 64-channel system presented in this article we were able to record extracellular signals from planar microelectrode chips. The sensor chips are reusable for up to 100 cultures and therefore very robust
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but inexpensive. With the almost dc-like amplification unit, we were able to interpret signal shapes of the recorded action potentials [16]. The system has a very good signal to noise ratio and by using an electrode array it was possible to map the propagation of electrical excitation-couplings inside the cell layer. By using a positive chronotrope, norepinephrine, which stimulates and increases the rate of the beating cardiac myocyte layer, we demonstrated the possible use of the system in pharmacological bioassays.
2. Materials and methods 2.1. Experimental set-up 2.1.1. Preamplifier headstage The extended gate electrode (EGE) preamplifier headstage was developed based on the knowledge gained from a former preamplifier unit [17]. The housing of the headstage consisted of a ground plate and a cover with an opening in the middle of the upper lid. It was designed for the use with either an inverted or an upright microscope. In the ground plate the electronic printed circuit board (PCB) with all electronic components in surface mount technique (SMD) were fixed (Fig. 1). On this board, a modified test socket (PLCC68 T/B IC51, YAMAICHI Inc.) was fixed, which provided 68 gilt spring contacts. The top cover of this test socket has been re-designed in black anodized aluminum. This cover acted as an additional shield and held an isolated Ag/AgCl ground electrode. The test socket provided a quick and reliable exchange of the EGE-chips. There were two connectors at the back of the EGE headstage. One connector (68 sub D) contained the data lines and one connector (50 sub D) was used for the voltage supply of the headstage.
Fig. 1. Preamplifier headstage with a mounted microelectrode array. The connectors of the external power supply and the signal lines of the microelectrodes are located at the backside.
2.1.2. Preamplifier circuit Essential elements of the first amplification circuit are low noise junction FETs (JFET PMBF5484 from Philips Semiconductors, USA). These transistors exhibited in the relevant frequency range (0.1 Hz /10 kHz) with a high signal strength and a very low inherent noise. The metal electrodes of the EGE array were directly connected to the gates of these JFETs [17]. The electrolyte solution was grounded with an Ag/AgCl electrode at the socket cover, which acted as ground potential when the gate-source and the drain-source voltages were applied to the JFETs. The voltage supply for the JFETs was located inside the main amplifier stage. The input at the metal microelectrode was converted into a current signal IDS by the JFET. An operational amplifier (OP-97F Analog Devices Inc., USA) was used for the I /V conversion with V /5 kOhm /I (Fig. 2). The converted current inputs were guided through the data lines as preamplified voltage signals. Therefore, the data lines did not have to be particularly shielded. In the predefined working point, a constant working current of the JFET in the 0.5 mA range superimposed the measurement signals Isignal, which were typically in the 10 nA range.
2.1.3. 64-Channel main amplifier The 64-channel main amplifier used a feedback circuitry for the compensation of this constant dc-part of the recorded signal (Fig. 2). The compensation was performed using a feedback loop, which subtracted the output signal from the baseline. The time constant of the feedback loop was in the second range and the 3 dB point of the set-up lied at 1.82 Hz. Therefore, the set-up was no longer a real-time dc measurement set-up compared to the set-up described in Ref. [17], but it left the shape of the slowest heart beat signals (up to 300 ms) unchanged. The advantages of it being that it compensated the very slow dc drifts of the FET and EGE devices caused by ionic and temperature changes. The set-up used an independent battery voltage supply for all electronic components. It is completely controlled with a standard PC (minimum requirements: PII with 400 MHz and a fast 10 GB hard disc), a PCI 6071 E data acquisition card from National Instruments, USA, and a 24-channel digital in- and output card (PIO-24 I/O card, BMC Messysteme, Germany). For data acquisition, the Panasonic MED64 Conductor 3.1 software was used. The front panel of the 64-channel main amplifier provided 64 plugs for additional control of the amplified signals with an oscilloscope. The almost dc-like measurement set-up without any active filtering elements offered the possibility of gaining deeper knowledge about signal shapes of the recorded action potentials [16 /18].
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Fig. 2. Block diagram of the extended gate amplification circuit. The 64 microelectrodes are connected to the gates of junction FETs. Current signals (modulated by the electrode potentials) are converted to voltages in the headstage, compensated and amplified in the main amplifier.
2.2. Sensor arrays The EGE arrays were manufactured using standard silicon planar technology. The electrodes were arranged in an 8 /8 matrix with a spacing of either 100 or 200 mm. Details of the sensor production process have been described [17]. In short, the gold interconnecting lanes, microelectrodes and bondpads were fabricated on 5ƒ glass wafers (¥ 125 mm, Borofloat 33, SCHOTT DESAG AG, Germany) using electron beam evaporation and a lift off process. For better adhesion of the 300nm gold on glass substrates, it was sandwiched between two 30 nm titanium layers. Three layers of 500 nm SiO2, 500 nm Si3N4 and 100 nm SiO2 were deposited using plasma enhanced chemical vapor deposition (PECVD) for stable passivation against the electrolyte. Sensor spots and outer electrode contact holes (bondpads) were opened with reactive ion technique (RIE) in CHF3 atmosphere and with wet etching of the upper titanium layer in a NH4F-mixture.
center and gold galvanized interconnecting lanes. In the first step, a conductive two-component epoxy glue (EPO-TEK H20E-PFC; Epoxy Technology Inc., USA) was printed on the contact areas with a screen printer (ESSEMTEC SP-002, ESSEMTEC AG, Switzerland). With the aid of a very precise xy -positioning system (Fineplacer 96, Finetech, Germany) the EGE arrays were glued onto the contact points of the PCB. After curing of the conductive glue, the gaps between the contacts were filled with a dielectric two component underfill (U300, Epoxy Technology Inc., USA) in order to prevent short-circuits between individual contacts. A petri-dish for cell culture was formed by gluing two glass rings with diameters of 7 mm and 16 mm and filling up the space between the rings with (poly)dimethylsiloxane (PDMS) (Sylgard 96-083, Dow Corning, Germany). The free area for cell culture is 38.5 mm2 and the sensor chip was able to hold 600 ml of culture medium. The fully encapsulated EGE array is shown in Fig. 3. 2.4. Chip cleaning and protein coating
2.3. Encapsulation of the EGE chips Compared to the sensor arrays used in previous studies [17] we tried to minimize cost and time of the encapsulation process. The single EGE chips (11 /11 mm2) were mounted with flip chip technology [19] on a 24 /24 mm2 and 1 mm thick printed circuit board (PCB). The PCB board has a 9 /9 mm2 opening in the
Before the culturing of living cells on the sensor surfaces, the EGE arrays have to be cleaned and primed with biocompatible protein layers. The encapsulated sensors are very robust and can be used several times for culturing of cells. Initially, mechanical cleaning of cell debris using cotton buds was performed. It was followed by ultrasonification for 10 min in 2 % detergent
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Fig. 3. EGE recording device. Two glass rings are forming the cell culture dish.
(Hellmanex II, Helma GmbH & Co. KG, Mu¨hlheim, Germany), a rinsing step with Milli-Q water and further ultrasonification in Milli-Q water. The main cleaning step was treating the cell culture containers with 20% sulphuric acid for 30 min at 80 8C followed by intensive rinsing with Milli-Q water. Immersing the cleaned sensor arrays in 70% ethanol solution was adequate for sterilisation. An intensive cleaning procedure for the sensors appeared to be crucial in maintaining optimal cell culture conditions. It is very important that the main cleaning materials, the detergent and the sulphuric acid, were completely washed off before the subsequent steps. The clean sensor surfaces were coated with fibronectin. The function of the fibronectin was to make the SiO2 surface biocompatible and to promote a close adherence of the cells onto the surface. All steps for coating the sensors with fibronectin were performed in a sterile clean bench. The sensors were removed from the storage ethanol and rinsed two times with autoclaved Milli-Q water followed by a third rinsing step in PBS-Buffer (Dulbeco, GibcoBRL). The sensitive areas of the chips were plated with 60 ml of 5 mg ml 1 fibronectin solution. The sensors were then incubated for 60 min at 37 8C in an atmosphere enriched with 5% CO2. After this incubation period, excess fibronectin solution was removed and the surfaces were gently rinsed three times with PBS.
matic dissociation was done with DNAse Type II solution (10 000 U ml 1). A solution containing HAMS F10 solution with 36% foetal calf serum (FCS), 0.5% insulin, transferrin, selenite, (ITS) solution, 6 mM L-glutamine and 2% penicillin/streptomycin mixture (5000 U per 5 mg ml 1) (all from Sigma, Germany) was used to stop trypsinization. The cell suspension was centrifuged five minutes at 1500 rpm to get rid of remaining blood or cell debris. The pellet was resuspended with HAMS F10 solution (10% FCS, 0.5% ITS, 6 mM glutamine and 2% antibiotic mixture) in cell culture flasks and incubated for two hours at 37 8C and 5% CO2. The incubation led to an accumulation of fibroblasts on the surface of the flasks preferentially over cardiac myocytes leaving a higher proportion of cardiac myocytes in suspension. The cells in the final suspension were adjusted to the desired cell concentration and transferred onto the fibronectin primed EGE arrays. One preparation resulted in 1/2 ml cell suspension of 1 million cells per ml. This quantity was sufficient to plate 25 /50 EGE arrays. After approximately three hours, the cell culture chambers were filled with medium (HAMS F10). Medium exchange was done every second day and recordings began after cells had formed a complete syncytium on the surface (2 /3 DIV). In Fig. 4 a cardiac myocyte layer (4 DIV) cultured on an EGE array is shown.
2.5. Cell culture
2.6. Pharmacology
The cell culture of embryonic cardiac myocytes was carried out following the protocol described in [20]. In short, hearts of about ten rat fetuses (Sprague Dawley; embryonic days 15 /18, E15 /E18) were removed under sterile conditions, minced and trypsinized. The enzy-
To validate the possible application of the EGE system in pharmacological bioassays, we used a wellestablished positive chronotrope, norepinephrine (NE), at three different concentrations (10 nM, 100 nM, 1mM). The different concentrations were made up in standard
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and the final concentrations were then prepared using the extracellular solution.
3. Results
Fig. 4. Cardiac myocytes cell-layer (4 DIV) being grown on a microelectrode chip.
extracellular solution (in mM: KCl, 5; NaCl, 150; MgCl2, 1; HEPES, 10; CaCl2, 2.5; glucose, 10; pH 7.35 at 22 8C, adjusted with 1 N NaOH). For the measurements, the culture medium was exchanged using extracellular solution and equilibrated in the incubator for 30 min before the experiments. The stock solution of NE (1 mM) was made up using 1/100 N HCl as vehicle
In Fig. 5, extracellular recorded signals from a cardiac myocyte layer (4 DIV) are shown. The signals were simultaneously measured from 64 differently localized gold microelectrodes (electrode diameter 30 mm; spacing 200 mm, sampling rate 10 kHz). Each signal represents the time course of the drain-source-current through the JFET connected to the microelectrode. In the time scale shown (10 s) three beats of the cell layer were detected. The signals were regular at approximately every four seconds. Some electrodes (e.g. the electrodes 30 and 46) did not show any signal, which was caused by an insufficient electrical contact between the microelectrode chip and the amplification unit. Some other electrodes showed only very weak signals e.g. the electrodes 41 and 48. The signal amplitude and the strength of the cell electrode contact were influenced by the position and the distance of the electrically active cell on the electrode [18]. Only an adequate seal of the microelectrode led to a good coupling. Best coupling in this experiment was
Fig. 5. Extracellular recordings of action potentials of a cardiac myocytes cell-layer (4 DIV) with a 64-channel EGE device.
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Fig. 6. Propagation of action potentials of cardiac myocytes cell-layer (4 DIV) recorded with 64-channel EGE device.
recorded with electrode 55 (amplitude 2 mV; signal-tonoise 11:1). The 64-channel recording can be used to localize the origin of the excitation (the so-called pacemaker center). From there, the electrical excitation spread out as an excitation wave within a few milliseconds over the entire cell layer. During the propagation of the excitation wave a temporal delay occurs in the measured signals at different places. This can clearly be seen in Fig. 6, which shows a single action potential of the cell layer is shown higher temporal resolution. The time window (20 ms) in Fig. 6 allows pursuing the signal propagation over the entire 8 /8 electrode matrix. The first signal was detected with electrode 8. Then the excitation wave-respectively the action potentials-propagates to the left and to the center. Subsequently, also the lower electrodes register the contraction of the cell layer. Therefore the pacemaker center must be located outside of the 8 /8 electrode matrix, but closest to electrode 8. The temporal difference of the signals between electrode 8 and electrode 57 is about 13 ms. The stimulative effect of the positive chronotrope norepinephrine in terms of changing the beating rate of the cell layer is shown in Fig. 7. The initial extracellular
solution and the drug-containing solutions were warmed to 37 8C before the experiment. Drugs were applied by completely exchanging the solution with a solution containing the next higher concentration. Traces of 10 s time period were shown for the initial state (no NE) resulting in a beating rate of 24 bpm, 10 nM NE resulting in 48 bpm, 100 nM NE resulting in 90 bpm and 1 mM NE resulting in 114 bpm. It can clearly be seen that the chronotrope stimulated the cells to higher beating rates.
4. Conclusion We developed a 64-channel amplifier system for the recording of extracellular signals with planar metal microelectrodes. The described extended gate electrode concept of having the metal microelectrodes directly connected to low noise JFETs offers the possibility to record extracellular signals using planar metal microelectrodes without modification of the electrode surface. With a special circuit for the compensation of dc offsets and baseline drifts, the system can sample all channels simultaneously at 10 kHz in real-time. We recorded
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tissue significantly. We have shown in a previous study [6] that the recordings obtained with the extracellular system can be used as reflection of the physiological responses of the whole organ in vivo. However there are still several refinements required in order to improve the sensitivity and durability of the sensor chips and of the recording system as a whole. The interpretation of the extracellular recorded signal shapes is still the subject of intense study [16,22]. The origin of the too high recorded signals amplitudes is still unclear and needs to be further investigated. Although several improvements and investigations are still required, the system at its present form is already capable of performing basic pharmacological experiments.
Acknowledgements
Fig. 7. Stimulation of a cardiac myocyte layer using a positive chronotropic agent, norepinephrine, at different concentrations.
signals from cardiac myocytes cultured on the sensor surfaces. The signal-to-noise ratios varied depending on the quality of the cell-electrode contacts. The best signalto-noise ratio was 11:1 and extracellular signal amplitudes of up to 2 mV were recorded. With this system, it is possible to pinpoint the focal point (or foci) of the cardiac myocyte layer and the development of the electrical excitation pattern can be followed over several days. In the first pharmacological bioassay experiment, we validated the system by using a positive chronotrope, norepinephrine, for the stimulation of the cell layer. We have chosen the cardiac myocyte culture for this study, because these cells provide auto-rhythmic extracellular signals to evaluate the EGE system without the use of artificial stimulation. Extracellular recording systems were used as additional tools in electrophysiology as a substitute for existing optical and intracellular methods. A review for cell-based biosensors has been published by Pancrazio et al. [21]. The concept of extracellular recording has several advantages compared to classical techniques such as patch-clamp recording or fluorescence methods. The recording is non-invasive, can be up-scaled to many different localized sensor spots and long-term measurements over several days can be performed as long as sterility of the cell culture is maintained. With a single cell culture up to 50 electrode chips can be prepared, which reduces the use of animal
We would like to acknowledge Prof. Dr. W. Knoll of the Max-Planck-Institute for Polymer Research, Mainz, with whom this project was initiated. We would like to acknowledge Dr. C.-K. Yeung of the Department of Ophthalmology and Visual Sciences, the Chinese University of Hong Kong, for establishing the experimental methods using cardiac stimulants and relaxants in this project and for the critical review of this manuscript. We thank Mr. Lacher, Dr. T. Zetterer and Mr. W. Staab from the Institute for Microtechnique in Mainz for the support during fabrication of the EGE sensor chips. We acknowledge the excellent technical support of Mr. Mu¨ller and the members of the electronic laboratory, and Mr. Gerstenberg and Mr. Christ from the mechanical workshop of the MPI for Polymer Research. We would like to thank Prof. Dr. M. J. Scho¨ning from the Research Center Ju¨elich and the Fachhochschule Ju¨elich for valuable discussions and support during this work. We would like to acknowledge Mrs. J. Hayashi from the RIKEN Institute, Frontier Research System, Wako-shi, Japan for the cell culture. All the measurements were done at the RIKEN institute. This work was partially funded by the Northrhine /Westphalian Ministry of Schools, Education, Science and Research (Project: ELMINOS).
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