- Email: [email protected]
Electrodeposition of anchored polypyrrole film on microelectrodes and stimulation of cultured cardiac myocytes
ARTICLE IN PRESS
Biomaterials 28 (2007) 1480–1485 www.elsevier.com/locate/biomaterials
Electrodeposition of anchored polypyrrole film on microelectrodes and stimulation of cultured cardiac myocytes Matsuhiko Nishizawa, Hyuma Nozaki, Hirokazu Kaji, Takahiro Kitazume, Noriyuki Kobayashi, Takeshi Ishibashi, Takashi Abe Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Aramaki-Aoba 6-6-01, Sendai 980-8579, Japan Received 21 September 2006; accepted 18 November 2006 Available online 12 December 2006
Abstract The electrically conducting polymer polypyrrole (PPy) was electrochemically deposited onto Pt microelectrodes on a polyimide (PI) substrate. Pre-modification of the PI surface with a self-assembled monolayer of octadecyltrichlorosilane-induced anisotropic lateral growth of PPy along the PI surface and enhanced adhesive strength of the PPy film. The lateral growth of PPy film around the electrode anchored the whole film to the substrate. External stimulation of cultured cardiac myocytes was carried out using the PPy-coated microelectrode. The myocytes on the microelectrode substrate were electrically conjugated to form a sheet, and showed synchronized beating upon stimulation. The threshold charge for effective stimulation of a 0.8 cm2 sheet of myocytes was around 0.2 mC, roughly corresponding to a membrane depolarization of 250 mV. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cardiomyocyte; Cell culture; Electrical stimulation; Electroactive polymer; Electrochemistry
1. Introduction Engineering the interfacial contact between electrodes and biological cells is of central importance to the advancement of cell-based bioelectronics [1–6] and the development of neural prosthetic devices [7–11]. Extracellular electrical stimulation is still often technically challenging, especially when using smaller-sized microelectrodes, due to limitations on the current value (charge value), which can be applied without causing a faradic reaction and gas evolution [12]. Although porous platinum black has been used as a high capacity electrode [13], its brittleness is a potential serious drawback in applications for design of flexible neural probes [11]. In addition, effective interaction with cells and tissues cannot be assumed using such metal electrodes. A conducting polymer such as polypyrrole (PPy) has a large surface area owing to its fibrous structure and thus is Corresponding author.
E-mail address: [email protected] (M. Nishizawa). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.11.034
a high-capacity electrode material. Furthermore, the ease of preparation, inherent electrical conductivity, controllability of surface biochemical properties, and biocompatibility make PPy a promising interfacial material for use in neural prosthetic devices. Biocompatibility [7,8] and impedance characteristics [9] of PPy have been studied aiming the use for implanted medical biodevices. In addition to in vivo applications, in vitro cellular engineering also requires suitable bio interfacing. Recent technical progress in cellular micropatterning [1–5,14–17] enables the bioassay of cellular networks combined with electrode arrays. For example, we have succeeded in preparing micropatterned neuronal PC12 cells on a microelectrode array substrate in a manner allowing alignment of the micropatterns of cells to that of electrodes [15]. Smart biointerfacing with conducting polymers will contribute to realization of these next-generation bioassay chips. In this paper, we will firstly present the method for formation of PPy films incorporating strong adhesion at microelectrodes on polyimide (PI) substrates. PI is a current attractive material for biodevices owing to its
ARTICLE IN PRESS M. Nishizawa et al. / Biomaterials 28 (2007) 1480–1485
flexibility, chemical stability and biocompatibility. Premodification of the PI surface with self-assembled alkylsilane monolayers induced anisotropic lateral growth of PPy films along the substrate surface and enhanced film adhesion. Using a PPy-coated microelectrode, we have achieved reproducible, noninvasive, external stimulation of a cultured excitatory cell, namely the chick embryonic cardiac myocyte. The myocytes on the microelectrode substrate were electrically conjugated through gap junctions, and showed synchronized beating upon pulsation with the underlying PPy-coated microelectrode. The threshold charge required to stimulate an 0.8 cm2 sheet of myocytes was estimated to be around 0.2 mC. 2. Experimental Pyrrole (Kanto Chemical Co.) was purified by distillation under reduced pressure before use. Octadecyltrichlorosilane (C18SiCl3, Shinetsu Chemical Industries), polydimethylsiloxane (PDMS, KE-106, Shin-Etsu Chem. Co., Ltd.), fluo-3 AM (Molecular Probes, Eugene, OR), and all other chemicals were used without further purification. Photosensitive PI (Toray, Photoneece) was spin-coated onto a glass plate (4000 rpm, 30 s), and baked at 150 1C for 90 min, resulting in a 2 mm thick PI film on the plate. A pair of microband electrodes was fabricated by photolithography with a sputter-deposited Pt film onto the PI-coated substrate. Each band-electrode was 20 mm wide and separated from the other by 10 mm, as illustrated in Fig. 1a. The electrode substrate was first treated with 2-propanol and then exposed to oxygen plasma (80 W, 1 min) for introduction of hydroxyl groups onto the PI surface. The substrate was then immediately immersed in a 0.2 mM C18SiCl3/heptane solution for 10 min to form a self-assembling monolayer of alkylsilane, followed by rinsing with pure heptane, ethanol and distilled water. A PDMS fence was used to set the electrochemical system on the substrate (Fig. 1a, b). The total electrode area exposed to the solution was ca. 1.6 10 3 cm2. The electropolymerization of pyrrole was conducted in an aqueous solution containing 0.1 M pyrrole and 0.1 M KNO3 under potentionstatic conditions. Using a bipotentiostat (Fig. 1b), the polymerization potential for one of the two bands was set at 660 mV and the other at 680 mV vs.
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Ag/AgCl, as carried out in previous studies of PPy growth on a glass substrate [18]. Since the potentials of the two bands are slightly different, the ohmic currents are superimposed on the polymerization currents after electrical interconnection of bands with the growing PPy films. It is, therefore, possible to detect the moment of the interconnection by the ohmic current, and to evaluate the lateral growth rate across the gap of the electrode substrate. Primary cardiac myocytes were prepared according to a previously described method [16,17]. Cells were isolated from the hearts of chick embryos at Humburger and Hamilton stage 34–35 (8–9 days) by trypsinization and suspended in a culture medium consisting of 90% Dulbecco’s modified eagle medium (DMEM) (Gibco), 10% fetal bovine serum (FBS) (Gibco), and a trace amount of penicillin–streptomycin (Gibco) solution. The cell suspension was pre-incubated in a tissue culture flask (Falcon) for 1 h to remove strongly adherent fibroblasts. The resulting myocyte-rich suspension (1 106 cells ml 1) was seeded onto the PPy-coated microelectrode substrate. The cardiac myocytes attached onto the substrate surface were cultured in serum-containing medium to promote cellular spreading, growth and formation of cell–cell junctions. Electrical stimulation of cardiac myocyte sheets was conducted with current pulses induced between the PPy-coated Pt microelectrode and the other Pt plate set in the culture chamber (Fig. 2), using an electronic stimulator (SEN-7203, Nihon Kohden) coupled with an isolator unit (SS-202J, Nihon Kohden). Intracellular Ca2+ imaging was performed during stimulation using a fluorescence microscope. Cultured myocytes were loaded with 10 mM fluo-3 AM for 30 min at 37 1C, and studied in a phosphate-buffered saline (PBS(+)) solution consisting of (mM) 0.90 CaCl2, 2.68 KCl, 1.47 KH2PO4, 0.49 MgCl2, 136.9 NaCl, 8.06 Na2HPO4, 5.55 glucose, and 0.327 sodium pyruvate (pH 7.4). FBS was removed during fluorescence measurements because it significantly increased background fluorescence and downgraded the S/N ratio. Cells were excited with irradiated light from a Xe lamp and fluorescence (excitation 488 nm/emission 530 nm) was detected with a digital CCD camera (HiSCA, C6790-81, Hamamatsu Photonics) through a barrier filter. Images were recorded on a computer and analyzed with Aquacosmos software (Hamamatsu Photonics).
3. Results and discussion 3.1. PPy growth at microelectrodes formed on PI substrates Fig. 3a shows typical variations of the currents during polymerization at the twin-microband electrode on PI substrates. The solid and dotted line curves correspond to electrodes set at higher (680 mV) and lower polymerization
Sputtered Pt Polyimide - coated Glass PDMS Chamber Polymerization Solution
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Fig. 1. (a) Schematic drawing of the Pt microelectrode substrate. A pair of Pt microband electrodes (20 mm width) were prepared on a polyimidecoated glass substrate. A PDMS chamber was placed over the substrate to define active electrode area to ca. 1.6 10 3 cm2. (b) Electrochemical apparatus for electropolymerization of pyrrole at the microelectrode array with a bipotentiostat (BPS).
Fig. 2. Schematic diagram of the setup for electrical extracellular stimulation of the sheet of cultured cardiac myocytes with a current pulse generator (PG). Negative current was induced at the stimulation electrode.
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compared to the normal growth on the electrode. Since the uniform lateral growth forms the contact as a line, the current after the interconnection steeply increases at the electrode element held at higher potential (or decreases at the element held at the lower potential). The anisotropy in PPy growth is more obvious in Fig. 3b-2 which shows the result of prolonged polymerization over the interconnection. By analogy with previous studies on a glass substrate, it can be concluded that the assembled alkyl chains would capture the intermediate oligomers, and increase polymerization efficiency at the lateral growth front [18]. Measurements of the film thickness with a surface profiler (Tencor P10) revealed the polymerization anisotropy (the ratio of lateral growth rate to vertical growth rate) to be ca. 20. The adhesive strength of the PPy film was evaluated with Scotch Tape (adhesion force: 3.7 N/cm). A 2 2 mm2 electrode on PI substrate was coated with PPy film (polymerization charge, 5.5 mC). The PPy formed on the untreated substrate was almost completely peeled off and transferred to the Scotch Tape as shown in Fig. 4a. From a practical standpoint, the adhesion of PPy film is often so weak as to be removed merely by washing. In contrast, the
Scotch Tape
a Fig. 3. (a) Variation of current during the controlled potential polymerization of pyrrole at the microelectrodes on the bare polyimide substrate (blue) and on the substrate treated with alkylsilane (red). The solid and dotted curves are for the electrodes set at higher polymerization potential (680 mV) and at lower potential (660 mV), respectively. (b) Photographs of polypyrrole films formed on the surface-treated substrate, taken at interconnection (b-1) and after prolonged (40 min) polymerization (b-2).
b potentials (660 mV), respectively. The bending points (shown by arrows) indicate the moment of the interconnection between band electrodes with the growing polymer. In the case of the untreated bare PI substrate (blue curves), polymer growth was modest and the bending point on the current profile was not clear probably due to the point-bypoint contact of irregularly growing polymer. The modification of the PI surface with alkylsilane monolayer made PPy growth active and the interconnection was readily detectable as a sharp change in the current profile at around 25 min (red curve). Fig. 3b-1 is a photograph of the PPy-coated electrode just at the interconnection. The polymer is very thin and semitransparent, indicating preferred lateral growth along the substrate surface
c
anchoring effect
Fig. 4. The Scotch Tape peeling test for PPy films (polymerization charge, 5.5 mC) formed on 2 2 mm2 Pt electrodes on (a) bare polyimide substrate and (b) surface-treated polyimide substrate. (c) Schematic illustration of the anchorage effect enhancing the adhesive strength of PPy film.
ARTICLE IN PRESS M. Nishizawa et al. / Biomaterials 28 (2007) 1480–1485
PPy film formed on the alkylsilane monolayer-modified substrates adhered strongly and was not peeled off by the Scotch Tape (Fig. 4b). The PPy film laterally grown around the electrode [19], as illustrated in Fig. 4c, and anchored the whole film. The stiffness of the polymer coating should be practically important for cell culturebased neuroengineering such as experiments in the next section. Furthermore, the technique can easily be applied to other types of microelectrodes such as the needle array electrode, and can be used for implanted neural devices [7–11]. 3.2. Stimulation of cultured cardiac myocytes by PPy-coated microelectrodes Confocal fluorescence Ca2+ imaging with a Nipkowtype high-speed confocal system was performed to analyze cellular communication within the myocytes sheet formed over the 0.1 0.1 mm2 microelectrode shown in Fig. 5a. The myocytes were loaded with 10 mM fluo-3 for 30 min at 37 1C, and studied in a PBS(+) solution at room temperature to suppress spontaneous contraction. Under this condition, the spontaneous beating of myocytes decreases frequency [17]. Fig. 5b shows the time course of fluorescence intensity changes corresponding to the changes in cytosolic Ca2+ concentration, in accordance with the Ca2+-induced Ca2+ release (CICR) mechanism [16,17]. Measurements were made at the sites 1 and 2 enclosed by dotted line in Fig. 5a, which shows the synchronized Ca2+ transients externally evoked by the current pulses delivered by the electrode (interval: 1 s, duration: 100 ms, amplitude: 10 mA). In fact, such transients were simultaneously observed throughout the culture substrate (Movie-1 in Supplementary Info.), indicating that the cardiac myocytes are electrically conjugated over the sheet through the gap junctions. These data were reproducibly obtained in at least 5 separate experiments, without
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any evidence of significant damage to cell cultures. The fabricated cell stimulating system worked stably during at least 1-week cultivation. The reproducible, noninvasive, external stimulation is a key technique for effective in vivo and in vitro neuroengineering, but has been still generally difficult, mainly due to the polarization of metal electrodes. As an example, Fig. 5c shows typical results for an experiment using a bare Pt microelectrode without PPy modification. Other experimental conditions are the same as for Fig. 5b. The data taken in the vicinity of the electrode (like site 1 in Fig. 5a) show an irreversible increase in the fluorescence signal, indicating breakdown of the CICR system in the myocytes. Polarization of the Pt electrode and the resulting faradic reaction dramatically changes surrounding conditions such as pH and temperature. In fact, gas evolution was often observed (Movie-2 in Supplementary Info.). Many kinds of porous electrode, such as platinum black [13] and PPy [9] have been applied to increase electric capacity of the cell/electrode interface and to prevent unfavorable polarization. In the present study, the surface capacity of the PPy-coated electrode (5.8 C/cm2) was 38 times larger than that of bare Pt, as judged from AC impedance spectra. In addition to electrical functions, the PPy electrode has advantages in its physical flexibility and biocompatibility with cells and tissues. The inflammatory response to PPy-coated implants is reported to be lower than that of PLGA [8], which is the FDA-approved polymer for in vivo studies. By using the present stimulation system with the PPy-coated microelectrode (0.01 mm2), the threshold conditions for excitation of a myocyte sheet were estimated. The amplitude of the current pulse was varied from 0.5 to 10 mA with altered pulse duration. Fig. 6 shows the excitation (O) and non-excitation (X) of cardiac myocytes, plotted in the current–duration format. The pulse with longer duration tends to constitute an effective
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Fig. 5. (a) Cardiac myocytes cultured over the polypyrrole-coated Pt microelectrode. The cells were stained with fluo-3 for visualizing cellular responses to electrical stimulation. (b) Time course of fluorescence intensity changes measured at the sites 1 and 2 in (a). Negative current pulses (interval: 1 s, duration: 100 ms, amplitude: 10 mA) were applied. (c) Time course of fluorescence intensity changes measured without polypyrrole coating. Other experimental conditions were as in (b).
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trode array, the micropatterned PPy [18,19], and cellular micropatterns [15–17] on a chip to realize advanced bioassays based on measuring cellular network activity.
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This study was supported by the Industrial Technology Research Grant Program from NEDO of Japan, by a Grant-in-Aid for Scientific Research B (No. 17310080), and by Scientific Research on Priority Areas (No. 18048004) from the Ministry of Education, Science, and Culture, Japan.
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Fig. 6. The plot of excitation (O) and non-excitation (X) of cardiac myocytes in the current–duration format. The dotted blue curve roughly shows the threshold duration for each current amplitude. Inset: the replot in the current–charge format.
stimulation condition regardless of current amplitude, and the threshold duration can be traced by the dotted curve in the figure. The inset shows a replot of these data in the current–charge format. It seems there is a threshold charge of around 0.2 mC needed to excite the myocyte sheet. This threshold charge did not significantly vary with size of the electrode, suggesting that the total charge is required for stimulation, rather than the charge density per electrode area. Similar observations were reported for perceptual thresholds for in vivo electrical stimulation of human retina [10]. The PDMS chamber (see Fig. 2) defined the area of myocyte sheet as ca. 0.8 cm2. If we assume that the induced charge of 0.2 mC was fully applied to the cellular membrane (1 mF/cm2 [20]) of the sheet, the resulting membrane polarization can be estimated as ca. 250 mV. More accurate analysis may require consideration of the currents shunted through cell–cell junctions. 4. Conclusion We have found that pre-treatment of an electrode substrate with alkylsilane enhanced the adhesive strength of PPy film. The stiffness of the polymer coating should be practically important for in vivo and in vitro neural engineering applications. In principle, the technique can easily be applied to other types of microelectrode such as the needle array electrode. The PPy coating was effective for reproducible, noninvasive stimulation of cultured cardiac myocytes. Although we propose that the advantage of the PPy electrode is based on its high surface capacity for noninvasive stimulation, the possible modification of surface biochemical properties [14,21] for controlling cell adhesion and differentiation is another attractive feature of PPy. It is planned to combine the PPy-coated microelec-
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials. 2006.11.034.
References [1] Fromherz P. Semiconductor chips with ion channels, nerve cells and brain. Physica A 2003;16:24–34. [2] Offenhausser A, Knoll W. Cell–transistor hybrid systems and their potential applications. Trends Biotechnol 2001;19:62–6. [3] Chang JC, Brewer GJ, Wheeler BC. Microelectrode array recordings of patterned hippocampal neurons for four weeks. Biomed Microdev 2000;2:245–53. [4] James CD, Davis R, Mayer A, Turner A, Turner S, Withers G, et al. Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays. IEEE Trans Biomed Eng 2000;47:17–21. [5] Vogt AK, Wrobel G, Meyer W, Knoll W, Offenhausser A. Synaptic plasticity in micropatterned neuronal networks. Biomaterials 2005;26:2549–57. [6] Klauke N, Smith GL, Cooper J. Stimulation of single isolated adult ventricular myocytes within a low volume using a planar microelectrode array. Byophys J 2003;85:1766–74. [7] George PM, Lyckman AW, LaVan DA, Hegde A, Leung Y, Avasare R, et al. Fabrication and biocompatibility of polypyrrole implants suitable for neural prosthetics. Biomaterials 2005;26: 3511–9. [8] Schmidt CE, Shastri VR, Vacanti JP, Langer R. Stimulation of neurite outgrowth using an electrically conducting polymer. Proc Natl Acad Sci 1997;94:8948–53. [9] Cui X, Hetke JF, Wiler JA, Anderson DJ, Martin DC. Electrochemical deposition and characterization of conducting polymer polypyrrole/PSS on multichannel neural probes. Sensor Actuator A 2001;93:8–18. [10] Rizzo JF, Wyatt J, Loewenstein J, Kelly S, Sbire D. Methods and perceptual thresholds for short-term electrical stimulation of human retina with microelectrode arrays. Invest Ophthalmol Vis Sci 2003;44: 5355–61. [11] Takeuchi S, Suzuki T, Mabuchi K, Fujita H. 3D flexible multichannel neural probe array. J Micromech Microeng 2004;14:104–7. [12] Rose TL, Robblee LS. Electrical stimulation with Pt electrodes, VIII. Electrochemically safe charge injection limits with 0.2 ms pulses. IEEE Trans Biomed Eng 1990;37:1118–20. [13] Helsen JA, Breme HJ. Metals as biomaterials. Chichester: Wiley; 1998. [14] Song HK, Toste B, Ahmann K, Hoffman-Kim D, Palmore GTR. Micropatterns of positive guidance cues anchored to polypyrrole doped with polyglutamic acid: a new platform for characterizing
ARTICLE IN PRESS M. Nishizawa et al. / Biomaterials 28 (2007) 1480–1485 neurite extension in complex environments. Biomaterials 2006;27: 473–84. [15] Takii Y, Kaji H, Matsue T, Nishizawa M. Microstamp-based micromachining for modulation of growth of cultured neuronal cells. JSME Int J Ser C 2004;47:956–61. [16] Kaji H, Nishizawa M, Matsue T. Localized chemical stimulation to micropatterned cells using multiple laminar fluidic flows. Lab Chip 2003;3:208–11. [17] Kaji H, Takii Y, Nishizawa M, Matsue T. Pharmacological characterization of micropatterned cardiac myocytes. Biomaterials 2003;24:4239–44.
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[18] Nishizawa M, Miwa Y, Matsue T, Uchida I. Surface pretreatment for electrochemical fabrication of ultrathin patterned conducting polymers. J Electrochem Soc 1993;140:1650–5. [19] Nishizawa M, Shibuya M, Sawaguchi T, Matsue T, Uchida I. Electrochemical preparation of ultrathin polypyrrole film at microarray electrodes. J Phys Chem 1991;95:9042–4. [20] Mattbews GG. Cellular physiology of nerve and muscle. 2nd ed. Boston: Blackwell scientific Publications; 1991. [21] Sanghvi AB, Miller KPH, Belcher AM, Schmidt CE. Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer. Nat Mater 2005;4:496–502.
Biomaterials 28 (2007) 1480–1485 www.elsevier.com/locate/biomaterials
Electrodeposition of anchored polypyrrole film on microelectrodes and stimulation of cultured cardiac myocytes Matsuhiko Nishizawa, Hyuma Nozaki, Hirokazu Kaji, Takahiro Kitazume, Noriyuki Kobayashi, Takeshi Ishibashi, Takashi Abe Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Aramaki-Aoba 6-6-01, Sendai 980-8579, Japan Received 21 September 2006; accepted 18 November 2006 Available online 12 December 2006
Abstract The electrically conducting polymer polypyrrole (PPy) was electrochemically deposited onto Pt microelectrodes on a polyimide (PI) substrate. Pre-modification of the PI surface with a self-assembled monolayer of octadecyltrichlorosilane-induced anisotropic lateral growth of PPy along the PI surface and enhanced adhesive strength of the PPy film. The lateral growth of PPy film around the electrode anchored the whole film to the substrate. External stimulation of cultured cardiac myocytes was carried out using the PPy-coated microelectrode. The myocytes on the microelectrode substrate were electrically conjugated to form a sheet, and showed synchronized beating upon stimulation. The threshold charge for effective stimulation of a 0.8 cm2 sheet of myocytes was around 0.2 mC, roughly corresponding to a membrane depolarization of 250 mV. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cardiomyocyte; Cell culture; Electrical stimulation; Electroactive polymer; Electrochemistry
1. Introduction Engineering the interfacial contact between electrodes and biological cells is of central importance to the advancement of cell-based bioelectronics [1–6] and the development of neural prosthetic devices [7–11]. Extracellular electrical stimulation is still often technically challenging, especially when using smaller-sized microelectrodes, due to limitations on the current value (charge value), which can be applied without causing a faradic reaction and gas evolution [12]. Although porous platinum black has been used as a high capacity electrode [13], its brittleness is a potential serious drawback in applications for design of flexible neural probes [11]. In addition, effective interaction with cells and tissues cannot be assumed using such metal electrodes. A conducting polymer such as polypyrrole (PPy) has a large surface area owing to its fibrous structure and thus is Corresponding author.
E-mail address: [email protected] (M. Nishizawa). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.11.034
a high-capacity electrode material. Furthermore, the ease of preparation, inherent electrical conductivity, controllability of surface biochemical properties, and biocompatibility make PPy a promising interfacial material for use in neural prosthetic devices. Biocompatibility [7,8] and impedance characteristics [9] of PPy have been studied aiming the use for implanted medical biodevices. In addition to in vivo applications, in vitro cellular engineering also requires suitable bio interfacing. Recent technical progress in cellular micropatterning [1–5,14–17] enables the bioassay of cellular networks combined with electrode arrays. For example, we have succeeded in preparing micropatterned neuronal PC12 cells on a microelectrode array substrate in a manner allowing alignment of the micropatterns of cells to that of electrodes [15]. Smart biointerfacing with conducting polymers will contribute to realization of these next-generation bioassay chips. In this paper, we will firstly present the method for formation of PPy films incorporating strong adhesion at microelectrodes on polyimide (PI) substrates. PI is a current attractive material for biodevices owing to its
ARTICLE IN PRESS M. Nishizawa et al. / Biomaterials 28 (2007) 1480–1485
flexibility, chemical stability and biocompatibility. Premodification of the PI surface with self-assembled alkylsilane monolayers induced anisotropic lateral growth of PPy films along the substrate surface and enhanced film adhesion. Using a PPy-coated microelectrode, we have achieved reproducible, noninvasive, external stimulation of a cultured excitatory cell, namely the chick embryonic cardiac myocyte. The myocytes on the microelectrode substrate were electrically conjugated through gap junctions, and showed synchronized beating upon pulsation with the underlying PPy-coated microelectrode. The threshold charge required to stimulate an 0.8 cm2 sheet of myocytes was estimated to be around 0.2 mC. 2. Experimental Pyrrole (Kanto Chemical Co.) was purified by distillation under reduced pressure before use. Octadecyltrichlorosilane (C18SiCl3, Shinetsu Chemical Industries), polydimethylsiloxane (PDMS, KE-106, Shin-Etsu Chem. Co., Ltd.), fluo-3 AM (Molecular Probes, Eugene, OR), and all other chemicals were used without further purification. Photosensitive PI (Toray, Photoneece) was spin-coated onto a glass plate (4000 rpm, 30 s), and baked at 150 1C for 90 min, resulting in a 2 mm thick PI film on the plate. A pair of microband electrodes was fabricated by photolithography with a sputter-deposited Pt film onto the PI-coated substrate. Each band-electrode was 20 mm wide and separated from the other by 10 mm, as illustrated in Fig. 1a. The electrode substrate was first treated with 2-propanol and then exposed to oxygen plasma (80 W, 1 min) for introduction of hydroxyl groups onto the PI surface. The substrate was then immediately immersed in a 0.2 mM C18SiCl3/heptane solution for 10 min to form a self-assembling monolayer of alkylsilane, followed by rinsing with pure heptane, ethanol and distilled water. A PDMS fence was used to set the electrochemical system on the substrate (Fig. 1a, b). The total electrode area exposed to the solution was ca. 1.6 10 3 cm2. The electropolymerization of pyrrole was conducted in an aqueous solution containing 0.1 M pyrrole and 0.1 M KNO3 under potentionstatic conditions. Using a bipotentiostat (Fig. 1b), the polymerization potential for one of the two bands was set at 660 mV and the other at 680 mV vs.
a
1481
Ag/AgCl, as carried out in previous studies of PPy growth on a glass substrate [18]. Since the potentials of the two bands are slightly different, the ohmic currents are superimposed on the polymerization currents after electrical interconnection of bands with the growing PPy films. It is, therefore, possible to detect the moment of the interconnection by the ohmic current, and to evaluate the lateral growth rate across the gap of the electrode substrate. Primary cardiac myocytes were prepared according to a previously described method [16,17]. Cells were isolated from the hearts of chick embryos at Humburger and Hamilton stage 34–35 (8–9 days) by trypsinization and suspended in a culture medium consisting of 90% Dulbecco’s modified eagle medium (DMEM) (Gibco), 10% fetal bovine serum (FBS) (Gibco), and a trace amount of penicillin–streptomycin (Gibco) solution. The cell suspension was pre-incubated in a tissue culture flask (Falcon) for 1 h to remove strongly adherent fibroblasts. The resulting myocyte-rich suspension (1 106 cells ml 1) was seeded onto the PPy-coated microelectrode substrate. The cardiac myocytes attached onto the substrate surface were cultured in serum-containing medium to promote cellular spreading, growth and formation of cell–cell junctions. Electrical stimulation of cardiac myocyte sheets was conducted with current pulses induced between the PPy-coated Pt microelectrode and the other Pt plate set in the culture chamber (Fig. 2), using an electronic stimulator (SEN-7203, Nihon Kohden) coupled with an isolator unit (SS-202J, Nihon Kohden). Intracellular Ca2+ imaging was performed during stimulation using a fluorescence microscope. Cultured myocytes were loaded with 10 mM fluo-3 AM for 30 min at 37 1C, and studied in a phosphate-buffered saline (PBS(+)) solution consisting of (mM) 0.90 CaCl2, 2.68 KCl, 1.47 KH2PO4, 0.49 MgCl2, 136.9 NaCl, 8.06 Na2HPO4, 5.55 glucose, and 0.327 sodium pyruvate (pH 7.4). FBS was removed during fluorescence measurements because it significantly increased background fluorescence and downgraded the S/N ratio. Cells were excited with irradiated light from a Xe lamp and fluorescence (excitation 488 nm/emission 530 nm) was detected with a digital CCD camera (HiSCA, C6790-81, Hamamatsu Photonics) through a barrier filter. Images were recorded on a computer and analyzed with Aquacosmos software (Hamamatsu Photonics).
3. Results and discussion 3.1. PPy growth at microelectrodes formed on PI substrates Fig. 3a shows typical variations of the currents during polymerization at the twin-microband electrode on PI substrates. The solid and dotted line curves correspond to electrodes set at higher (680 mV) and lower polymerization
Sputtered Pt Polyimide - coated Glass PDMS Chamber Polymerization Solution
b W1
PDMS Chamber PG Cardiac Myocyte
BPS RE
CE
W2
-
+ Pt
Pt
PPy
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Fig. 1. (a) Schematic drawing of the Pt microelectrode substrate. A pair of Pt microband electrodes (20 mm width) were prepared on a polyimidecoated glass substrate. A PDMS chamber was placed over the substrate to define active electrode area to ca. 1.6 10 3 cm2. (b) Electrochemical apparatus for electropolymerization of pyrrole at the microelectrode array with a bipotentiostat (BPS).
Fig. 2. Schematic diagram of the setup for electrical extracellular stimulation of the sheet of cultured cardiac myocytes with a current pulse generator (PG). Negative current was induced at the stimulation electrode.
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compared to the normal growth on the electrode. Since the uniform lateral growth forms the contact as a line, the current after the interconnection steeply increases at the electrode element held at higher potential (or decreases at the element held at the lower potential). The anisotropy in PPy growth is more obvious in Fig. 3b-2 which shows the result of prolonged polymerization over the interconnection. By analogy with previous studies on a glass substrate, it can be concluded that the assembled alkyl chains would capture the intermediate oligomers, and increase polymerization efficiency at the lateral growth front [18]. Measurements of the film thickness with a surface profiler (Tencor P10) revealed the polymerization anisotropy (the ratio of lateral growth rate to vertical growth rate) to be ca. 20. The adhesive strength of the PPy film was evaluated with Scotch Tape (adhesion force: 3.7 N/cm). A 2 2 mm2 electrode on PI substrate was coated with PPy film (polymerization charge, 5.5 mC). The PPy formed on the untreated substrate was almost completely peeled off and transferred to the Scotch Tape as shown in Fig. 4a. From a practical standpoint, the adhesion of PPy film is often so weak as to be removed merely by washing. In contrast, the
Scotch Tape
a Fig. 3. (a) Variation of current during the controlled potential polymerization of pyrrole at the microelectrodes on the bare polyimide substrate (blue) and on the substrate treated with alkylsilane (red). The solid and dotted curves are for the electrodes set at higher polymerization potential (680 mV) and at lower potential (660 mV), respectively. (b) Photographs of polypyrrole films formed on the surface-treated substrate, taken at interconnection (b-1) and after prolonged (40 min) polymerization (b-2).
b potentials (660 mV), respectively. The bending points (shown by arrows) indicate the moment of the interconnection between band electrodes with the growing polymer. In the case of the untreated bare PI substrate (blue curves), polymer growth was modest and the bending point on the current profile was not clear probably due to the point-bypoint contact of irregularly growing polymer. The modification of the PI surface with alkylsilane monolayer made PPy growth active and the interconnection was readily detectable as a sharp change in the current profile at around 25 min (red curve). Fig. 3b-1 is a photograph of the PPy-coated electrode just at the interconnection. The polymer is very thin and semitransparent, indicating preferred lateral growth along the substrate surface
c
anchoring effect
Fig. 4. The Scotch Tape peeling test for PPy films (polymerization charge, 5.5 mC) formed on 2 2 mm2 Pt electrodes on (a) bare polyimide substrate and (b) surface-treated polyimide substrate. (c) Schematic illustration of the anchorage effect enhancing the adhesive strength of PPy film.
ARTICLE IN PRESS M. Nishizawa et al. / Biomaterials 28 (2007) 1480–1485
PPy film formed on the alkylsilane monolayer-modified substrates adhered strongly and was not peeled off by the Scotch Tape (Fig. 4b). The PPy film laterally grown around the electrode [19], as illustrated in Fig. 4c, and anchored the whole film. The stiffness of the polymer coating should be practically important for cell culturebased neuroengineering such as experiments in the next section. Furthermore, the technique can easily be applied to other types of microelectrodes such as the needle array electrode, and can be used for implanted neural devices [7–11]. 3.2. Stimulation of cultured cardiac myocytes by PPy-coated microelectrodes Confocal fluorescence Ca2+ imaging with a Nipkowtype high-speed confocal system was performed to analyze cellular communication within the myocytes sheet formed over the 0.1 0.1 mm2 microelectrode shown in Fig. 5a. The myocytes were loaded with 10 mM fluo-3 for 30 min at 37 1C, and studied in a PBS(+) solution at room temperature to suppress spontaneous contraction. Under this condition, the spontaneous beating of myocytes decreases frequency [17]. Fig. 5b shows the time course of fluorescence intensity changes corresponding to the changes in cytosolic Ca2+ concentration, in accordance with the Ca2+-induced Ca2+ release (CICR) mechanism [16,17]. Measurements were made at the sites 1 and 2 enclosed by dotted line in Fig. 5a, which shows the synchronized Ca2+ transients externally evoked by the current pulses delivered by the electrode (interval: 1 s, duration: 100 ms, amplitude: 10 mA). In fact, such transients were simultaneously observed throughout the culture substrate (Movie-1 in Supplementary Info.), indicating that the cardiac myocytes are electrically conjugated over the sheet through the gap junctions. These data were reproducibly obtained in at least 5 separate experiments, without
a
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any evidence of significant damage to cell cultures. The fabricated cell stimulating system worked stably during at least 1-week cultivation. The reproducible, noninvasive, external stimulation is a key technique for effective in vivo and in vitro neuroengineering, but has been still generally difficult, mainly due to the polarization of metal electrodes. As an example, Fig. 5c shows typical results for an experiment using a bare Pt microelectrode without PPy modification. Other experimental conditions are the same as for Fig. 5b. The data taken in the vicinity of the electrode (like site 1 in Fig. 5a) show an irreversible increase in the fluorescence signal, indicating breakdown of the CICR system in the myocytes. Polarization of the Pt electrode and the resulting faradic reaction dramatically changes surrounding conditions such as pH and temperature. In fact, gas evolution was often observed (Movie-2 in Supplementary Info.). Many kinds of porous electrode, such as platinum black [13] and PPy [9] have been applied to increase electric capacity of the cell/electrode interface and to prevent unfavorable polarization. In the present study, the surface capacity of the PPy-coated electrode (5.8 C/cm2) was 38 times larger than that of bare Pt, as judged from AC impedance spectra. In addition to electrical functions, the PPy electrode has advantages in its physical flexibility and biocompatibility with cells and tissues. The inflammatory response to PPy-coated implants is reported to be lower than that of PLGA [8], which is the FDA-approved polymer for in vivo studies. By using the present stimulation system with the PPy-coated microelectrode (0.01 mm2), the threshold conditions for excitation of a myocyte sheet were estimated. The amplitude of the current pulse was varied from 0.5 to 10 mA with altered pulse duration. Fig. 6 shows the excitation (O) and non-excitation (X) of cardiac myocytes, plotted in the current–duration format. The pulse with longer duration tends to constitute an effective
b
100µm
c
Site 1
Fluorescence Intensity / a.u.
Sit e 1 Sit e 2
Fluorescence Intensity/ a.u.
Current Pulse
Site 2
0
5
10 15 Time/sec
20
25
0
5
10 15 Time / sec
20
25
Fig. 5. (a) Cardiac myocytes cultured over the polypyrrole-coated Pt microelectrode. The cells were stained with fluo-3 for visualizing cellular responses to electrical stimulation. (b) Time course of fluorescence intensity changes measured at the sites 1 and 2 in (a). Negative current pulses (interval: 1 s, duration: 100 ms, amplitude: 10 mA) were applied. (c) Time course of fluorescence intensity changes measured without polypyrrole coating. Other experimental conditions were as in (b).
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trode array, the micropatterned PPy [18,19], and cellular micropatterns [15–17] on a chip to realize advanced bioassays based on measuring cellular network activity.
600 0.6
Charge/µC
500
Duration / µs
400
0.4
Acknowledgments 0.2
This study was supported by the Industrial Technology Research Grant Program from NEDO of Japan, by a Grant-in-Aid for Scientific Research B (No. 17310080), and by Scientific Research on Priority Areas (No. 18048004) from the Ministry of Education, Science, and Culture, Japan.
300 0
200
0
4
8
12
Current / mA
100
Appendix A. Supplementary Materials 0 0
2
4
6 current / mA
8
10
12
Fig. 6. The plot of excitation (O) and non-excitation (X) of cardiac myocytes in the current–duration format. The dotted blue curve roughly shows the threshold duration for each current amplitude. Inset: the replot in the current–charge format.
stimulation condition regardless of current amplitude, and the threshold duration can be traced by the dotted curve in the figure. The inset shows a replot of these data in the current–charge format. It seems there is a threshold charge of around 0.2 mC needed to excite the myocyte sheet. This threshold charge did not significantly vary with size of the electrode, suggesting that the total charge is required for stimulation, rather than the charge density per electrode area. Similar observations were reported for perceptual thresholds for in vivo electrical stimulation of human retina [10]. The PDMS chamber (see Fig. 2) defined the area of myocyte sheet as ca. 0.8 cm2. If we assume that the induced charge of 0.2 mC was fully applied to the cellular membrane (1 mF/cm2 [20]) of the sheet, the resulting membrane polarization can be estimated as ca. 250 mV. More accurate analysis may require consideration of the currents shunted through cell–cell junctions. 4. Conclusion We have found that pre-treatment of an electrode substrate with alkylsilane enhanced the adhesive strength of PPy film. The stiffness of the polymer coating should be practically important for in vivo and in vitro neural engineering applications. In principle, the technique can easily be applied to other types of microelectrode such as the needle array electrode. The PPy coating was effective for reproducible, noninvasive stimulation of cultured cardiac myocytes. Although we propose that the advantage of the PPy electrode is based on its high surface capacity for noninvasive stimulation, the possible modification of surface biochemical properties [14,21] for controlling cell adhesion and differentiation is another attractive feature of PPy. It is planned to combine the PPy-coated microelec-
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials. 2006.11.034.
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