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Surface functionalization of amorphous silicon and silicon suboxides for biological applications
Thin Solid Films 427 (2003) 201–207
Surface functionalization of amorphous silicon and silicon suboxides for biological applications C. Dahmena, A. Janottab, D. Dimova-Malinovskac, S. Marxd, B. Jeschkee, B. Niese, H. Kesslera, M. Stutzmannb,* a
¨ Munchen, ¨ Institute for Organic Chemistry and Biochemistry II, Technische Universitat Lichtenbergstraße 4, 85748 Garching, Germany b ¨ Munchen, ¨ Walter Schottky Institut, Technische Universitat Am Coulombwall 3, 85748 Garching, Germany c Central Laboratory for Solar Energy and New Energy Resources, Bulgarian Academy of Sciences, Tzarigradsko Chaussee 72, 1784 Sofia, Bulgaria d ¨ Munchen, ¨ Physics Department E22, Technische Universitat James-Franck-Straße, 85748 Garching, Germany e Merck Biomaterial GmbH, Frankfurter Str. 250, 64271 Darmstadt, Germany
Abstract We have studied the chemical modification and functionalization of the surface of hydrogenated amorphous silicon (a-Si:H) and silicon suboxides (a-SiOx:H) for the purpose of selective cell adhesion. The preparation of hydrophobicyhydrophilic contrast on the amorphous surfaces has been investigated systematically as a function of the oxygen content by contact angle measurements. Successful functionalization of a-Si:H by hydrosilylation with methyl acrylate is reported and analyzed by thermal desorption studies as well as enzyme linked immunosorbent assays using RGD-peptide derivatives. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Amorphous silicon; Silicon suboxides; Surface functionalization; Hydrosilylation; Cell adhesion; RGD peptides
1. Introduction Amorphous hydrogenated silicon (a-Si:H) and its alloys with oxygen, nitrogen, or carbon possess a lot of physical properties which would make these thin film materials ideal candidates for biological applications, e.g. in advanced biosensors: (i) they can be deposited on large areas and at low temperatures on almost any substrate material by standard plasma-enhanced chemical vapour deposition (PECVD) processes. (ii) They are very flexible with respect to processing and structural modifications, including nanopatterning, and enjoy an overall compatibility with the established silicon technology. (iii) Their electronic and optical properties can be defined by doping and alloying to meet a broad range of requirements concerning electrical conductivity and optical transparency. (iv) Advanced applications such as photosensors, photovoltaic cells, thin film transistors in active matrix displays, or micro-electro-
mechanical systems are by now well established on an industrial scale or are under investigation at the prototype level. (v) Compared to crystalline silicon, amorphous silicon has the additional advantage of a strongly reduced thermal carrier generation under normal biocompatible conditions because of its larger band gap. It is, therefore, quite surprising that the use of amorphous or nanocrystalline silicon as a substrate or functional material for biological applications is still in its infancy. Very few research groups have addressed this problem, and even less systematic work has been done so far. Although amorphous SiC-alloys have been used to enhance the biocompatibility of artificial heart valves for more than 10 years w1x, almost nothing is known about why this actually happens. Similarly, a-Si:H solar cells are being considered as optical receptors in artificial retinas w2x, however, without a basic understanding of bioelectronic processes at the a-Si:Hyorganic interface. 2. Experimental details
*Corresponding author. Tel.: q49-89-2891-2760; fax: q49-892891-2737. E-mail address: [email protected] (M. Stutzmann).
Samples of a-Si:H or hydrogenated amorphous silicon suboxides (a-SiOx:H) were deposited by r.f. (13.56
0040-6090/03/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 0 - 6 0 9 0 Ž 0 2 . 0 1 2 2 8 - 2
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C. Dahmen et al. / Thin Solid Films 427 (2003) 201–207
MHz) PECVD in a capacitively coupled reactor. Silane and CO2 were used as the source gases and were diluted in H2 with a ratio of approximately 1:5. For comparison, also samples prepared by hot-wire deposition or by reactive sputtering in AryH2 atmosphere were investigated. Crystalline silicon wafers, quartz, and glass (Corning 7059) were used as substrates. Usual deposition temperatures were between 200 and 350 8C. Further details of the deposition conditions can be found in w3x. For surface functionalization of a-Si:H and a-SiOx:H, thin film samples (500–1000 nm) on glass substrates were used in order to avoid spurious hydrosilylation of crystalline Si substrates. All samples were first cleaned by dipping in water and acetone. The native oxide was removed by either exposing the surface to the vapour of 50% HF for 1 min or by dipping in 10% HF for 30 s. This simple procedure was sufficient to obtain a hydrogen-terminated, hydrophobic surface with contact angles between 80 and 908. Static contact angles were determined under ambient conditions using deionized water with a pipette-controlled drop volume of a few ml and observation with a back-illuminated CCD microscope system. Advancing and receding contact angles were also investigated, but will not be discussed here. For the preparation of hydrophilic surfaces, precleaned samples were exposed to a stream of ozone (O3) for several minutes at temperatures between 20 and 110 8C, following a proposal given in w4x. Ozone was obtained from a commercial ozonizer using pure O2 as the starting gas. In addition, the HF-treated, hydrogen-terminated surfaces of pure a-Si:H films were further functionalized by hydrosilylation, using one of the following two routes: (i) The hydrogen-terminated silicon surface was exposed to methyl acrylate at 80 8C for 2.5 h, followed by washing 5 times with petrol ether (b.p. 60–80 8C), methanol, and sonication in dichloromethane in order to remove methyl acrylate which was not covalently bonded to Si atoms at the film surface. Afterwards, the samples were dried in a stream of Ar gas. The same procedure was also performed on a bare glass substrate in order to exclude unwanted binding of functional groups to the substrate material. (ii) Several a-Si:H samples were also functionalized with cyclic RGDfK-peptides (where ‘RGD’ symbolizes the amino acid sequence ‘Arg–Gly–Asp’ known to promote binding with many integrins in living cells) using aminohexanoic acid as spacer moieties (3–4) and pent-4-enoic acid for anchoring. Hydrosilylation was performed at 50 8C for several hours in toluene with or without Wilkinson-catalyst. RGD-peptide modified surfaces are known to promote cell adhesion via integrin interactions which improve biocompatibility w5–7x. Successful functionalization of a-Si:H by RGD-peptides was
investigated by enzyme linked immunosorbent assay (ELISA). 3. Results and discussion 3.1. Hydrophilicyhydrophobic contrast of a-SiOx:H surfaces The macroscopic biocompatibility of a solid surface is strongly linked to its specific wetting behaviour with respect to water. In the particular case of a-Si:H, it has previously been shown by Smith et al. that cell adhesion and proliferation was much more pronounced for hydrophilic, oxidized a-Si:H surfaces than for hydrophobic surfaces obtained by functionalization of a-Si:H with octyltrichlorosilane w4x. The unique chemical variability of amorphous thin films allows one to change the chemical composition continuously from e.g. Si to SiO2 without major changes in the surface morphology. Therefore, we have investigated the influence of the oxygen content, wOx, of amorphous silicon suboxide films (a-SiOx:H) on the wetting behaviour of water. A significant dependence was expected based on the usual hydrophobic behaviour of hydrogenated silicon on one side and the mostly hydrophilic characteristics of stoichiometric SiO2 on the other side w8,9x. However, as shown in Fig. 1, the experimentally observed dependence of the water wetting angle on oxygen content is very weak. In the as-deposited state, a-SiOx:H samples with wOx between 0 and 44 at.% are mainly hydrophobic, with wetting angles between 60 and 808. Although there is a slight tendency of lower wetting angles for increasing oxygen content, this tendency certainly does not follow in a linear fashion the density of heteropolar Si–O bonds in the random alloy. It is, therefore, assumed that the surface properties of our samples are dominated by excess Si–H bonds as a result of the hydrogen-rich growth conditions. Moreover, we find that the as-deposited state of the surface is very stable, showing no significant signs of alteration even after 14 days of storage in a rough vacuum (10y2 mbar). In order to learn more about the wetting behaviour of a-SiOx:H in comparison to crystalline silicon or pure aSi:H, we have studied in a systematic way the influence of common surface treatments and cleaning procedures on the hydrophilicity of four different samples: a crystalline Si sample with (1 0 0) surface orientation, two a-Si:H samples prepared by sputtering or by PECVD, and an a-SiOx:H sample with an oxygen content of 44 at.%. The water contact angles of these four samples were determined in the as-deposited state and after the following surface treatments: dipping in 10% HF in water for 30 s, cleaning in acetone for 30 min, cleaning in acetone followed by washing in isopropanole for 10 min, ‘RCA’ cleaning for 10 min, and ozonization for
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Fig. 1. Water wetting angle of a-SiOx:H in the as-deposited state (solid squares) and after storage in an inert nitrogen atmosphere for 14 days (open sqaures).
10 min at 300 or 380 K, respectively. The results, which are summarized in Fig. 2, are qualitatively similar for all samples. The most hydrophobic surfaces are obtained after hydrogen termination by exposure to hydrofluoric acid. Surprisingly, this even works best for the suboxide sample. All other chemical treatments or ozonization at room temperature gave rise to intermediate contact angles between 20 and 708. On the other hand, a brief ozonization at 100 8C for 10 min rendered all surfaces completely hydrophilic, with contact angles below 108 and very little dispersion among the different samples. The influence of the ozonization temperature on contact angle for an a-SiOx:H sample with 40 at.% oxygen is shown in more detail in Fig. 3. Starting from
a contact angle of 658 in the as-deposited state, HFetching increases the contact angle to more than 908. Ozonization for 10 min at successively higher temperatures gives rise to a rapid decrease of the wetting angle, which stabilizes at approximately 58 for ozonization temperatures above 70 8C. As summarized in Fig. 4, the same behaviour is also obtained for crystalline silicon and pure a-Si:H, so that ozonization emerges as a gentle, convenient, and low-temperature process for the reliable hydrophilization of a large variety of different silicon surfaces. Preliminary cell adhesion tests with T3T fibroblast cells were performed and indicate a significantly higher cell survival rate during a period of 3 days for the hydrophilic, ozonized surfaces of a-Si:H and a-
Fig. 2. Wetting angle for four different silicon and suboxide samples as a function of surface treatment. See text for details.
C. Dahmen et al. / Thin Solid Films 427 (2003) 201–207
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Fig. 3. Dependence of the wetting angle of water on a-SiOx:H with an oxygen content of 40 at.% on ozonization temperature. Ozonization was performed for 10 min with an oxygen flow of 100 lyh. The inset shows photographs of the wetting droplets.
SiOx:H compared to the hydrophobic surfaces obtained after HF exposure. However, a more systematic biocompatibility test still needs to be done. Also, the microscopic reason for the much better wetting properties of ozonized samples still is unknown, as well as the influence of this particular surface treatment on the electronic properties of Si- and SiO-surfaces. Investigations to resolve these questions are currently underway. 3.2. Hydrosilylation of a-Si:H It has been known for quite some time that the hydrogen-terminated surface of a-Si:H is much less
susceptible to spontaneous oxidation under ambient conditions than that of crystalline silicon w10,11x. This, in addition to other advantages of disordered silicon phases mentioned in the introduction, has led us to the investigation of a-Si:H as a promising substrate material for functionalization via hydrosilylation reactions, which so far, to the best of our knowledge, have been exclusively studied for crystalline silicon substrates w12,13x. However, in the case of crystalline silicon, the almost immediate formation of a natural substoichiometric oxide upon exposure to air turns out to be a difficult practical problem for the defect free hydrosilylation of the surface. Ideally, a hydrosilylation reaction proceeds as indicated in Fig. 5a. Starting from a fully hydrogen-terminated silicon surface, thermally or optically induced dissociation of a Si–H bond at the surface gives rise to a reactive Si dangling bond, which can form a Si–C bond with the C,C double bond at the end of alkanes (v-alkenes). The ensuing transformation of the double to a single C–C bond gives rise to the appearance of a carbon dangling bond orbital at the second carbon atom of the former C_ C bond, which can abstract a neighbouring hydrogen atom from the hydrogenated silicon surface, giving rise to a new Si dangling bond at the surface. In this way, the hydrosilylation reaction proceeds serially across the Si–H surface and, thus, can be expected to be very sensitive to local disturbances, e.g. caused by weak oxidation of the surface. In the present investigation, hydrosilylation of hydrogen-terminated a-Si:H was performed using methyl acrylate or pent-4-enoic acid derivative as described in Section 2. Successful hydrosilylation with methyl acrylate was investigated with the help of thermally stimu-
Fig. 4. Wetting angle vs. ozonization temperature for the four samples of Fig. 2.
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Fig. 5. Hydrosilylation of a-Si:H: (a) schematic picture of the hydrosilylation reaction with methyl acrylate; (b) structure of the RGDfK peptide used for functionalization of a-Si:H. See Section 2 for a description of the various functional units; (c) ELISA results, as discussed in the text.
lated effusion measurements (Fig. 6). To this end, the hydrosilylated sample is placed into a quartz tube connected to an ultra high vacuum system equipped with a quadrupole mass analyzer. Upon heating of the quartz tube with a linear temperature ramp (20 Kymin), chemisorbed species in the a-Si:H thin film are thermally desorbed and can be identified according to their masses. The partial pressure at a given mass is a measure for the desorption rate at the respective temperature. In Fig. 6, three different masses have been monitored. H2O molecules with a mass of 18 amu are a usual background signal in thermal effusion and indicate the general quality of the vacuum used during the measurement. The H2 signal at mass 2 amu is partially due to
the thermal dissociation of water molecules at the quadrupole analyzer filament. In addition, the effusion peak at 550 8C is caused by the thermal emission of bonded hydrogen atoms from the bulk of the a-Si:H substrate w14x. Of main importance here is the effusion signal at mass 31 amu, which is due to the CH3O— endgroup of the methyl acrylate molecules used for surface functionalization (solid squares in Fig. 6). We observe a small shoulder of the effusion spectrum at 120 8C, which is assigned to the evaporation of physisorbed molecules. In contrast, the main effusion peak at 410 8C is due to covalently bonded methyl acrylate molecules, which loose their CH3O—endgroup at this particular temperature. The integrated intensity of the
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Fig. 6. Thermal effusion spectra of a-Si:H functionalized with methyl acrylate. The reference trace shows the signal at mass 31 amu for an untreated a-Si-H sample.
effusion peak is a measure for the surface density of chemisorbed methyl acrylate molecules. Using the known hydrogen concentration of the underlying a-Si:H substrate, a comparison of the integrated H2- and CH3Oeffusion signals can be used to estimate the surface coverage by methyl acrylate molecules. The effusion peak in Fig. 6 indeed is of the order of magnitude expected for a monolayer coverage, however, a more careful quantitative investigation based on different samples will be needed to substantiate this rough estimate. Also shown in Fig. 6 is the CH3O—background signal obtained from a sample not subjected to the hydrosilylation treatment, clearly showing a much lower effusion pressure, as expected. In addition to the hydrosilylation by simple methyl acrylate, hydrogen-terminated a-Si:H was also functionalized with the much more complex molecules shown in Fig. 5b. These molecules again consist of an anchor group with a C,C double bond for hydrosilylation, followed by a spacer group (aminohexanoic acid, repeated ns3 or 4 times), and finally a specific cyclic ‘RGD’ peptide group which is known to promote the adhesion of cells on a large number of substrate materials w5–7x. The successful functionalization of a-Si:H with this molecule was investigated by ELISA. In this method, optical marker molecules with a specific absorption spectrum are bound to molecules involved in the surface functionalization of interest by a selective biochemical reaction. Therefore, the optical absorption at the specific wavelength of the marker molecule is a direct, albeit relative measure for the degree of surface functionalization achieved. As depicted in Fig. 5c, indeed a
significant difference between functionalized and virgin a-Si:H surfaces is observed, indicating a successful biofunctionalization with the RGD-peptide. However, this qualitative result will have to be followed up by a more quantitative investigation in the future. 4. Conclusions a-Si:H and a-SiOx:H are interesting alternative substrate materials for biological applications. Exposure to dilute hydrofluoric acid, on one hand, and ozonization at moderate temperatures, on the other hand, can be used to generate a high hydrophilicyhydrophobic contrast, irrespective of the specific chemical composition of the sample. Hydrophilic samples of amorphous silicon or silicon suboxides have been found to be largely biocompatible. Surface functionalization of these materials by hydrosilylation reactions have been explored and appear to be very promising for biological applications. Acknowledgments This work was supported by Deutsche Forschungsgemeinschaft (SFB 563). References w1x A. Bolz, M. Schaldach, Artif. Organs 14 (1990) 260. w2x M. Rojahn, M.B. Schubert, Mat. Res. Soc. Symp. Proc. 609 (2000) A21.4.1. w3x R. Janssen, A. Janotta, D. Dimova-Malinovska, M. Stutzmann, Phys. Rev. B 60 (1999) 13561.
C. Dahmen et al. / Thin Solid Films 427 (2003) 201–207 w4x L.L. Smith, K. Wang, G.N. Parsons, R. Hernandez, D.T. Brown, Mat. Res. Soc. Symp. Proc. 609 (2000) A21.5.1. w5x M. Kantlehner, P. Schaffner, D. Finsinger, J. Meyer, A. Jon¨ czyk, B. Diefenbach, B. Nies, G. Holzemann, S.L. Goodman, H. Kessler, Chem. Biochem. 1 (2000) 107. w6x M. Kantlehner, D. Finsinger, J. Meyer, P. Schaffner, A. Jonczyk, B. Diefenbach, B. Nies, H. Kessler, Angew. Chem. Eng. Int. 38 (1999) 560–562. w7x R.G. Lebaron, K.A. Athanasiou, Tissue Eng. 6 (2000) 85. w8x R. Williams, A.M. Goodman, Appl. Phys. Lett. 25 (1974) 531.
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w9x R.R. Thomas, F.B. Kaufman, J.T. Kirleis, R.A. Belsky, J. Electrochem. Soc. 143 (1996) 643. w10x J.P. Ponpon, B. Bourdon, Solid State Electron. 25 (1982) 875. w11x L. Ley, H. Richter, R. Karcher, ¨ R.L. Johnson, J. Reichhardt, J. Phys. C4 (1981) 753, Paris. w12x M.R. Lindford, P. Fenter, P. Eisenberger, C. Chidsey, J. Am. Chem. Soc. 117 (1995) 3145. w13x A. Bansal, X. Li, S.I. Yi, W.H. Weinberg, N.S. Lewis, J. Phys. Chem. B105 (2001) 10266. w14x W. Beyer, J. Non-Cryst. Solids 198–200 (1996) 40.
Surface functionalization of amorphous silicon and silicon suboxides for biological applications C. Dahmena, A. Janottab, D. Dimova-Malinovskac, S. Marxd, B. Jeschkee, B. Niese, H. Kesslera, M. Stutzmannb,* a
¨ Munchen, ¨ Institute for Organic Chemistry and Biochemistry II, Technische Universitat Lichtenbergstraße 4, 85748 Garching, Germany b ¨ Munchen, ¨ Walter Schottky Institut, Technische Universitat Am Coulombwall 3, 85748 Garching, Germany c Central Laboratory for Solar Energy and New Energy Resources, Bulgarian Academy of Sciences, Tzarigradsko Chaussee 72, 1784 Sofia, Bulgaria d ¨ Munchen, ¨ Physics Department E22, Technische Universitat James-Franck-Straße, 85748 Garching, Germany e Merck Biomaterial GmbH, Frankfurter Str. 250, 64271 Darmstadt, Germany
Abstract We have studied the chemical modification and functionalization of the surface of hydrogenated amorphous silicon (a-Si:H) and silicon suboxides (a-SiOx:H) for the purpose of selective cell adhesion. The preparation of hydrophobicyhydrophilic contrast on the amorphous surfaces has been investigated systematically as a function of the oxygen content by contact angle measurements. Successful functionalization of a-Si:H by hydrosilylation with methyl acrylate is reported and analyzed by thermal desorption studies as well as enzyme linked immunosorbent assays using RGD-peptide derivatives. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Amorphous silicon; Silicon suboxides; Surface functionalization; Hydrosilylation; Cell adhesion; RGD peptides
1. Introduction Amorphous hydrogenated silicon (a-Si:H) and its alloys with oxygen, nitrogen, or carbon possess a lot of physical properties which would make these thin film materials ideal candidates for biological applications, e.g. in advanced biosensors: (i) they can be deposited on large areas and at low temperatures on almost any substrate material by standard plasma-enhanced chemical vapour deposition (PECVD) processes. (ii) They are very flexible with respect to processing and structural modifications, including nanopatterning, and enjoy an overall compatibility with the established silicon technology. (iii) Their electronic and optical properties can be defined by doping and alloying to meet a broad range of requirements concerning electrical conductivity and optical transparency. (iv) Advanced applications such as photosensors, photovoltaic cells, thin film transistors in active matrix displays, or micro-electro-
mechanical systems are by now well established on an industrial scale or are under investigation at the prototype level. (v) Compared to crystalline silicon, amorphous silicon has the additional advantage of a strongly reduced thermal carrier generation under normal biocompatible conditions because of its larger band gap. It is, therefore, quite surprising that the use of amorphous or nanocrystalline silicon as a substrate or functional material for biological applications is still in its infancy. Very few research groups have addressed this problem, and even less systematic work has been done so far. Although amorphous SiC-alloys have been used to enhance the biocompatibility of artificial heart valves for more than 10 years w1x, almost nothing is known about why this actually happens. Similarly, a-Si:H solar cells are being considered as optical receptors in artificial retinas w2x, however, without a basic understanding of bioelectronic processes at the a-Si:Hyorganic interface. 2. Experimental details
*Corresponding author. Tel.: q49-89-2891-2760; fax: q49-892891-2737. E-mail address: [email protected] (M. Stutzmann).
Samples of a-Si:H or hydrogenated amorphous silicon suboxides (a-SiOx:H) were deposited by r.f. (13.56
0040-6090/03/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 0 - 6 0 9 0 Ž 0 2 . 0 1 2 2 8 - 2
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C. Dahmen et al. / Thin Solid Films 427 (2003) 201–207
MHz) PECVD in a capacitively coupled reactor. Silane and CO2 were used as the source gases and were diluted in H2 with a ratio of approximately 1:5. For comparison, also samples prepared by hot-wire deposition or by reactive sputtering in AryH2 atmosphere were investigated. Crystalline silicon wafers, quartz, and glass (Corning 7059) were used as substrates. Usual deposition temperatures were between 200 and 350 8C. Further details of the deposition conditions can be found in w3x. For surface functionalization of a-Si:H and a-SiOx:H, thin film samples (500–1000 nm) on glass substrates were used in order to avoid spurious hydrosilylation of crystalline Si substrates. All samples were first cleaned by dipping in water and acetone. The native oxide was removed by either exposing the surface to the vapour of 50% HF for 1 min or by dipping in 10% HF for 30 s. This simple procedure was sufficient to obtain a hydrogen-terminated, hydrophobic surface with contact angles between 80 and 908. Static contact angles were determined under ambient conditions using deionized water with a pipette-controlled drop volume of a few ml and observation with a back-illuminated CCD microscope system. Advancing and receding contact angles were also investigated, but will not be discussed here. For the preparation of hydrophilic surfaces, precleaned samples were exposed to a stream of ozone (O3) for several minutes at temperatures between 20 and 110 8C, following a proposal given in w4x. Ozone was obtained from a commercial ozonizer using pure O2 as the starting gas. In addition, the HF-treated, hydrogen-terminated surfaces of pure a-Si:H films were further functionalized by hydrosilylation, using one of the following two routes: (i) The hydrogen-terminated silicon surface was exposed to methyl acrylate at 80 8C for 2.5 h, followed by washing 5 times with petrol ether (b.p. 60–80 8C), methanol, and sonication in dichloromethane in order to remove methyl acrylate which was not covalently bonded to Si atoms at the film surface. Afterwards, the samples were dried in a stream of Ar gas. The same procedure was also performed on a bare glass substrate in order to exclude unwanted binding of functional groups to the substrate material. (ii) Several a-Si:H samples were also functionalized with cyclic RGDfK-peptides (where ‘RGD’ symbolizes the amino acid sequence ‘Arg–Gly–Asp’ known to promote binding with many integrins in living cells) using aminohexanoic acid as spacer moieties (3–4) and pent-4-enoic acid for anchoring. Hydrosilylation was performed at 50 8C for several hours in toluene with or without Wilkinson-catalyst. RGD-peptide modified surfaces are known to promote cell adhesion via integrin interactions which improve biocompatibility w5–7x. Successful functionalization of a-Si:H by RGD-peptides was
investigated by enzyme linked immunosorbent assay (ELISA). 3. Results and discussion 3.1. Hydrophilicyhydrophobic contrast of a-SiOx:H surfaces The macroscopic biocompatibility of a solid surface is strongly linked to its specific wetting behaviour with respect to water. In the particular case of a-Si:H, it has previously been shown by Smith et al. that cell adhesion and proliferation was much more pronounced for hydrophilic, oxidized a-Si:H surfaces than for hydrophobic surfaces obtained by functionalization of a-Si:H with octyltrichlorosilane w4x. The unique chemical variability of amorphous thin films allows one to change the chemical composition continuously from e.g. Si to SiO2 without major changes in the surface morphology. Therefore, we have investigated the influence of the oxygen content, wOx, of amorphous silicon suboxide films (a-SiOx:H) on the wetting behaviour of water. A significant dependence was expected based on the usual hydrophobic behaviour of hydrogenated silicon on one side and the mostly hydrophilic characteristics of stoichiometric SiO2 on the other side w8,9x. However, as shown in Fig. 1, the experimentally observed dependence of the water wetting angle on oxygen content is very weak. In the as-deposited state, a-SiOx:H samples with wOx between 0 and 44 at.% are mainly hydrophobic, with wetting angles between 60 and 808. Although there is a slight tendency of lower wetting angles for increasing oxygen content, this tendency certainly does not follow in a linear fashion the density of heteropolar Si–O bonds in the random alloy. It is, therefore, assumed that the surface properties of our samples are dominated by excess Si–H bonds as a result of the hydrogen-rich growth conditions. Moreover, we find that the as-deposited state of the surface is very stable, showing no significant signs of alteration even after 14 days of storage in a rough vacuum (10y2 mbar). In order to learn more about the wetting behaviour of a-SiOx:H in comparison to crystalline silicon or pure aSi:H, we have studied in a systematic way the influence of common surface treatments and cleaning procedures on the hydrophilicity of four different samples: a crystalline Si sample with (1 0 0) surface orientation, two a-Si:H samples prepared by sputtering or by PECVD, and an a-SiOx:H sample with an oxygen content of 44 at.%. The water contact angles of these four samples were determined in the as-deposited state and after the following surface treatments: dipping in 10% HF in water for 30 s, cleaning in acetone for 30 min, cleaning in acetone followed by washing in isopropanole for 10 min, ‘RCA’ cleaning for 10 min, and ozonization for
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Fig. 1. Water wetting angle of a-SiOx:H in the as-deposited state (solid squares) and after storage in an inert nitrogen atmosphere for 14 days (open sqaures).
10 min at 300 or 380 K, respectively. The results, which are summarized in Fig. 2, are qualitatively similar for all samples. The most hydrophobic surfaces are obtained after hydrogen termination by exposure to hydrofluoric acid. Surprisingly, this even works best for the suboxide sample. All other chemical treatments or ozonization at room temperature gave rise to intermediate contact angles between 20 and 708. On the other hand, a brief ozonization at 100 8C for 10 min rendered all surfaces completely hydrophilic, with contact angles below 108 and very little dispersion among the different samples. The influence of the ozonization temperature on contact angle for an a-SiOx:H sample with 40 at.% oxygen is shown in more detail in Fig. 3. Starting from
a contact angle of 658 in the as-deposited state, HFetching increases the contact angle to more than 908. Ozonization for 10 min at successively higher temperatures gives rise to a rapid decrease of the wetting angle, which stabilizes at approximately 58 for ozonization temperatures above 70 8C. As summarized in Fig. 4, the same behaviour is also obtained for crystalline silicon and pure a-Si:H, so that ozonization emerges as a gentle, convenient, and low-temperature process for the reliable hydrophilization of a large variety of different silicon surfaces. Preliminary cell adhesion tests with T3T fibroblast cells were performed and indicate a significantly higher cell survival rate during a period of 3 days for the hydrophilic, ozonized surfaces of a-Si:H and a-
Fig. 2. Wetting angle for four different silicon and suboxide samples as a function of surface treatment. See text for details.
C. Dahmen et al. / Thin Solid Films 427 (2003) 201–207
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Fig. 3. Dependence of the wetting angle of water on a-SiOx:H with an oxygen content of 40 at.% on ozonization temperature. Ozonization was performed for 10 min with an oxygen flow of 100 lyh. The inset shows photographs of the wetting droplets.
SiOx:H compared to the hydrophobic surfaces obtained after HF exposure. However, a more systematic biocompatibility test still needs to be done. Also, the microscopic reason for the much better wetting properties of ozonized samples still is unknown, as well as the influence of this particular surface treatment on the electronic properties of Si- and SiO-surfaces. Investigations to resolve these questions are currently underway. 3.2. Hydrosilylation of a-Si:H It has been known for quite some time that the hydrogen-terminated surface of a-Si:H is much less
susceptible to spontaneous oxidation under ambient conditions than that of crystalline silicon w10,11x. This, in addition to other advantages of disordered silicon phases mentioned in the introduction, has led us to the investigation of a-Si:H as a promising substrate material for functionalization via hydrosilylation reactions, which so far, to the best of our knowledge, have been exclusively studied for crystalline silicon substrates w12,13x. However, in the case of crystalline silicon, the almost immediate formation of a natural substoichiometric oxide upon exposure to air turns out to be a difficult practical problem for the defect free hydrosilylation of the surface. Ideally, a hydrosilylation reaction proceeds as indicated in Fig. 5a. Starting from a fully hydrogen-terminated silicon surface, thermally or optically induced dissociation of a Si–H bond at the surface gives rise to a reactive Si dangling bond, which can form a Si–C bond with the C,C double bond at the end of alkanes (v-alkenes). The ensuing transformation of the double to a single C–C bond gives rise to the appearance of a carbon dangling bond orbital at the second carbon atom of the former C_ C bond, which can abstract a neighbouring hydrogen atom from the hydrogenated silicon surface, giving rise to a new Si dangling bond at the surface. In this way, the hydrosilylation reaction proceeds serially across the Si–H surface and, thus, can be expected to be very sensitive to local disturbances, e.g. caused by weak oxidation of the surface. In the present investigation, hydrosilylation of hydrogen-terminated a-Si:H was performed using methyl acrylate or pent-4-enoic acid derivative as described in Section 2. Successful hydrosilylation with methyl acrylate was investigated with the help of thermally stimu-
Fig. 4. Wetting angle vs. ozonization temperature for the four samples of Fig. 2.
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Fig. 5. Hydrosilylation of a-Si:H: (a) schematic picture of the hydrosilylation reaction with methyl acrylate; (b) structure of the RGDfK peptide used for functionalization of a-Si:H. See Section 2 for a description of the various functional units; (c) ELISA results, as discussed in the text.
lated effusion measurements (Fig. 6). To this end, the hydrosilylated sample is placed into a quartz tube connected to an ultra high vacuum system equipped with a quadrupole mass analyzer. Upon heating of the quartz tube with a linear temperature ramp (20 Kymin), chemisorbed species in the a-Si:H thin film are thermally desorbed and can be identified according to their masses. The partial pressure at a given mass is a measure for the desorption rate at the respective temperature. In Fig. 6, three different masses have been monitored. H2O molecules with a mass of 18 amu are a usual background signal in thermal effusion and indicate the general quality of the vacuum used during the measurement. The H2 signal at mass 2 amu is partially due to
the thermal dissociation of water molecules at the quadrupole analyzer filament. In addition, the effusion peak at 550 8C is caused by the thermal emission of bonded hydrogen atoms from the bulk of the a-Si:H substrate w14x. Of main importance here is the effusion signal at mass 31 amu, which is due to the CH3O— endgroup of the methyl acrylate molecules used for surface functionalization (solid squares in Fig. 6). We observe a small shoulder of the effusion spectrum at 120 8C, which is assigned to the evaporation of physisorbed molecules. In contrast, the main effusion peak at 410 8C is due to covalently bonded methyl acrylate molecules, which loose their CH3O—endgroup at this particular temperature. The integrated intensity of the
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Fig. 6. Thermal effusion spectra of a-Si:H functionalized with methyl acrylate. The reference trace shows the signal at mass 31 amu for an untreated a-Si-H sample.
effusion peak is a measure for the surface density of chemisorbed methyl acrylate molecules. Using the known hydrogen concentration of the underlying a-Si:H substrate, a comparison of the integrated H2- and CH3Oeffusion signals can be used to estimate the surface coverage by methyl acrylate molecules. The effusion peak in Fig. 6 indeed is of the order of magnitude expected for a monolayer coverage, however, a more careful quantitative investigation based on different samples will be needed to substantiate this rough estimate. Also shown in Fig. 6 is the CH3O—background signal obtained from a sample not subjected to the hydrosilylation treatment, clearly showing a much lower effusion pressure, as expected. In addition to the hydrosilylation by simple methyl acrylate, hydrogen-terminated a-Si:H was also functionalized with the much more complex molecules shown in Fig. 5b. These molecules again consist of an anchor group with a C,C double bond for hydrosilylation, followed by a spacer group (aminohexanoic acid, repeated ns3 or 4 times), and finally a specific cyclic ‘RGD’ peptide group which is known to promote the adhesion of cells on a large number of substrate materials w5–7x. The successful functionalization of a-Si:H with this molecule was investigated by ELISA. In this method, optical marker molecules with a specific absorption spectrum are bound to molecules involved in the surface functionalization of interest by a selective biochemical reaction. Therefore, the optical absorption at the specific wavelength of the marker molecule is a direct, albeit relative measure for the degree of surface functionalization achieved. As depicted in Fig. 5c, indeed a
significant difference between functionalized and virgin a-Si:H surfaces is observed, indicating a successful biofunctionalization with the RGD-peptide. However, this qualitative result will have to be followed up by a more quantitative investigation in the future. 4. Conclusions a-Si:H and a-SiOx:H are interesting alternative substrate materials for biological applications. Exposure to dilute hydrofluoric acid, on one hand, and ozonization at moderate temperatures, on the other hand, can be used to generate a high hydrophilicyhydrophobic contrast, irrespective of the specific chemical composition of the sample. Hydrophilic samples of amorphous silicon or silicon suboxides have been found to be largely biocompatible. Surface functionalization of these materials by hydrosilylation reactions have been explored and appear to be very promising for biological applications. Acknowledgments This work was supported by Deutsche Forschungsgemeinschaft (SFB 563). References w1x A. Bolz, M. Schaldach, Artif. Organs 14 (1990) 260. w2x M. Rojahn, M.B. Schubert, Mat. Res. Soc. Symp. Proc. 609 (2000) A21.4.1. w3x R. Janssen, A. Janotta, D. Dimova-Malinovska, M. Stutzmann, Phys. Rev. B 60 (1999) 13561.
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