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On the chemical modification of pacemaker electrodes and patterned surface functionalization of planar substrates
Biosensors & Bioelectronics Vol. 12. No. 8, pp. 853–865, 1997 1997 Published by Elsevier Science Limited All rights reserved. Printed in Great Britain 0956–5663/97/$17.00 PII: S0956–5663(97)00050-X
On the chemical modification of pacemaker electrodes and patterned surface functionalization of planar substrates Martin Stelzle,*† Roland Wagner,§ Wilfried Nisch,† Wolfram Ja¨germann,얏 Ronald Fro¨hlich‡ & Max Schaldach‡ †Naturwissenschaftliches und Medizinisches Institut an der Universita¨t Tu¨bingen, Eberhardstraße 29, D-72762 Reutlingen, Reutlingen, Germany ‡Zentralinstitut fu¨r Biomedizinische Technik der Universita¨t Erlangen-Nu¨rnberg, Turnstraße 5, 91058 Erlangen, Erlangen, Germany §Max-Planck-Institut fu¨r Kolloid- und Grenzfla¨chenforschung, Rudower Chaussee 5, 12489 Berlin, Berlin, Germany 얏Hahn-Meitner-Institut, Glienicker Straße 100, 14109 Berlin, Berlin, Germany
Abstract: We report on experiments towards the chemical modification of metal electrodes in order to enhance biocompatibility or improve cell adhesion properties. In the first example pacemaker electrodes were modified with a thin polysiloxane network which allowed for further derivatization with a poly(ethylene glycol) layer. The primary goal was to suppress inflammatory response of tissue after implantation of electrodes. FTIR, ESCA and a.c.impedance spectroscopy show the integrity of the ultrathin membrane. No significant reduction of the electrode capacitance was observed, providing further proof for the deposition of a homogeneously thin membrane. The second example deals with the patterned chemical modification of planar surfaces. The goal was to eventually effect selective adhesion of electrosensitive cells above microelectrodes for stimulation and/or recording. First results demonstrate the compatibility of monolayer deposition techniques with common photolithography. It is thus possible to create surfaces with patterned chemical functionality. A gas-phase silylation process was developed in order to control more precisely surface hydration and reaction parameters than is possible with common solution-based silylation procedures. 1997 Elsevier Science Limited Keywords: chemical surface modification, pacemaker electrodes, gas-phase silylation, monolayer lithography, cell adhesion, biocompatibility, photolithography
INTRODUCTION The interface between electrodes and tissue has been widely studied (Bolz, 1995; Fro¨hlich et al., *To whom correspondence should be addressed.
1995; Mond & Stokes, 1992; Tanghe et al., 1990). Biocompatibility in this context usually means non-toxicity of the materials employed (Williams, 1981), the tendency of cells and tissue to adhere to the electrode surface, the absence of inflammatory responses after implantation of the 853
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electrodes (Meachim & Pedley, 1981; Mond et al., 1988; Beyersdorfer et al., 1988) and good electrical stimulation capability of the electrodes for extended periods of time (Weidlich et al., 1980). After implantation of pacemaker electrodes one observes that over a period of several weeks after implantation a relatively high stimulation amplitude is required to stimulate effectively myocardic tissue. This is most probably owing to tissue irritation and an inflammatory response. Since a high stimulation amplitude results in increased consumption of limited battery power, one goal in electrode design is to avoid this condition. In the past, sophisticated electrodes have been developed that suppress tissue inflammation by leaking steroid medication through a porous electrode head (Mond & Stokes, 1992). Our approach was different, however. Many reports have shown that poly(ethylen glycol) (PEG) grafted surfaces prevent protein adsorption which in turn is considered a prerequisite of any cell/interface interaction (Mrksich et al., 1995; Prime & Whitesides, 1993; Bergsto¨m et al., 1992; Gombotz et al., 1991). We thus concluded that it should be possible to limit tissue response by covering the electrode with a PEGcontaining membrane. On the other hand, the membrane must not reduce the electrode capacitance significantly, i.e. the membrane must be very thin. In order to cover an electrode exhibiting fractal surface topology with a very thin layer we devised a two-step reaction scheme and evaluated membrane properties and electrode capactitance with ESCA, FTIR and a.c.-impedance measurements. Planar microelectrodes have been used in the past in so-called microelectrode arrays to stimulate single cells or slices of tissue (Gross, 1979; Pine, 1980; Nisch et al., 1994) and the electrical properties of the microelectrode/cell interface have been discussed (Robinson, 1968; Ranck, 1981; Grattarola & Martinoia, 1993). These studies revealed that a close contact between cell and microelectrode, including high seal resistance towards the surrounding electrolyte, is the main prerequisite for recording high signal-to-noise data and for effective stimulation. Thus far, however, the experimentalist had either to rely on chance to position some cells properly on the microelectrodes or to use micromanipulation techniques for this purpose (Fromherz & Stett, 1995). Attempts have been made to create surfaces with 854
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patterned surface chemical functionality in order to effect guided growth of cells and promote cell attachment on surfaces at pre-selected sites (Ranieri et al., 1993; Singhvi et al., 1994; Lopez et al., 1993; Cle´mence et al., 1995). Our goal is to devise a versatile reaction scheme to create surfaces with patterned affinity towards cells using common photolithography processes. First successful steps in this direction were reported by Kleinfeld et al. (1988). Such surfaces would comprise areas with low or high affinity, with or without chemically reactive groups, respectively. Biomolecules or synthetic molecules may then be covalently attached to this surface which is thus tailored to interact with the target cell or tissue in the desired fashion.
EXPERIMENTAL DETAILS Chemical procedures for surface functionalization In order to control surface hydration and reaction parameters more precisely than in common solution-based reaction schemes a gas-phase reactor was used for silylation of surfaces whenever the molecules to be used exhibited a sufficient vapor pressure at temperatures below 150°C. Hexadecyltrichlorosilane (HTS, Cl3Si(CH2)15CH3) and aminopropyltrimethoxysilane (APS, H2N(CH2)2Si(OCH3)3) were obtained from ABCR, Germany, and used as received. The chemical structures of trichlorosilane (TCS), allylpoly(ethylene glycol) (allyl-PEG), allyl-glycidether (AGE) and bis-amino-poly(ethylene glycol) (BAP) are shown in Fig. 1. With the gas-phase reactor setup (Fig. 3) it is possible to avoid build-up of three-dimensional polymer networks, which frequently occurs when polyfunctional silanes are used for surface modification and residual moisture is present. The reactor setup including valves and inlets for the silylating agent is heatable to allow for effective water desorption prior to the reaction. Furthermore, sufficient vapor pressure of the silylating agent may be achieved and reaction speed also is increased at elevated temperatures. In the case of silylation with hexadecyltrichlorosilane (HTS) the reaction was performed in a 1% solution of HTS in dry n-hexane (Roth GmbH, Germany, water ⬍ 0·02%, in some instances further dryed on 3 Å molecular sieve)
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Chemical modification of pacemaker electrodes
Fig. 1. Derivatization scheme for chemical modification of iridium pacemaker electrodes with a two-step (I) reaction scheme using allyl-PEG or a three-step reaction scheme (IIa and IIb) with subsequent reaction of allyl-glycidether (AGE) and bis-amino-PEG (BAP) involving silylation and hydrosilylation reactions. In each case the first layer consists of a polysiloxane network deposited from trichlorosilane (TCS) in the gas phase.
using a glove box providing dry inert gas conditions. After silylation and rinsing in n-hexane the substrates were baked under N2 at ⬇ 100°C for 30 min. Electrochemical techniques for evaluation of thin organic films A Schlumberger Si1260 impedance analyzer in combination with a home-built potentiostat was employed for electrochemical measurements. A Ag/AgCl reference electrode and a Pt counter electrode was used. As redox-couple Fe(CN)36 − /4 − was employed (2 mM K3Fe(CN)6 and K4Fe(CN)6 each in 150 mM NaCl/5 mM
HEPES, pH 7·4). The drop in cell impedance due to Faradic currents around the Nernst potential of the redox-couple was used as an indicator for the integrity and homogeneity of the membrane deposited on pacemaker electrodes. In case of an electrode covered homogeneously with a membrane, electron transfer between electrode and the dissolved redox-couple is hindered and as a result the drop of impedance around the Nernst potential is small compared to the case of a bare electrode. FTIR-ATR measurements FTIR-ATR measurements were performed with a Bruker IFS66 instrument which was equipped 855
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with a MCT detector and purged with nitrogen. The spectrometer was run at 4 cm−1 resolution, and between 200 and 500 scans were usually acquired for each spectrum. Germanium ATR crystals were used as internal reflection elements and as substrates on which membranes were deposited in order to evaluate the surface chemistry occurring during the two-step reaction scheme. ESCA analysis The ESCA experiments were performed with a Leybold multitechnique surface analysis system (base pressure below 10−10 mbar) equipped with a hemispherical analyzer and multichannel detector. Mg K␣ was used as excitation source. The electrons were analyzed in normal detection with an acceptance cone of 30°. The spectrometer was calibrated with sputtered Au and Cu foils with respect to the Fermi edge and to binding energies of 932·6 eV (Cu 2p3/2) and 83·8 eV (Au 4f7/2), respectively. The samples were mechanically mounted to the sample holder ensuring an electric back contact, as is evident from the lack of charging of the sample surfaces. No further pretreatment was applied to the sample surfaces before analyis. However, in some cases the outgassing of the samples took several hours before reasonable pressure below 10−9 mbar was reached. Fluorescent labeling of amino-reactive surfaces A 1 mg/ml solution of rhodamine isothiocyanate (RITC) (obtained from Sigma, St Louis, MO) in dry DMSO (stored on molecular sieve) was prepared freshly prior to the labeling procedure. Substrates were immersed in PBS buffer, pH 8·5, and 1% (vol/vol) of the RITC solution was added. The solution was stirred for at least 10 min. The samples were rinsed and sonicated in ultrapure water, isopropanol and—if necessary to remove unspecifically adsorbed RITC—also in DMSO. Test samples of glass without aminosilane and glass derivatized with aminosilane were usually processed along with the other samples in order to check the derivatization and subsequent rinsing procedures with respect to their selectivity towards labeling of amino residues. Substrates used for FITR-ATR, ESCA and electrochemical measurements The primary goal of the analysis was to establish the composition and integrity of the thin organic 856
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films formed by two- or three-step chemical processes. Owing to the geometry of the pacemaker electrodes with their curved surface and small (approximately 2 mm) diameter, only the electrochemical measurements could be performed directly on the electrodes themselves. Owing to instrumental limitations we had to use larger samples for the ESCA analysis. We therefore fabricated steel disks (10 mm diameter) and deposited iridium films (thickness 5 m) onto them with the same procedure used for deposition of fractal iridium films on pacemaker electrodes. Thus, samples with a surface texture comparable to that found on the pacemaker electrodes (except for the slight surface curvature) were available for derivatization and subsequent ESCA analysis. We suggest that no significant differences are to be expected between the flat samples and the pacemaker electrodes with respect to composition and integrity of the thin organic films deposited on both types of samples. Our purpose in performing the FTIR-ATR analysis was to characterize further the hydrosilylation reaction (step 2 of the chemical modification), and in particular ensure proper formation of covalent Si–C bonds. Here, Ge-ATR crystals were employed as samples. On both the Ge crystals and on the iridium substrates the presence (or absence) of the polysiloxane network can be independently established. The Si–H stretching vibration at 2250 cm−1 provides a suitable measure for the conversion from Si–H to Si–C bonds during hydrosilylation. In contrast, it is difficult to prove the formation of these bonds by ESCA. There remains, however, some uncertainty concerning the influence of surface roughness on efficiency of the hydrosilylation.
RESULTS AND DISCUSSION Chemical modification of pacemaker electrodes with fractal surface topology In Fig. 1 the derivatization scheme employed in the chemical modification of pacemaker electrodes is shown (Stelzle et al., 1996; Wagner et al., 1996). The electrodes were fabricated using a plasma deposition process which results in the formation of layers with a fractal microstructure (Bolz, 1995; Fro¨hlich et al., 1995; Bolz et al., 1995) as is demonstrated in Fig. 2. A first layer of trichlorosilane (TCS) serves as reactive
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Chemical modification of pacemaker electrodes
Fig. 2. SEM image of an iridium electrode deposited by high pressure PE-CVD. The high degree of surface texture is evident.
counterpart for the immobilization of the ethylene glycol derivative via C–Si bonds. TCS has the ability to crosslink and form a network like layer on the electrode surface. ESCA measurements (cf. detailed discussion below) even appear to show evidence for the existence of some surface oxide or hydroxyl groups on the iridium electrodes. At this point it is not yet clear if chemical bonds between the silane and the iridium surface may be formed or if the high tenacity of the polysiloxane layer is solely due to intercalation with the electrode surface. In a recent publication it has been reported that stable well-ordered monolayers may be formed from trifunctional long chain alkyl silanes without significant degree of covalent bonding to the surface being involved (Allara et al., 1995). The second reaction step of this versatile modification scheme (Fig. 1) involves a hydrosilylation reaction employing a Pt catalyst with either an allyl-PEO derivative or a modified epoxy-allyl derivative. In the latter case, the PEO-chain is introduced by reaction of the epoxy residue with the amino groups of an ␣--amino-terminated PEO chain.
Fig. 3. Gas-phase reactor setup used in this study. The whole setup, including recipient, valves, inlets and the container for the silylating agent, may be heated, thus allowing for removal of residual moisture prior to the silylation step. In addition, sufficient vapor pressure of the silylating agent and increased reaction speed may be achieved at elevated temperatures.
Analysis of thin organic layers on pacemaker electrodes ESCA analysis For ESCA analysis samples were prepared on flat substrates along with pacemaker electrodes in the 857
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same deposition and derivatization process. Thus, the iridium layer exhibited the same fractal surface structure as the electrodes. The electrodes themselves were too small to be directly analyzed with our ESCA instrument. Fig. 4 shows spectra of samples at each stage of the derivatization process. In the present case the route via trichlorosilane, allyl-glycidyl-ether (AGE) and bis-amino-PEO (BAP) was chosen (IIa and IIb in Fig. 1). The main core level emission lines of Ir, O, Si and C after different steps of film preparation are shown. Spectra were acquired (from bottom to top) of (P1) the bare iridium substrate, (P2) after derivatization with trichlorosilane, (P3) after hydrosilylation with AGE and (P4) after reaction with BAP. The main binding energy lines are summarized in Table 1. The metallic Ir support shows, besides the metallic Ir line, a small amount of chemisorbed oxide/hydroxide (asymmetric peak), which is also evident from the O 1s line (Muilenberg, 1979; Augustynski et al., 1984; Peuckert, 1984). Based on the O/Ir intensity ratio the surface coverage of O is well below one monolayer. In addition, small amounts of C contamination but no Si or Cl was detected. After treatment with trichlorosilane (P2) the shape of the Ir emission remains unchanged but is reduced in intensity. The O 1s line is drastically increased in intensity and the observed binding energy (Table 1) indicates the existence of hydroxide residues. In addition, Cl (binding energy (BE) 2p around 199 eV) (spectrum not shown) and Si in its oxidized form (BE 2p = 102·6 eV) are present on the surface (Muilenberg, 1979). An estimate of the surface coverage using theoretical cross-sections suggests about one monolayer of the silane on the surface. After treatment with AGE (P3) the surface becomes covered by C. The C 1s lines show a contribution of aliphatic character (BE = 285 eV) and at least one additional line at BE = 286·4 eV of oxidized C (Muilenberg, 1979) as is expected from the chemical structure of AGE. The Ir and Si lines remain unchanged but are attenuated in intensity owing to the C-containing overlayer. The O emission is shifted to higher BE due to additionally adsorbed H2O; Cl is lost from the surface indicating completion of Si–O– Si bond formation or hydrolysis of Si–Cl residues, respectively. The intensity of the Cl 2p line is decreased at least by a factor of 10 and is hardly detected in the background noise. The spectra measured for sample P4 (after attachment of 858
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BAP) remain almost unchanged except for an increase of the Ir and Si intensity, indicating a thinner C-containing overlayer. Also, the relative intensities of the aliphatic and C–O component are changed. Only a very weak emission of N, which is a label for BAP, is found. An estimate of the N/C intensity ratio (considering theoretical cross-sections) yields a value below ⬇ 3%. This, however, is probably due to the fact that the BAP used here has a molecular weight of 1900, which corresponds to about 40 ethylen oxide monomers, with two C atoms per monomer and a total of two nitrogen atoms per BAP molecule. Hence, one would expect a nitrogen/carbon intensity ratio of ⬇ 2·5%. Taking into account contributions from additional carbon present at the surface, the observed low nitrogen intensity is in agreement with the expected value. In summary, the ESCA results suggest that the polysiloxane network is not only strongly physisorbed and bound by intercalation with the rough surface but may also be partly covalently bound by Ir–O–Si bridging groups to the surfaceoxidized Ir support. However, further investigations are necessary to clarify this issue. The network remains unchanged during subsequent functionalization of the Si–H group. The measured C 1s spectra are in good agreement with the changing composition of the respective overlayer. The relative attenuation of the Ir and Si emission intensities can be qualitatively related to the different thickness of the overlayer. A precise calculation of the overlayer thickness is not possible due to an uncontrolled amount of H2O adsorbed or incorporated into the film. It is nevertheless evident from the data that no three-dimensional build-up of material occurs and that the membrane is actually very thin, since the iridium and silicon peaks are still clearly detected after the final derivatization step. Assuming an escape depth in organic films on the order of 2·5 nm one can roughly estimate that the membrane thickness is also of that order. FTIR spectroscopy FTIR-ATR spectra were acquired from membranes prepared on Ge-ATR crystals which were derivatized along with the iridium samples in the same processes. In Fig. 5 an example of the derivatization along process I (Fig. 1) with trichlorosilane (TCS) and allyl-PEG is shown. The isotropic spectrum of Allyl-PEG (Fig. 5, bottom) shows a double band at 1110 and
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Chemical modification of pacemaker electrodes
Fig. 4. ESCA spectra of samples in different stages of the derivatization process. Traces (from bottom to top): (1) bare iridium substrate; (2) after derivatization with trichlorosilane; (3) after hydrosilylation with allyl-glycidylether; and (4) after reaction with bis-amino-PEO. For details see text. 859
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TABLE 1 Core level binding energies (in eV) of the main emission lines on stepwise membrane built up according to the procedure described in Fig. 1 (II)* Sample Bare Ir (P1) Ir/TCS (P2) Ir/TCS/AGE (P3) Ir/TCS/AGE/BAP (P4)
Ir 4f7/2
Si 2p
C 1s
O 1s
Cl 2p3/2
N 1s
60·5 60·5 60·5 60·5
— 102·7 102·8 103
284·1 284·4 285; 286·4 285·3; 286·3
531·8 532·4 533·2 533·2
— 197·9 199 —
— — — 398
*TCS, trichlorosilane; AGE, allyl-glycid-ether; BAP, bis-amino-poly(ethylene oxide).
loxane network. A broad band containing multiple bands from the Si–O–Si backbone and C–O stretching vibrations from the PEG is observed between 1000 and 1250 cm−1. These findings demonstrate the feasibility of formation of an ultrathin TCS/allyl-PEG membrane along the proposed reaction scheme I (Fig. 1) in situ on a planar surface.
Fig. 5. FTIR spectra of an isotropic sample of allylPEG (bottom trace) and ATR spectra of a germanium ATR crystal derivatized with trichlorosilane (TCS) (middle trace) and TCS/allyl-PEG (top trace). Note the presence of the Si–H stretching vibration at 2250 cm−1 on the sample with TCS. Around 1140 cm−1 the Si– O–Si stretching vibrations are visible, indicating the formation of a polysiloxane network on the crystal. Upon hydrosilylation the Si–H vibration vanishes almost completely indicating successful coupling of the allyl-PEG to the polysiloxane network.
1143 cm−1 which can be assigned to the C–O stretching vibration of the PEG chain. Further, there is a strong band between 2850 and 2960 cm−1 due to the CH2 stretching vibration. The Ge-ATR/TCS sample shows Si–O–Si bands at 苲 1140 and 1070 cm−1 (Fig. 5, middle). In addition the Si–H stretching vibration at 2250 cm−1 is clearly visible. After hydrosilylation with Allyl-PEG (Fig. 5, top trace) this band vanishes almost completely, indicating efficient covalent binding of the PEG chains to the polysi860
A.C.-Impedance analysis ESCA and FTIR-ATR spectroscopy provide evidence for the successful formation of the ultrathin membrane during the two- or three-step process proposed in Fig. 1. However, little or no information can be obtained with these methods about whether or not the membranes cover the surfaces homogeneously and densely. This analysis is certainly rendered more difficult by the fact that the surface of the iridium electrodes is very rough. A.C.-impedance analysis is a very sensitive method to probe the electron transfer between a metal electrode and redox species dissolved in the electrolyte in situ even on a rough surface. An in-depth discussion of the electrical properties of the interface electrode/electrolyte can for example be found in Bard & Faulkner (1980). Briefly, the interface can be described by a parallel connection of the so-called double-layer capacitance and the Faraday impedance. The latter accounts for electrochemical processes occurring at the electrode/electrolyte interface. At high frequency the double-layer capacitance dominates the impedance, Z, while at low frequency the measured impedance is mainly determined by Faradic contributions. If an electrode is covered with a thin membrane the double-layer capacitance, Cdl, will be lower than in case of the bare electrode, since the measured overall capacitance, Ct, consists of a series connection of Cdl and the membrane capacitance, Cm. A large decrease of the capacitance would indicate the deposition of
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rather thick membrane which in the case of the pacemaker electrodes is certainly undesired. Maintaining a high interface capacitance is necessary to be able to stimulate effectively myocard tissue. As a second effect of the membrane one expects reduced Faradic currents because of hindered electron transfer. Thus, the degree of reduction in Faradic contributions to the overall electrode impedance is a sensitive indicator for the integrity and homogeneity of the membrane. In Fig. 6 a.c.-impedance measurements of pacemaker electrodes are shown at different stages of surface derivatization. The measurement frequency was chosen quite low (0·1 Hz) so that the Faradic contributions to the measured impedance were dominant. The bare electrode shows a quite drastic drop of impedance around the Nernst potential E0 of the redox-couple ( ⬇ 0·3 V). Interestingly, the electrode derivatized with TCS only does not exhibit a significantly reduced electron transfer. This finding supports the interpretation of the formation of a monolayer polysiloxane network in the first step. Obviously, it does not pose a dense barrier to electron transfer, yet allows for the secure attachment of the allyl-PEG derivative. Also shown are impedance measure-
Fig. 6. A.C.-impedance measurements on pacemaker electrodes. The drop in impedance around the Nernst potential is due to the electron transfer between the electrode and the redox-couple dissolved in the buffer solution. The impedance drop is very pronounced in the case of the bare electrode (•) and reduced in the case of electrodes covered with different membranes. For details see text.
Chemical modification of pacemaker electrodes
ments on electrodes derivatized with TCS/AGE/BAP and TCS/allyl-PEG. The best results in terms of blocking of electron transfer were obtained with electrodes covered with TCS/allyl-PEG: only a very small drop of impedance was observed at 0·3 V. Although efficient blocking of electron transfer indicates the formation of a dense and homogeneous membrane, on the other hand in all cases the overall capacitance of electrodes covered with these membranes was not significantly lower than that of bare electrodes, indicating that these membranes must indeed be very thin. Monolayer lithography towards guided cell adhesion and growth Cell adhesion to surfaces is a central issue for application and long-term maintenance of cell cultures in neurotechnology and for pharmaceutical studies. Many groups have studied surface modification with silane or thiol derivatives (Massia & Hubbell, 1991; Sukenik et al., 1990; Cheng et al., 1994) and peptides (Massia & Hubbell, 1990; Vargo et al., 1995) in order to promote cell adhesion and growth of cells. Some groups have reported guided cell adhesion and outgrowth on patterned surfaces (Kleinfeld et al., 1988; Cle´mence et al., 1995; Ranieri et al., 1993). While Kleinfeld and coworkers reported the application of standard photolithography processes (Kleinfeld et al., 1988), the molecules they used for surface modification were relatively simple silane derivatives, i.e. not particularly adapted to their task of inducing a specific cell/surface interaction. Nevertheless, guided outgrowth of neurons on surfaces patterned in this way was observed. In constrast, Cle´mence et al. (1995) investigated photochemically patterned surfaces which used the cell adhesion protein laminin to induce specific cell/surface interaction. Or purpose in this ongoing study is to combine both standard photolithography and semiconductor fabrication processes and a versatile surface derivatization scheme to produce laterally patterned surfaces with covalently attached biomolecules such as, for example, laminin or peptide fragments. As in the example reported above, again a two- or three-step process will be employed such that the first step is optimized to provide covalent bonding to the surface while the second and third step are geared towards the attachment of the appropriate biomolecule. Here 861
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we report first results on the fabrication of patterned surfaces with the first layer exhibiting a reactivity pattern or surface energy pattern. The process developed for fabrication of patterned surfaces with photolithographic techniques is shown schematically in Fig. 7. At first the substrate is silylated with alkyltrichlorosilane. Subsequently, a layer of photoresist is deposited with a spin coater and photolithographically structured. Thus, after development of the photoresist, part of the monolayer of alkyl chains is exposed and can be removed by dry etching in an argon/oxygen (10 sccm/30 sccm) plasma. After rinsing with isopropylalcohol and drying under an infrared lamp the samples are further dried in the gas-phase reactor at 100°C for 1–2 h in order to remove all but the first monolayer of water molecules from the surface. After this drying step the silylating agent for the second silylation is evaporated and the samples are incubated at 80– 150°C for about 30–60 min. Finally, merely physisorbed molecules of the silylating agent are removed by evacuating the samples at elevated temperatures for several hours. A surface of patterned reactivity is formed by this process. In order to assess the feasibility of this approach and to demonstrate the resolution achievable thereof, a glass surface was derivatized with a pattern of non-reactive alkyl chains and NH2-terminated silane, respectively. The NH2 residues were labeled with rhodamine isothiocyanate using standard procedures. A typical
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example is shown in Fig. 8 indicating a high contrast in surface reactivity towards the fluorescent label and a lateral resolution limited only by the photolithographic tools employed. Instead of the fluorescent label versatile derivatization schemes with biomolecules are conceivable and are presently under investigation. Fig. 9 demonstrates the patterning of surface wetting behavior on a sample fabricated with the process described above. On a sample with low and high surface energy areas, water condenses when the cooled sample is exposed to ambient atmosphere, demonstrating again the high resolution and sharp contrast between the different areas.
CONCLUSION Versatile two- and three-step surface modification procedures have been developed and applied to surface functionalization of pacemaker electrodes and fabrication of surfaces with patterned surface reactivity or wettability, respectively. In particular, the benefits of employing a gas-phase silylation process have been demonstrated. It allows for reliable deposition of homogeneous monolayers on planar and nanostructured surfaces. Monolayer lithography using standard photolithography technology has also been demonstrated. The results obtained will be used to modify further surfaces with biomolecules in such a way that
Fig. 7. Schematic process for fabrication of chemically patterned surfaces employing monolayer deposition by silylation and standard photolithography. 862
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Fig. 8. Surface reactivity pattern visualized by fluorescence microscopy. Areas consisting of a NH2-terminated silane were labeled with rhodamine isothiocyanate and appear bright in this image. The areas covered with a monolayer of hexadecyltrichlorosilane are inert towards labeling and therefore appear dark.
Fig. 9. Patterned surface wetting behavior as observed with microscopy. Water condenses on a sample in ambient atmosphere upon cooling, thus visualizing the pattern of surface energy created by monolayer lithography. The chemical composition of the different surface areas is indicated. 863
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adhesion and growth of cells on artificial surfaces may be controlled.
ACKNOWLEDGEMENTS M. S. gratefully acknowledges the grant of a postdoctoral fellowship from the Max-Planck Society and support from and helpful discussions with H. Mo¨hwald. We thank S. Kubala for very skillfully performing the ESCA measurements.
REFERENCES Bolz, A. (1995). Die Bedeutung der Phasengrenze zwischen alloplastischen Festko¨rpern und biologischen Geweben fu¨r die Elektrostimulation. Schiele & Scho¨n, Berlin. Fro¨hlich, R., Wetzig, T., Bolz, A. and Schaldach, M. (1995) Messung und Analyse monophasischer Aktionspotentiale mit Hilfe fraktal beschichteter Elektroden—Teil I. Biomedizinische Technik 40, 154–159. Mond, H. G. and Stokes, K. B. (1992) The electrode– tissue interface: the revolutionary role of steroid elution. Pace 15, 95–197. Tanghe, S. J., Najafi, K. and Wise, K. D. (1990) A planar IrO multichannel stimulating electrode for use in neural prostheses. Sensors & Actuators B1, 464–467. Williams, D. F. (1981). Biomaterials and Biocompatibility. In Fundamental Aspects of Biocompatibility. CRC Press, Boca Raton, FL, Vol. 1, pp. 1–11. Meachim, G. & Pedley, R. B. (1981). The tissue response at implant sites. In Fundamental Aspects of Biocompatibility. CRC Press, Boca Raton, FL, Vol. 1, pp. 107–145. Mond, H., Stokes, K., Helland, J., Grigg, L., Kertes, P., Pate, B. and Hunt, D. (1988) The porous titanium steroid eluting electrode. Pace 11, 214– 219. Beyersdorfer, F., Schneider, M., Kreuzer, J., Falk, S., Zegelmann, M. and Satter, P. (1988) Studies of the tissue reaction induced by transvenous pacemaker electrodes. I. Microscopic examination of the extent of connective tissue around the electrode tip in the human right ventricle. Pace 11, 1753–1759. Weidlich, E., Richter, G. J., Mund, K., Sturm, F. V., David, E. and Brandt, G. (1980) Threshold measurement using stimulating electrodes of different materials in the skeletal muscles of cats. Med. Progr. Technol. 7, 11–19. Mrksich, M., Sigal, G. B. and Whitesides, G. M. (1995) Surface plasmon resonance permits in situ measurement of protein adsorption on self864
Biosensors & Bioelectronics assembled monolayers of alkanethiolates on gold. Langmuir 11, 4383–4385. Prime, K. L. and Whitesides, G. M. (1993) Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using selfassembled monolayers. J. Am. Chem. Soc. 115, 10714–10721. Bergsto¨m, K., Holmberg, K., Safranj, A., Hoffman, A. S., Edgell, M. J., Kozlowski, A., Hovanes, B. A. and Harris, J. M. (1992) Reduction of fibrinogen adsorption of PEG-coated polystyrene surfaces. J. Biomed. Mater. Res. 26, 779–790. Gombotz, W. R., Guanghui, W., Horbett, T. A. and Hoffman, A. S. (1991) Protein adsorption to poly(ethylen oxide) surfaces. J. Biomed. Mater. Res. 25, 1547–1562. Gross, G. W. (1979) Simultaneously single unit recording in vitro with a photoetched laser deinsulated gold multimicroelectrode surface. IEEE Trans. Biomed. Eng. 26, 273–279. Pine, J. (1980) Recording action potentials from cultured neurons with extracellular microcircuit electrodes. J. Neurosci. Meth. 2, 19–31. Nisch, W., Bo¨ck, J., Egert, U., Ha¨mmerle, H. and Mohr, A. (1994) A thin film microelectrode array for monitoring extracellular neuronal activity in vitro. Biosensors & Bioelectronics 9, 737–741. Robinson, D. A. (1968) The electrical properties of metal microelectrodes. Proc. IEEE 56, 1065–1071. Ranck, J. B., Jr (1981). Extracellular stimulation. In Electrical Stimulation Research Techniques, ed. M. M. Patterson & R. P. Kesner. Academic Press, New York, pp. 1–36. Grattarola, M. and Martinoia, S. (1993) Modeling the neuron–microtransducer junction: from extracellular to patch recording. IEEE Trans. Biomed. Eng. 40, 35–41. Fromherz, P. and Stett, A. (1995) Silicon–neuron junction: capacitive stimulation of an individual neuron on a silicon chip. Phys. Rev. Lett. 75, 1670–1673. Ranieri, J. P., Bellamkonda, R., Jacob, J., Vargo, T. G., Gardella, J. A. and Aebischer, P. (1993) Selective neuronal cell attachment to a covalently patterned monoamine on fluorinated ethylene propylene films. J. Biomed. Mater. Res. 27, 917–925. Singhvi, R., Kumar, A., Lopez, G. P., Stephanopoulos, G. N., Wang, D. I. C., Whitesides, G. M. and Ingber, D. E. (1994) Engineering cell shape and function. Science 264, 696–698. Lopez, G. P., Albers, M. W., Schreiber, S. L., Carroll, R., Peralta, E. and Whitesides, G. M. (1993) Convenient methods for patterning the adhesion of mammalian cells to surfaces using self-assembled monolayers of alkanethiolates on gold. J. Am. Chem. Soc. 115, 5877–5878. Cle´mence, J. -F., Ranieri, J. P., Aebischer, P. and Sigrist, H. (1995) Photoimmobilization of a bioac-
Biosensors & Bioelectronics tive laminin fragment and pattern-guided selective neronal cell attachment. Bioconjugate Chem. 6, 411–417. Kleinfeld, D., Kahler, K. H. and Hockberger, P. E. (1988) Controlled outgrowth of dissociated neurons on patterned substrates. J. Neurosci. 8, 4098–4120. Stelzle, M., Diederich, A., Schmitt, J., Cassier, T., Wagner, R. & Fro¨hlich, R. (1996). Biokompatible implantierbare Niedrig-Impedanz Elektroden mit chemisch funktionalisierten Polyelektrolytschichten. Patent application: DE 19613002.6, Germany. Wagner, R., Stelzle, M., Diederich, A., Schmitt, J., Cassier, T. & Fro¨hlich, R. (1996). Biokompatible implantierbare Niedrig-Impedanz Elektroden mit chemisch funktionalisierten Polyelektrolytschichten. Patent application (extension): DE 196 30 563.2, Germany. Bolz, A., Fro¨hlich, R., Schmidt, K. and Schaldach, M. (1995) Effect of smooth, porous and fractal surface on the properties of an interface. J. Mater. Sci.: Mater. Med. 6, 844–848. Allara, D. L., Parikh, A. N. and Rondelez, F. (1995) Evidence for a unique chain organization in long chain silane monolayers deposited on two widely different solid substrates. Langmuir 11, 2357–2360. Muilenberg, G. E. (1979). Handbook of X-ray Photoelectron Spectroscopy. Perkin-Elmer, Eden Prairie. Augustynski, J., Koudelka, M., Sanchez, J. and Conway, B. E. (1984) ESCA study of the state of
Chemical modification of pacemaker electrodes iridium oxide in electrochemically and thermally formed iridium oxide films. J. Electroanal. Chem. 160, 233–248. Peuckert, M. (1984) XPS study on thermally and electrochemically prepared oxidic adlayers on iridium. Surface Sci. 144, 451–464. Bard, A. J. & Faulkner, L. R. (1980). Electrochemical Methods, Fundamentals and Applications. John Wiley, New York. Massia, S. P. and Hubbell, J. A. (1991) Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials. J. Biomed. Mater. Res. 25, 223–242. Sukenik, C. N., Balachander, N., Culp, L. A., Lewandowska, K. and Merritt, K. (1990) Modulation of cell adhesion by modification of titanium surfaces with covalently attached self-assembled monolayers. J. Biomed. Mater. Res. 24, 1307–1323. Cheng, S. -S., Chittur, K. K., Sukenik, C. N., Culp, L. A. and Lewandowska, K. (1994) The conformation of fibronectin on self-assembled monolayers with different surface composition: an FTIR/ATR study. J. Colloid Interface Sci. 162, 135–143. Massia, S. P. and Hubbell, J. A. (1990) Covalent surface immobilization of Arg-Gly-Asp- and TyrIle-Gly-Ser-Arg-containing peptides to obtain welldefined cell-adhesive substrates. Analyt. Biochem. 187, 292–301.
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On the chemical modification of pacemaker electrodes and patterned surface functionalization of planar substrates Martin Stelzle,*† Roland Wagner,§ Wilfried Nisch,† Wolfram Ja¨germann,얏 Ronald Fro¨hlich‡ & Max Schaldach‡ †Naturwissenschaftliches und Medizinisches Institut an der Universita¨t Tu¨bingen, Eberhardstraße 29, D-72762 Reutlingen, Reutlingen, Germany ‡Zentralinstitut fu¨r Biomedizinische Technik der Universita¨t Erlangen-Nu¨rnberg, Turnstraße 5, 91058 Erlangen, Erlangen, Germany §Max-Planck-Institut fu¨r Kolloid- und Grenzfla¨chenforschung, Rudower Chaussee 5, 12489 Berlin, Berlin, Germany 얏Hahn-Meitner-Institut, Glienicker Straße 100, 14109 Berlin, Berlin, Germany
Abstract: We report on experiments towards the chemical modification of metal electrodes in order to enhance biocompatibility or improve cell adhesion properties. In the first example pacemaker electrodes were modified with a thin polysiloxane network which allowed for further derivatization with a poly(ethylene glycol) layer. The primary goal was to suppress inflammatory response of tissue after implantation of electrodes. FTIR, ESCA and a.c.impedance spectroscopy show the integrity of the ultrathin membrane. No significant reduction of the electrode capacitance was observed, providing further proof for the deposition of a homogeneously thin membrane. The second example deals with the patterned chemical modification of planar surfaces. The goal was to eventually effect selective adhesion of electrosensitive cells above microelectrodes for stimulation and/or recording. First results demonstrate the compatibility of monolayer deposition techniques with common photolithography. It is thus possible to create surfaces with patterned chemical functionality. A gas-phase silylation process was developed in order to control more precisely surface hydration and reaction parameters than is possible with common solution-based silylation procedures. 1997 Elsevier Science Limited Keywords: chemical surface modification, pacemaker electrodes, gas-phase silylation, monolayer lithography, cell adhesion, biocompatibility, photolithography
INTRODUCTION The interface between electrodes and tissue has been widely studied (Bolz, 1995; Fro¨hlich et al., *To whom correspondence should be addressed.
1995; Mond & Stokes, 1992; Tanghe et al., 1990). Biocompatibility in this context usually means non-toxicity of the materials employed (Williams, 1981), the tendency of cells and tissue to adhere to the electrode surface, the absence of inflammatory responses after implantation of the 853
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electrodes (Meachim & Pedley, 1981; Mond et al., 1988; Beyersdorfer et al., 1988) and good electrical stimulation capability of the electrodes for extended periods of time (Weidlich et al., 1980). After implantation of pacemaker electrodes one observes that over a period of several weeks after implantation a relatively high stimulation amplitude is required to stimulate effectively myocardic tissue. This is most probably owing to tissue irritation and an inflammatory response. Since a high stimulation amplitude results in increased consumption of limited battery power, one goal in electrode design is to avoid this condition. In the past, sophisticated electrodes have been developed that suppress tissue inflammation by leaking steroid medication through a porous electrode head (Mond & Stokes, 1992). Our approach was different, however. Many reports have shown that poly(ethylen glycol) (PEG) grafted surfaces prevent protein adsorption which in turn is considered a prerequisite of any cell/interface interaction (Mrksich et al., 1995; Prime & Whitesides, 1993; Bergsto¨m et al., 1992; Gombotz et al., 1991). We thus concluded that it should be possible to limit tissue response by covering the electrode with a PEGcontaining membrane. On the other hand, the membrane must not reduce the electrode capacitance significantly, i.e. the membrane must be very thin. In order to cover an electrode exhibiting fractal surface topology with a very thin layer we devised a two-step reaction scheme and evaluated membrane properties and electrode capactitance with ESCA, FTIR and a.c.-impedance measurements. Planar microelectrodes have been used in the past in so-called microelectrode arrays to stimulate single cells or slices of tissue (Gross, 1979; Pine, 1980; Nisch et al., 1994) and the electrical properties of the microelectrode/cell interface have been discussed (Robinson, 1968; Ranck, 1981; Grattarola & Martinoia, 1993). These studies revealed that a close contact between cell and microelectrode, including high seal resistance towards the surrounding electrolyte, is the main prerequisite for recording high signal-to-noise data and for effective stimulation. Thus far, however, the experimentalist had either to rely on chance to position some cells properly on the microelectrodes or to use micromanipulation techniques for this purpose (Fromherz & Stett, 1995). Attempts have been made to create surfaces with 854
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patterned surface chemical functionality in order to effect guided growth of cells and promote cell attachment on surfaces at pre-selected sites (Ranieri et al., 1993; Singhvi et al., 1994; Lopez et al., 1993; Cle´mence et al., 1995). Our goal is to devise a versatile reaction scheme to create surfaces with patterned affinity towards cells using common photolithography processes. First successful steps in this direction were reported by Kleinfeld et al. (1988). Such surfaces would comprise areas with low or high affinity, with or without chemically reactive groups, respectively. Biomolecules or synthetic molecules may then be covalently attached to this surface which is thus tailored to interact with the target cell or tissue in the desired fashion.
EXPERIMENTAL DETAILS Chemical procedures for surface functionalization In order to control surface hydration and reaction parameters more precisely than in common solution-based reaction schemes a gas-phase reactor was used for silylation of surfaces whenever the molecules to be used exhibited a sufficient vapor pressure at temperatures below 150°C. Hexadecyltrichlorosilane (HTS, Cl3Si(CH2)15CH3) and aminopropyltrimethoxysilane (APS, H2N(CH2)2Si(OCH3)3) were obtained from ABCR, Germany, and used as received. The chemical structures of trichlorosilane (TCS), allylpoly(ethylene glycol) (allyl-PEG), allyl-glycidether (AGE) and bis-amino-poly(ethylene glycol) (BAP) are shown in Fig. 1. With the gas-phase reactor setup (Fig. 3) it is possible to avoid build-up of three-dimensional polymer networks, which frequently occurs when polyfunctional silanes are used for surface modification and residual moisture is present. The reactor setup including valves and inlets for the silylating agent is heatable to allow for effective water desorption prior to the reaction. Furthermore, sufficient vapor pressure of the silylating agent may be achieved and reaction speed also is increased at elevated temperatures. In the case of silylation with hexadecyltrichlorosilane (HTS) the reaction was performed in a 1% solution of HTS in dry n-hexane (Roth GmbH, Germany, water ⬍ 0·02%, in some instances further dryed on 3 Å molecular sieve)
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Chemical modification of pacemaker electrodes
Fig. 1. Derivatization scheme for chemical modification of iridium pacemaker electrodes with a two-step (I) reaction scheme using allyl-PEG or a three-step reaction scheme (IIa and IIb) with subsequent reaction of allyl-glycidether (AGE) and bis-amino-PEG (BAP) involving silylation and hydrosilylation reactions. In each case the first layer consists of a polysiloxane network deposited from trichlorosilane (TCS) in the gas phase.
using a glove box providing dry inert gas conditions. After silylation and rinsing in n-hexane the substrates were baked under N2 at ⬇ 100°C for 30 min. Electrochemical techniques for evaluation of thin organic films A Schlumberger Si1260 impedance analyzer in combination with a home-built potentiostat was employed for electrochemical measurements. A Ag/AgCl reference electrode and a Pt counter electrode was used. As redox-couple Fe(CN)36 − /4 − was employed (2 mM K3Fe(CN)6 and K4Fe(CN)6 each in 150 mM NaCl/5 mM
HEPES, pH 7·4). The drop in cell impedance due to Faradic currents around the Nernst potential of the redox-couple was used as an indicator for the integrity and homogeneity of the membrane deposited on pacemaker electrodes. In case of an electrode covered homogeneously with a membrane, electron transfer between electrode and the dissolved redox-couple is hindered and as a result the drop of impedance around the Nernst potential is small compared to the case of a bare electrode. FTIR-ATR measurements FTIR-ATR measurements were performed with a Bruker IFS66 instrument which was equipped 855
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with a MCT detector and purged with nitrogen. The spectrometer was run at 4 cm−1 resolution, and between 200 and 500 scans were usually acquired for each spectrum. Germanium ATR crystals were used as internal reflection elements and as substrates on which membranes were deposited in order to evaluate the surface chemistry occurring during the two-step reaction scheme. ESCA analysis The ESCA experiments were performed with a Leybold multitechnique surface analysis system (base pressure below 10−10 mbar) equipped with a hemispherical analyzer and multichannel detector. Mg K␣ was used as excitation source. The electrons were analyzed in normal detection with an acceptance cone of 30°. The spectrometer was calibrated with sputtered Au and Cu foils with respect to the Fermi edge and to binding energies of 932·6 eV (Cu 2p3/2) and 83·8 eV (Au 4f7/2), respectively. The samples were mechanically mounted to the sample holder ensuring an electric back contact, as is evident from the lack of charging of the sample surfaces. No further pretreatment was applied to the sample surfaces before analyis. However, in some cases the outgassing of the samples took several hours before reasonable pressure below 10−9 mbar was reached. Fluorescent labeling of amino-reactive surfaces A 1 mg/ml solution of rhodamine isothiocyanate (RITC) (obtained from Sigma, St Louis, MO) in dry DMSO (stored on molecular sieve) was prepared freshly prior to the labeling procedure. Substrates were immersed in PBS buffer, pH 8·5, and 1% (vol/vol) of the RITC solution was added. The solution was stirred for at least 10 min. The samples were rinsed and sonicated in ultrapure water, isopropanol and—if necessary to remove unspecifically adsorbed RITC—also in DMSO. Test samples of glass without aminosilane and glass derivatized with aminosilane were usually processed along with the other samples in order to check the derivatization and subsequent rinsing procedures with respect to their selectivity towards labeling of amino residues. Substrates used for FITR-ATR, ESCA and electrochemical measurements The primary goal of the analysis was to establish the composition and integrity of the thin organic 856
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films formed by two- or three-step chemical processes. Owing to the geometry of the pacemaker electrodes with their curved surface and small (approximately 2 mm) diameter, only the electrochemical measurements could be performed directly on the electrodes themselves. Owing to instrumental limitations we had to use larger samples for the ESCA analysis. We therefore fabricated steel disks (10 mm diameter) and deposited iridium films (thickness 5 m) onto them with the same procedure used for deposition of fractal iridium films on pacemaker electrodes. Thus, samples with a surface texture comparable to that found on the pacemaker electrodes (except for the slight surface curvature) were available for derivatization and subsequent ESCA analysis. We suggest that no significant differences are to be expected between the flat samples and the pacemaker electrodes with respect to composition and integrity of the thin organic films deposited on both types of samples. Our purpose in performing the FTIR-ATR analysis was to characterize further the hydrosilylation reaction (step 2 of the chemical modification), and in particular ensure proper formation of covalent Si–C bonds. Here, Ge-ATR crystals were employed as samples. On both the Ge crystals and on the iridium substrates the presence (or absence) of the polysiloxane network can be independently established. The Si–H stretching vibration at 2250 cm−1 provides a suitable measure for the conversion from Si–H to Si–C bonds during hydrosilylation. In contrast, it is difficult to prove the formation of these bonds by ESCA. There remains, however, some uncertainty concerning the influence of surface roughness on efficiency of the hydrosilylation.
RESULTS AND DISCUSSION Chemical modification of pacemaker electrodes with fractal surface topology In Fig. 1 the derivatization scheme employed in the chemical modification of pacemaker electrodes is shown (Stelzle et al., 1996; Wagner et al., 1996). The electrodes were fabricated using a plasma deposition process which results in the formation of layers with a fractal microstructure (Bolz, 1995; Fro¨hlich et al., 1995; Bolz et al., 1995) as is demonstrated in Fig. 2. A first layer of trichlorosilane (TCS) serves as reactive
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Chemical modification of pacemaker electrodes
Fig. 2. SEM image of an iridium electrode deposited by high pressure PE-CVD. The high degree of surface texture is evident.
counterpart for the immobilization of the ethylene glycol derivative via C–Si bonds. TCS has the ability to crosslink and form a network like layer on the electrode surface. ESCA measurements (cf. detailed discussion below) even appear to show evidence for the existence of some surface oxide or hydroxyl groups on the iridium electrodes. At this point it is not yet clear if chemical bonds between the silane and the iridium surface may be formed or if the high tenacity of the polysiloxane layer is solely due to intercalation with the electrode surface. In a recent publication it has been reported that stable well-ordered monolayers may be formed from trifunctional long chain alkyl silanes without significant degree of covalent bonding to the surface being involved (Allara et al., 1995). The second reaction step of this versatile modification scheme (Fig. 1) involves a hydrosilylation reaction employing a Pt catalyst with either an allyl-PEO derivative or a modified epoxy-allyl derivative. In the latter case, the PEO-chain is introduced by reaction of the epoxy residue with the amino groups of an ␣--amino-terminated PEO chain.
Fig. 3. Gas-phase reactor setup used in this study. The whole setup, including recipient, valves, inlets and the container for the silylating agent, may be heated, thus allowing for removal of residual moisture prior to the silylation step. In addition, sufficient vapor pressure of the silylating agent and increased reaction speed may be achieved at elevated temperatures.
Analysis of thin organic layers on pacemaker electrodes ESCA analysis For ESCA analysis samples were prepared on flat substrates along with pacemaker electrodes in the 857
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same deposition and derivatization process. Thus, the iridium layer exhibited the same fractal surface structure as the electrodes. The electrodes themselves were too small to be directly analyzed with our ESCA instrument. Fig. 4 shows spectra of samples at each stage of the derivatization process. In the present case the route via trichlorosilane, allyl-glycidyl-ether (AGE) and bis-amino-PEO (BAP) was chosen (IIa and IIb in Fig. 1). The main core level emission lines of Ir, O, Si and C after different steps of film preparation are shown. Spectra were acquired (from bottom to top) of (P1) the bare iridium substrate, (P2) after derivatization with trichlorosilane, (P3) after hydrosilylation with AGE and (P4) after reaction with BAP. The main binding energy lines are summarized in Table 1. The metallic Ir support shows, besides the metallic Ir line, a small amount of chemisorbed oxide/hydroxide (asymmetric peak), which is also evident from the O 1s line (Muilenberg, 1979; Augustynski et al., 1984; Peuckert, 1984). Based on the O/Ir intensity ratio the surface coverage of O is well below one monolayer. In addition, small amounts of C contamination but no Si or Cl was detected. After treatment with trichlorosilane (P2) the shape of the Ir emission remains unchanged but is reduced in intensity. The O 1s line is drastically increased in intensity and the observed binding energy (Table 1) indicates the existence of hydroxide residues. In addition, Cl (binding energy (BE) 2p around 199 eV) (spectrum not shown) and Si in its oxidized form (BE 2p = 102·6 eV) are present on the surface (Muilenberg, 1979). An estimate of the surface coverage using theoretical cross-sections suggests about one monolayer of the silane on the surface. After treatment with AGE (P3) the surface becomes covered by C. The C 1s lines show a contribution of aliphatic character (BE = 285 eV) and at least one additional line at BE = 286·4 eV of oxidized C (Muilenberg, 1979) as is expected from the chemical structure of AGE. The Ir and Si lines remain unchanged but are attenuated in intensity owing to the C-containing overlayer. The O emission is shifted to higher BE due to additionally adsorbed H2O; Cl is lost from the surface indicating completion of Si–O– Si bond formation or hydrolysis of Si–Cl residues, respectively. The intensity of the Cl 2p line is decreased at least by a factor of 10 and is hardly detected in the background noise. The spectra measured for sample P4 (after attachment of 858
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BAP) remain almost unchanged except for an increase of the Ir and Si intensity, indicating a thinner C-containing overlayer. Also, the relative intensities of the aliphatic and C–O component are changed. Only a very weak emission of N, which is a label for BAP, is found. An estimate of the N/C intensity ratio (considering theoretical cross-sections) yields a value below ⬇ 3%. This, however, is probably due to the fact that the BAP used here has a molecular weight of 1900, which corresponds to about 40 ethylen oxide monomers, with two C atoms per monomer and a total of two nitrogen atoms per BAP molecule. Hence, one would expect a nitrogen/carbon intensity ratio of ⬇ 2·5%. Taking into account contributions from additional carbon present at the surface, the observed low nitrogen intensity is in agreement with the expected value. In summary, the ESCA results suggest that the polysiloxane network is not only strongly physisorbed and bound by intercalation with the rough surface but may also be partly covalently bound by Ir–O–Si bridging groups to the surfaceoxidized Ir support. However, further investigations are necessary to clarify this issue. The network remains unchanged during subsequent functionalization of the Si–H group. The measured C 1s spectra are in good agreement with the changing composition of the respective overlayer. The relative attenuation of the Ir and Si emission intensities can be qualitatively related to the different thickness of the overlayer. A precise calculation of the overlayer thickness is not possible due to an uncontrolled amount of H2O adsorbed or incorporated into the film. It is nevertheless evident from the data that no three-dimensional build-up of material occurs and that the membrane is actually very thin, since the iridium and silicon peaks are still clearly detected after the final derivatization step. Assuming an escape depth in organic films on the order of 2·5 nm one can roughly estimate that the membrane thickness is also of that order. FTIR spectroscopy FTIR-ATR spectra were acquired from membranes prepared on Ge-ATR crystals which were derivatized along with the iridium samples in the same processes. In Fig. 5 an example of the derivatization along process I (Fig. 1) with trichlorosilane (TCS) and allyl-PEG is shown. The isotropic spectrum of Allyl-PEG (Fig. 5, bottom) shows a double band at 1110 and
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Fig. 4. ESCA spectra of samples in different stages of the derivatization process. Traces (from bottom to top): (1) bare iridium substrate; (2) after derivatization with trichlorosilane; (3) after hydrosilylation with allyl-glycidylether; and (4) after reaction with bis-amino-PEO. For details see text. 859
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TABLE 1 Core level binding energies (in eV) of the main emission lines on stepwise membrane built up according to the procedure described in Fig. 1 (II)* Sample Bare Ir (P1) Ir/TCS (P2) Ir/TCS/AGE (P3) Ir/TCS/AGE/BAP (P4)
Ir 4f7/2
Si 2p
C 1s
O 1s
Cl 2p3/2
N 1s
60·5 60·5 60·5 60·5
— 102·7 102·8 103
284·1 284·4 285; 286·4 285·3; 286·3
531·8 532·4 533·2 533·2
— 197·9 199 —
— — — 398
*TCS, trichlorosilane; AGE, allyl-glycid-ether; BAP, bis-amino-poly(ethylene oxide).
loxane network. A broad band containing multiple bands from the Si–O–Si backbone and C–O stretching vibrations from the PEG is observed between 1000 and 1250 cm−1. These findings demonstrate the feasibility of formation of an ultrathin TCS/allyl-PEG membrane along the proposed reaction scheme I (Fig. 1) in situ on a planar surface.
Fig. 5. FTIR spectra of an isotropic sample of allylPEG (bottom trace) and ATR spectra of a germanium ATR crystal derivatized with trichlorosilane (TCS) (middle trace) and TCS/allyl-PEG (top trace). Note the presence of the Si–H stretching vibration at 2250 cm−1 on the sample with TCS. Around 1140 cm−1 the Si– O–Si stretching vibrations are visible, indicating the formation of a polysiloxane network on the crystal. Upon hydrosilylation the Si–H vibration vanishes almost completely indicating successful coupling of the allyl-PEG to the polysiloxane network.
1143 cm−1 which can be assigned to the C–O stretching vibration of the PEG chain. Further, there is a strong band between 2850 and 2960 cm−1 due to the CH2 stretching vibration. The Ge-ATR/TCS sample shows Si–O–Si bands at 苲 1140 and 1070 cm−1 (Fig. 5, middle). In addition the Si–H stretching vibration at 2250 cm−1 is clearly visible. After hydrosilylation with Allyl-PEG (Fig. 5, top trace) this band vanishes almost completely, indicating efficient covalent binding of the PEG chains to the polysi860
A.C.-Impedance analysis ESCA and FTIR-ATR spectroscopy provide evidence for the successful formation of the ultrathin membrane during the two- or three-step process proposed in Fig. 1. However, little or no information can be obtained with these methods about whether or not the membranes cover the surfaces homogeneously and densely. This analysis is certainly rendered more difficult by the fact that the surface of the iridium electrodes is very rough. A.C.-impedance analysis is a very sensitive method to probe the electron transfer between a metal electrode and redox species dissolved in the electrolyte in situ even on a rough surface. An in-depth discussion of the electrical properties of the interface electrode/electrolyte can for example be found in Bard & Faulkner (1980). Briefly, the interface can be described by a parallel connection of the so-called double-layer capacitance and the Faraday impedance. The latter accounts for electrochemical processes occurring at the electrode/electrolyte interface. At high frequency the double-layer capacitance dominates the impedance, Z, while at low frequency the measured impedance is mainly determined by Faradic contributions. If an electrode is covered with a thin membrane the double-layer capacitance, Cdl, will be lower than in case of the bare electrode, since the measured overall capacitance, Ct, consists of a series connection of Cdl and the membrane capacitance, Cm. A large decrease of the capacitance would indicate the deposition of
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rather thick membrane which in the case of the pacemaker electrodes is certainly undesired. Maintaining a high interface capacitance is necessary to be able to stimulate effectively myocard tissue. As a second effect of the membrane one expects reduced Faradic currents because of hindered electron transfer. Thus, the degree of reduction in Faradic contributions to the overall electrode impedance is a sensitive indicator for the integrity and homogeneity of the membrane. In Fig. 6 a.c.-impedance measurements of pacemaker electrodes are shown at different stages of surface derivatization. The measurement frequency was chosen quite low (0·1 Hz) so that the Faradic contributions to the measured impedance were dominant. The bare electrode shows a quite drastic drop of impedance around the Nernst potential E0 of the redox-couple ( ⬇ 0·3 V). Interestingly, the electrode derivatized with TCS only does not exhibit a significantly reduced electron transfer. This finding supports the interpretation of the formation of a monolayer polysiloxane network in the first step. Obviously, it does not pose a dense barrier to electron transfer, yet allows for the secure attachment of the allyl-PEG derivative. Also shown are impedance measure-
Fig. 6. A.C.-impedance measurements on pacemaker electrodes. The drop in impedance around the Nernst potential is due to the electron transfer between the electrode and the redox-couple dissolved in the buffer solution. The impedance drop is very pronounced in the case of the bare electrode (•) and reduced in the case of electrodes covered with different membranes. For details see text.
Chemical modification of pacemaker electrodes
ments on electrodes derivatized with TCS/AGE/BAP and TCS/allyl-PEG. The best results in terms of blocking of electron transfer were obtained with electrodes covered with TCS/allyl-PEG: only a very small drop of impedance was observed at 0·3 V. Although efficient blocking of electron transfer indicates the formation of a dense and homogeneous membrane, on the other hand in all cases the overall capacitance of electrodes covered with these membranes was not significantly lower than that of bare electrodes, indicating that these membranes must indeed be very thin. Monolayer lithography towards guided cell adhesion and growth Cell adhesion to surfaces is a central issue for application and long-term maintenance of cell cultures in neurotechnology and for pharmaceutical studies. Many groups have studied surface modification with silane or thiol derivatives (Massia & Hubbell, 1991; Sukenik et al., 1990; Cheng et al., 1994) and peptides (Massia & Hubbell, 1990; Vargo et al., 1995) in order to promote cell adhesion and growth of cells. Some groups have reported guided cell adhesion and outgrowth on patterned surfaces (Kleinfeld et al., 1988; Cle´mence et al., 1995; Ranieri et al., 1993). While Kleinfeld and coworkers reported the application of standard photolithography processes (Kleinfeld et al., 1988), the molecules they used for surface modification were relatively simple silane derivatives, i.e. not particularly adapted to their task of inducing a specific cell/surface interaction. Nevertheless, guided outgrowth of neurons on surfaces patterned in this way was observed. In constrast, Cle´mence et al. (1995) investigated photochemically patterned surfaces which used the cell adhesion protein laminin to induce specific cell/surface interaction. Or purpose in this ongoing study is to combine both standard photolithography and semiconductor fabrication processes and a versatile surface derivatization scheme to produce laterally patterned surfaces with covalently attached biomolecules such as, for example, laminin or peptide fragments. As in the example reported above, again a two- or three-step process will be employed such that the first step is optimized to provide covalent bonding to the surface while the second and third step are geared towards the attachment of the appropriate biomolecule. Here 861
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we report first results on the fabrication of patterned surfaces with the first layer exhibiting a reactivity pattern or surface energy pattern. The process developed for fabrication of patterned surfaces with photolithographic techniques is shown schematically in Fig. 7. At first the substrate is silylated with alkyltrichlorosilane. Subsequently, a layer of photoresist is deposited with a spin coater and photolithographically structured. Thus, after development of the photoresist, part of the monolayer of alkyl chains is exposed and can be removed by dry etching in an argon/oxygen (10 sccm/30 sccm) plasma. After rinsing with isopropylalcohol and drying under an infrared lamp the samples are further dried in the gas-phase reactor at 100°C for 1–2 h in order to remove all but the first monolayer of water molecules from the surface. After this drying step the silylating agent for the second silylation is evaporated and the samples are incubated at 80– 150°C for about 30–60 min. Finally, merely physisorbed molecules of the silylating agent are removed by evacuating the samples at elevated temperatures for several hours. A surface of patterned reactivity is formed by this process. In order to assess the feasibility of this approach and to demonstrate the resolution achievable thereof, a glass surface was derivatized with a pattern of non-reactive alkyl chains and NH2-terminated silane, respectively. The NH2 residues were labeled with rhodamine isothiocyanate using standard procedures. A typical
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example is shown in Fig. 8 indicating a high contrast in surface reactivity towards the fluorescent label and a lateral resolution limited only by the photolithographic tools employed. Instead of the fluorescent label versatile derivatization schemes with biomolecules are conceivable and are presently under investigation. Fig. 9 demonstrates the patterning of surface wetting behavior on a sample fabricated with the process described above. On a sample with low and high surface energy areas, water condenses when the cooled sample is exposed to ambient atmosphere, demonstrating again the high resolution and sharp contrast between the different areas.
CONCLUSION Versatile two- and three-step surface modification procedures have been developed and applied to surface functionalization of pacemaker electrodes and fabrication of surfaces with patterned surface reactivity or wettability, respectively. In particular, the benefits of employing a gas-phase silylation process have been demonstrated. It allows for reliable deposition of homogeneous monolayers on planar and nanostructured surfaces. Monolayer lithography using standard photolithography technology has also been demonstrated. The results obtained will be used to modify further surfaces with biomolecules in such a way that
Fig. 7. Schematic process for fabrication of chemically patterned surfaces employing monolayer deposition by silylation and standard photolithography. 862
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Fig. 8. Surface reactivity pattern visualized by fluorescence microscopy. Areas consisting of a NH2-terminated silane were labeled with rhodamine isothiocyanate and appear bright in this image. The areas covered with a monolayer of hexadecyltrichlorosilane are inert towards labeling and therefore appear dark.
Fig. 9. Patterned surface wetting behavior as observed with microscopy. Water condenses on a sample in ambient atmosphere upon cooling, thus visualizing the pattern of surface energy created by monolayer lithography. The chemical composition of the different surface areas is indicated. 863
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adhesion and growth of cells on artificial surfaces may be controlled.
ACKNOWLEDGEMENTS M. S. gratefully acknowledges the grant of a postdoctoral fellowship from the Max-Planck Society and support from and helpful discussions with H. Mo¨hwald. We thank S. Kubala for very skillfully performing the ESCA measurements.
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