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Surface modification of poly(dimethylsiloxane) for controlling biological cells’ adhesion using a scanning radical microjet
Thin Solid Films 515 (2007) 5172 – 5178 www.elsevier.com/locate/tsf
Surface modification of poly(dimethylsiloxane) for controlling biological cells’ adhesion using a scanning radical microjet Helen M.L. Tan a , H. Fukuda b , T. Akagi c,d , T. Ichiki c,d,⁎ a
Department of Materials Engineering, School of Engineering, The University of Tokyo, 2–11–16 Yayoi, Bunkyo-ku, 113–8656, Japan b Department of Electrical and Electronics Engineering, Toyo University, 2100 Kujirai, Kawagoe, 350–8585, Japan c Department of Bioengineering, School of Engineering, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, 113–8656, Japan d Center for NanoBio Integration, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, 113–8656, Japan Available online 30 November 2006
Abstract A scanning radical microjet (SRMJ) equipment using oxygen microplasma has been developed and successfully applied for controlling biological cells’ attachment on biocompatible polymer material, poly(dimethylsiloxane) (PDMS). The radical microjet has advantages in localized and high-rate surface treatment. Moreover, maskless hydrophilic patterning using SRMJ has been demonstrated to be applicable to patterned cell cultivation which is useful in emerging biotechnological field such as tissue engineering and cell-based biosensors. Since control of PDMS surface properties is an indispensable prerequisite for cells’ attachment, effects of oxygen flow rates and treatment time on localized hydrophilic patterning of PDMS surfaces were first investigated for controlling HeLa cells’ (human epitheloid carcinoma cell line) attachment. Relationships between surface conditions of treated PDMS films and attached cell density are also discussed based on surface properties analyzed using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). © 2006 Elsevier B.V. All rights reserved. Keywords: Scanning radical microjet equipment; Poly(dimethylsiloxane) (PDMS); Surface modifications; Cell adhesion
1. Introduction In recent years, rapid growth has been seen in research and development of biomicrosystems such as microreactors and bioanalytical devices, which are driven by aggressive fusion of nano/microfabrication technology and modern biotechnology. In such a field, silicone elastomer, poly(dimethylsiloxane) (PDMS) has been widely used as a fabrication material due to ease in micropattern fabrication at low cost, resistance to various chemicals and pH environments, excellent transparency in the UV–vis range [1,2]. Its remarkable gas permeability and biocompatibility have in particular attracted the integration of biological applications into PDMS devices [3]. A recent review by Sia and Whitesides [4] describes microfluidic systems in PDMS for biological studies. It also reports on bioanalytical applications comprising of immunoassays, separation of biomolecules, as well as sorting and manipulation of cells. ⁎ Corresponding author. Tel./fax: +81 3 5841 1180. E-mail address: [email protected] (T. Ichiki). 0040-6090/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2006.10.026
Despite these advantages, the extreme hydrophobicity of PDMS often faces certain difficulties in most biological applications. Therefore, various solutions have been proposed for tailoring PDMS surface properties, e.g., oxygen plasma treatment [5], silanization [6], adsorbed coatings [7], UV curing [8], and plasma grafting [9]. Among these, surface modification with oxygen plasma is mostly used in rapid prototyping of microfluidic devices. On the other hand, in recent years, cell patterning on a chip has been intensively studied for regulating configuration of cultivated cells. Conventional methods such as photolithography, contact printing and ink-jet printing have provided feasible patterning of various kinds of biomolecules and cells [10–14]. For more precise control of cells’ behavior, however, physical effect, especially topographic or mechanical effects should be involved for cell patterning. Recent reports demonstrated that application of a mechanical factor to cultured cells is important for understanding cell growth and proliferation [15]. Some researchers attempted it using elastic materials such as PDMS. Although silicone elastomer substrate is of great interest as an
H.M.L. Tan et al. / Thin Solid Films 515 (2007) 5172–5178
alternative to glass- or plastic-based substrates, problems of insufficient cell attachment and growth remain unsettled [16] and are also to be solved by adopting proper surface modification for controlling adsorption of proteins which dominate cell attachment on extracellular matrix. In a recent paper by Rhee et al. [17], they reported molecular patterning on a PDMS surface using dry process. Although their method was successful in generating patterned cell culture inside microfluidic devices, the patterning process required time consuming steps in preparing stencil masks specially fabricated with PDMS. In this paper, we used a scanning radical microjet (SRMJ) instead of direct plasma exposure onto PDMS surface in order to avoid damage to the PDMS surface. Also, we carried out maskless patterning for treatment on the PDMS surface. Effects of oxygen flow rates and scanning speeds on localized hydrophilic patterning on the PDMS surface were first investigated for controlling HeLa cells’ attachment using an SRMJ. Since cell attachment to the material is closely related to wettability of its surface [18,19], contact angle measurements on PDMS were conducted and effects of wettability of treated PDMS were investigated biologically for better understanding of cell–surface interactions. By using SMRJ, we have achieved patterned HeLa cells’ cultivation in a single line according to the radical-treated line. 2. Experimental 2.1. Apparatus Fig. 1 shows a schematic of the SMRJ apparatus employed in this study. The compact plasma source is comprised of a 250-μmthick copper antenna deposited on an alumina plate and a silica discharge tube with a 0.9 mm inner diameter. A 144-MHz VHF power supplied to the plasma source was kept constant at 50 W [20,21]. A fine argon plasma jet was emitted through the pinched end of the discharge tube which has an inner diameter of 0.1 mm. Another small cylindrical tube was connected to the end of the discharge tube for the introduction and subsequent dissociation of oxygen. The oxygen radicals were emitted out through a 100 μm
5173
hole at the end of the cylindrical tube. The flow rate of argon used in this study was kept constant at 180 ccm. In addition, another low-pressure ICP plasma apparatus was also adopted for investigating the relationship between wettability and cell adhesion. Low-pressure ICP plasma was used since it could achieve wide-range wettability control of polymer surface as compared to radical treatment. Oxygen plasma was produced in a stainless steel chamber 260 mm in diameter by supplying 13.5-MHz rf power to a single-turn antenna set on a quartz window at one end. A sample was placed on the stage set in the downstream region 36 cm away from the antenna. The plasma was generated at a total pressure of 6.5 × 10− 3 Pa and rf power of 400 W. 2.2. Sample preparation and surface characterizations SYLGARD 184 (Dow Corning), base and curing agent, was used to prepare thin PDMS films on glass substrates using a spin-coater and cured at 100 °C for 2 min. Line scanning in x- or y-direction was then performed for hydrophilization using SRMJ equipment and water contact angles distributions of microscopic-size water droplets obtained from condensed steam of boiling water were measured across the scanned line using a self-made contact angle measurement system. This system consists of a humidity chamber and microscope with a long focal length. With such system, rapid evaporation of small droplets of approximately 50 μm size can be prevented and contact angle can be measured more precisely. Working parameters such as oxygen flow rates and scanning speed of SRMJ were first investigated to find out their effects on hydrophilic patterning of PDMS surface and subsequent cell patterning. Also, surface properties such as surface roughness and chemical states were also analyzed using atomic force microscope (AFM: Shimadzu SPM-9500) and X-ray photoelectron spectroscope (XPS: Shimadzu-Kratos Axis His), respectively. In the latter analysis, monochromatic Mg Kα was used as an X-ray source. 2.3. Cell cultivation on surface modified PDMS films As a feasibility study, HeLa cells (human epitheloid carcinoma cell line) were cultured on treated PDMS films in Dulbecco's modified Eagle's medium supplemented with 10% fatal bovine serum, L-glutamine and penicillin–streptomycin, and incubated for 72 h (3 days) at 37 °C in 5% CO2 in air atmosphere. Initial area density of seeded cells was constant at 3 × 103/cm2 in all the experiments. Cell density on treated PDMS surfaces was counted using an optical microscope. 3. Results 3.1. Effects of scanning speed
Fig. 1. A schematic diagram of the scanning radical microjet (SRMJ) employed in this study.
3.1.1. Wettability of treated PDMS Effect of scanning speed on hydrophilic patterning was first investigated. Fig. 2 shows water contact angles distribution measured across the scanned line. Subsequently from these profiles,
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3.1.3. Cell adhesion analysis Fig. 6 illustrates the cell proliferation profiles for four different scan speeds used in treating PDMS surfaces for DIV (day in vitro) 2 of the cell culture process. From the profiles, scan speed of 0.5 mm/s shows highest cell density of approximately 420 cells/mm2 among other scan speeds. Here, in order to highlight the cell density profile across the treated line, cell density was measured at 0.1 mm interval from the center axis of the treated line. Namely, total number of cells on the segmented area of 0.1 mm width was divided by this area. From Fig. 7, it can be observed that as scanning speed increases, cell densities for both DIV 1 and 2 decrease. In this case, average cell density is defined as the cell density on the treated line for each individual day. This was calculated by dividing the total number of cells on the treated line over the whole area of the treated line. The results from Fig. 7 imply that lower scan speed promotes better cell adhesion to the oxygen radicaltreated surfaces. 3.2. Effects of oxygen flow rates
Fig. 2. Scanned line width profiles plotted from water contact angles measurements for different scanning speeds. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
minimal contact angle, θmin and treated line width, W are plotted in Fig. 3. W is defined here as the width at 10% of the maximum valley depth of the water contact angles’ profile. Contact angle for non-treated PDMS surface was approximately 112°. It is observed that as scanning speed increases from 0.5 to 6 mm/s, minimal contact angle increases from 72 to 79° while the treated line width decreases from 2.2 to 1.5 mm. This phenomenon could be ascribed to the shorter treatment time by the oxygen radicals on the PDMS surface as scanning speed increases, hence resulting in decreasing hydrophilicity and thus narrower line width and higher water contact angles. 3.1.2. Surface morphology of treated PDMS Since oxygen radicals are likely to etch polymer surface, surface morphology of treated PDMS was analyzed using AFM. Fig. 4 shows surface morphologies for 3 different scan speeds of 0.5, 3 and 6 mm/s. The surface roughness values were then plotted as shown in Fig. 5. Both root mean square (Rrms) and average roughness (Ra) values show decreasing phenomena with the increase of scanning speed. Such tendency can be ascribed to decreasing treatment time as scanning speed is increased. Ra and Rrms of non-treated PDMS surface were measured and found to be 2.2 nm and 2.9 nm, respectively.
Similarly to Section 3.1, effects of oxygen flow rates on wettability and surface morphology of treated PDMS and cell adhesion properties were also investigated. Fig. 8(a) shows the effects of oxygen flow rates on water contact angles of PDMS surfaces treated by SRMJ. As oxygen flow rate increases from 20 to 40 ccm, θmin shows gradual decrease from 83 and then remains almost constant at 69°. However, when oxygen flow rate increases further from 40 ccm to 110 ccm, the hydrophilization effect on the PDMS surface is saturated and resulted in almost constant contact angles. Similarly, with increasing oxygen flow rate from 20 to 60 ccm, line width, W increases and reaches constant value when flow rate is further increased from 80 to 110 ccm. Fig. 8(b) shows the results on the AFM images of PDMS surfaces treated by oxygen radicals using SRMJ. Both Ra and Rrms values show gradual increase in roughness values with the increase of oxygen flow rates. In Fig. 8(c), cell density shows monotonic increase as oxygen flow rates increase from 20 to 110 ccm. Since cell density increases
Fig. 3. Relationship between minimal contact angle, treated line width and scanning speeds of radical jet used. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
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Fig. 4. 3D profiles of treated PDMS surface morphology of different scan speeds taken by AFM. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
exponentially through the proliferation, high density of cell adhesion was observed on the treated surface for the long cultivation period of DIV 3. 4. Discussion It is commonly known that biological cells adhere easily to the surface with water contact angle of 60–70°. Thus, we conducted an additional experiment which adopted lowpressure oxygen-based ICP plasma treatment instead of using SRMJ, since it can achieve wide-range wettability control of polymer surface compared with radical treatment. As shown in Fig. 9, the relationship between HeLa cell attachment and wettability on PDMS surface is in good accordance with the reported values, namely, good cell adhesion occurs at approximately 60°. PDMS surfaces treated by low-pressure ICP and radical microjet may not show identical phenomena, for example, oxide layer thickness or surface roughness might be different. We still, however, believe the relationship between cell adhesion and hydrophilization effect for SRMJ is achieved in a similar way qualitatively. On the other hand, cell-adhesive protein, so-called cell attachment factor, such as fibronectin and collagen has a characteristic to combine with a receptor on the cell membrane. Thus, cell adhesion control by surface treatment is actually the result of controlling protein adhesion which works as extracellular matrix. The contact angle of approxi-
mately 60–70° is reported to be preferable for protein adsorption on solid surfaces. Subsequently, we further discuss about the chemical and physical change on PDMS surface by SMRJ treatment. It has been reported that hydrophilic change of PDMS surface by plasma- or UV-enhanced oxidation occurs as a result of successive substitution of methyl (\CH3) group on PDMS surface by −OH group [22]. As similar surface reactions are likely to occur in our samples, XPS analysis of Si 2p spectrum
Fig. 5. Relationship between surface roughness of treated PDMS and different scanning speeds used in SRMJ. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
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2p3/2 levels due to the spin–orbit interaction, for spectra analysis, they were decomposed into the Si2p1/2 and Si2p3/2 spectra. Si2p3/2 spectra from PDMS films could be decomposed into three chemically shifted peaks, resulted from structures denoted as D[(CH3)2Si O2/2], T[(CH3)SiO3/2], and Q[SiO4/2] siloxy units. The binding-energy shifts for D, T, and Q were determined by spectra fitting of a mixed Gaussian–Lorentzian product function with the equally retained FWHM for each peak, and the binding-energy values obtained were 102.3, 102.9, and 103.8 eV, respectively. Also as shown in Fig. 10, the percentage composition of T-structure also shows an increase from 4.2% for the untreated sample to 13.8% after treatment by
Fig. 6. Cell proliferation profile of different scanning speeds for DIV 2 of cell culture process. Surface treatment conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
was conducted to investigate change in chemical state of Si near the top surface. Chemical states of Si on PDMS surface, Si2p photoelectron spectra were further analyzed in detail as shown in Fig. 10. Since the Si2p XPS spectra are composed of 2p1/2 and
Fig. 7. Average cell density over total proliferated area for DIV 1 and DIV 2 of cell culture process. Surface treatment conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
Fig. 8. (a) Relationship between minimal contact angle, θmin and treated line width, W for different oxygen flow rates; (b) root mean square (Rrms) value and average roughness (Ra) value taken by AFM for different oxygen flow rates measured; (c) average cell density over total proliferated area of HeLa cells cultured for 3 consecutive days (DIV 1, DIV 2, DIV 3) for each oxygen flow rate measured. Surface treatment conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
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Fig. 9. Attachment characteristics of HeLa cells on PDMS films treated in oxygen inductively coupled plasmas operated at 400 W and 6.5 × 10− 3 Pa. Fig. 11. A microphotograph of cells cultivated in line on treated PDMS surfaces using scan speed of 0.5 mm/s and oxygen flow rate of 90 ccm.
SRMJ. Thus, we confirm that surface oxidation had occurred on the PDMS surface after SRMJ treatment. Moreover, changes in surface morphology by SRMJ could also be considered as another factor which may affect cell attachment onto the PDMS film. Our results of AFM observation show that cells attach more easily when surface roughness values are higher. Although the result might be ascribed to stronger cell–surface interactions when surface is rough, further study is needed to clarify the cause-and-effect relationship between surface roughness and cell adhesion in nano-meter scale. Finally, based on our experimental results, we could demonstrate linear patterned cell cultivation of several hundred micro-meter width as shown in Fig. 11. Optimized surface treatment conditions of high oxygen flow rate of 90 ccm and
low scan speed of 0.5 mm/s were adopted. Such controlled cell cultivation will be useful for research and development of biomicrosystems in the near future. 5. Summary A scanning radical microjet has been developed and hydrophilic patterning and enhanced cell attachment on PDMS films have been successfully achieved. Effects of experimental parameters such as scanning speed and oxygen flow rate on hydrophilization were comprehensively investigated. By controlling these operation parameters, surface characteristics of PDMS could be modified accordingly, thus changing it from hydrophobic to hydrophilic nature; approximately 70° of water contact angle. HeLa cells could adhere much more easily on the treated PDMS surface at higher flow rate and lower scanning speed (longer treatment time on PDMS surface) due to higher \OH concentration which resulted in increasing hydrophilicity. In addition, cells adhered more easily to surfaces which indicated higher roughness values. Changes in surface morphology might also be another factor that affected cell–surface interactions, though the cause-and-effect relationship is still not clear. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References
Fig. 10. Si2p3/2 spectra of the PDMS samples for untreated (top) and oxygen plasma-treated (bottom) samples. XPS spectra were obtained using Mg Kα Xray source and the take-off angle of 35°.
[1] P. Lenz, C.M. Ajo-Franklin, S.G. Boxer, Langmuir 20 (2004) 11092. [2] J.C. McDonald, G.M. Whitesides, Acc. Chem. Res. 35 (2002) 491. [3] W. Hellmich, J. Regtmeier, T.T. Duong, R. Ros, D. Anselmetti, A. Ros, Langmuir 21 (2005) 7551. [4] S.K. Sia, G.M. Whitesides, Electrophoresis 24 (2003) 3563. [5] D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Anal. Chem. 70 (1998) 4974.
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[6] B.A. Grzybowski, R. Haag, N. Bowden, G.M. Whitesides, Anal. Chem. 70 (1998) 4645. [7] Y. Liu, J.C. Fanguy, J.M. Bledsoe, C.S. Henry, Anal. Chem. 72 (2000) 5939. [8] S. Hu, X. Ren, M. Bachman, C.E. Sims, G.P. Li, N. Allbritton, Anal. Chem. 74 (2002) 4117. [9] Q. He, Z. Liu, P. Xiao, R. Liang, N. He, Z. Lu, Langmuir 19 (2003) 6982. [10] E.A. Roth, T. Xu, M. Das, C. Gregory, J.J. Hickman, T. Boland, Biomaterials 25 (2004) 3707. [11] G. Csucs, R. Michel, J.W. Lussi, M. Textor, G. Danuser, Biomaterials 24 (2003) 1713. [12] M.R. Dusseiller, D. Schlaepfer, M. Koch, R. Kroschewski, M. Textor, Biomaterials 26 (2005) 5917. [13] T. Xu, J. Jin, C. Gregory, J.J. Hickman, T. Boland, Biomaterials 26 (2005) 93.
[14] R.G. Flemming, C.J. Murphy, G.A. Abrams, S.L. Goodman, P.F. Nealey, Biomaterials 20 (1999) 573. [15] X.Q. Brown, K. Ookawa, J.Y. Wong, Biomaterials 26 (2005) 3123. [16] J.J. Cunningham, J.J. Linderman, D.J. Mooney, BioTechniques 32 (2002) 876. [17] S.W. Rhee, A.M. Taylor, C.H. Tu, D.H. Cribbs, C.W. Cotman, N.L. Jeon, Lab. Chip. 5 (2005) 102. [18] H. Kawahara, M. Nakamura, J. Chem. Rev. 21 (1978) 13. [19] H. Matsumoto, J. Chem. Rev. 21 (1978) 35. [20] T. Ideno, T. Ichiki, J. Photopolym. Sci. Tech. 17 (2004) 173. [21] H.M.L. Tan, T. Ideno, T. Ichiki, J. Photopolym. Sci. Tech. 18 (2005) 237. [22] V.-M. Graubner, R. Jordan, O. Nuyken, Macromolecules 37 (2004) 5936.
Surface modification of poly(dimethylsiloxane) for controlling biological cells’ adhesion using a scanning radical microjet Helen M.L. Tan a , H. Fukuda b , T. Akagi c,d , T. Ichiki c,d,⁎ a
Department of Materials Engineering, School of Engineering, The University of Tokyo, 2–11–16 Yayoi, Bunkyo-ku, 113–8656, Japan b Department of Electrical and Electronics Engineering, Toyo University, 2100 Kujirai, Kawagoe, 350–8585, Japan c Department of Bioengineering, School of Engineering, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, 113–8656, Japan d Center for NanoBio Integration, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, 113–8656, Japan Available online 30 November 2006
Abstract A scanning radical microjet (SRMJ) equipment using oxygen microplasma has been developed and successfully applied for controlling biological cells’ attachment on biocompatible polymer material, poly(dimethylsiloxane) (PDMS). The radical microjet has advantages in localized and high-rate surface treatment. Moreover, maskless hydrophilic patterning using SRMJ has been demonstrated to be applicable to patterned cell cultivation which is useful in emerging biotechnological field such as tissue engineering and cell-based biosensors. Since control of PDMS surface properties is an indispensable prerequisite for cells’ attachment, effects of oxygen flow rates and treatment time on localized hydrophilic patterning of PDMS surfaces were first investigated for controlling HeLa cells’ (human epitheloid carcinoma cell line) attachment. Relationships between surface conditions of treated PDMS films and attached cell density are also discussed based on surface properties analyzed using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). © 2006 Elsevier B.V. All rights reserved. Keywords: Scanning radical microjet equipment; Poly(dimethylsiloxane) (PDMS); Surface modifications; Cell adhesion
1. Introduction In recent years, rapid growth has been seen in research and development of biomicrosystems such as microreactors and bioanalytical devices, which are driven by aggressive fusion of nano/microfabrication technology and modern biotechnology. In such a field, silicone elastomer, poly(dimethylsiloxane) (PDMS) has been widely used as a fabrication material due to ease in micropattern fabrication at low cost, resistance to various chemicals and pH environments, excellent transparency in the UV–vis range [1,2]. Its remarkable gas permeability and biocompatibility have in particular attracted the integration of biological applications into PDMS devices [3]. A recent review by Sia and Whitesides [4] describes microfluidic systems in PDMS for biological studies. It also reports on bioanalytical applications comprising of immunoassays, separation of biomolecules, as well as sorting and manipulation of cells. ⁎ Corresponding author. Tel./fax: +81 3 5841 1180. E-mail address: [email protected] (T. Ichiki). 0040-6090/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2006.10.026
Despite these advantages, the extreme hydrophobicity of PDMS often faces certain difficulties in most biological applications. Therefore, various solutions have been proposed for tailoring PDMS surface properties, e.g., oxygen plasma treatment [5], silanization [6], adsorbed coatings [7], UV curing [8], and plasma grafting [9]. Among these, surface modification with oxygen plasma is mostly used in rapid prototyping of microfluidic devices. On the other hand, in recent years, cell patterning on a chip has been intensively studied for regulating configuration of cultivated cells. Conventional methods such as photolithography, contact printing and ink-jet printing have provided feasible patterning of various kinds of biomolecules and cells [10–14]. For more precise control of cells’ behavior, however, physical effect, especially topographic or mechanical effects should be involved for cell patterning. Recent reports demonstrated that application of a mechanical factor to cultured cells is important for understanding cell growth and proliferation [15]. Some researchers attempted it using elastic materials such as PDMS. Although silicone elastomer substrate is of great interest as an
H.M.L. Tan et al. / Thin Solid Films 515 (2007) 5172–5178
alternative to glass- or plastic-based substrates, problems of insufficient cell attachment and growth remain unsettled [16] and are also to be solved by adopting proper surface modification for controlling adsorption of proteins which dominate cell attachment on extracellular matrix. In a recent paper by Rhee et al. [17], they reported molecular patterning on a PDMS surface using dry process. Although their method was successful in generating patterned cell culture inside microfluidic devices, the patterning process required time consuming steps in preparing stencil masks specially fabricated with PDMS. In this paper, we used a scanning radical microjet (SRMJ) instead of direct plasma exposure onto PDMS surface in order to avoid damage to the PDMS surface. Also, we carried out maskless patterning for treatment on the PDMS surface. Effects of oxygen flow rates and scanning speeds on localized hydrophilic patterning on the PDMS surface were first investigated for controlling HeLa cells’ attachment using an SRMJ. Since cell attachment to the material is closely related to wettability of its surface [18,19], contact angle measurements on PDMS were conducted and effects of wettability of treated PDMS were investigated biologically for better understanding of cell–surface interactions. By using SMRJ, we have achieved patterned HeLa cells’ cultivation in a single line according to the radical-treated line. 2. Experimental 2.1. Apparatus Fig. 1 shows a schematic of the SMRJ apparatus employed in this study. The compact plasma source is comprised of a 250-μmthick copper antenna deposited on an alumina plate and a silica discharge tube with a 0.9 mm inner diameter. A 144-MHz VHF power supplied to the plasma source was kept constant at 50 W [20,21]. A fine argon plasma jet was emitted through the pinched end of the discharge tube which has an inner diameter of 0.1 mm. Another small cylindrical tube was connected to the end of the discharge tube for the introduction and subsequent dissociation of oxygen. The oxygen radicals were emitted out through a 100 μm
5173
hole at the end of the cylindrical tube. The flow rate of argon used in this study was kept constant at 180 ccm. In addition, another low-pressure ICP plasma apparatus was also adopted for investigating the relationship between wettability and cell adhesion. Low-pressure ICP plasma was used since it could achieve wide-range wettability control of polymer surface as compared to radical treatment. Oxygen plasma was produced in a stainless steel chamber 260 mm in diameter by supplying 13.5-MHz rf power to a single-turn antenna set on a quartz window at one end. A sample was placed on the stage set in the downstream region 36 cm away from the antenna. The plasma was generated at a total pressure of 6.5 × 10− 3 Pa and rf power of 400 W. 2.2. Sample preparation and surface characterizations SYLGARD 184 (Dow Corning), base and curing agent, was used to prepare thin PDMS films on glass substrates using a spin-coater and cured at 100 °C for 2 min. Line scanning in x- or y-direction was then performed for hydrophilization using SRMJ equipment and water contact angles distributions of microscopic-size water droplets obtained from condensed steam of boiling water were measured across the scanned line using a self-made contact angle measurement system. This system consists of a humidity chamber and microscope with a long focal length. With such system, rapid evaporation of small droplets of approximately 50 μm size can be prevented and contact angle can be measured more precisely. Working parameters such as oxygen flow rates and scanning speed of SRMJ were first investigated to find out their effects on hydrophilic patterning of PDMS surface and subsequent cell patterning. Also, surface properties such as surface roughness and chemical states were also analyzed using atomic force microscope (AFM: Shimadzu SPM-9500) and X-ray photoelectron spectroscope (XPS: Shimadzu-Kratos Axis His), respectively. In the latter analysis, monochromatic Mg Kα was used as an X-ray source. 2.3. Cell cultivation on surface modified PDMS films As a feasibility study, HeLa cells (human epitheloid carcinoma cell line) were cultured on treated PDMS films in Dulbecco's modified Eagle's medium supplemented with 10% fatal bovine serum, L-glutamine and penicillin–streptomycin, and incubated for 72 h (3 days) at 37 °C in 5% CO2 in air atmosphere. Initial area density of seeded cells was constant at 3 × 103/cm2 in all the experiments. Cell density on treated PDMS surfaces was counted using an optical microscope. 3. Results 3.1. Effects of scanning speed
Fig. 1. A schematic diagram of the scanning radical microjet (SRMJ) employed in this study.
3.1.1. Wettability of treated PDMS Effect of scanning speed on hydrophilic patterning was first investigated. Fig. 2 shows water contact angles distribution measured across the scanned line. Subsequently from these profiles,
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3.1.3. Cell adhesion analysis Fig. 6 illustrates the cell proliferation profiles for four different scan speeds used in treating PDMS surfaces for DIV (day in vitro) 2 of the cell culture process. From the profiles, scan speed of 0.5 mm/s shows highest cell density of approximately 420 cells/mm2 among other scan speeds. Here, in order to highlight the cell density profile across the treated line, cell density was measured at 0.1 mm interval from the center axis of the treated line. Namely, total number of cells on the segmented area of 0.1 mm width was divided by this area. From Fig. 7, it can be observed that as scanning speed increases, cell densities for both DIV 1 and 2 decrease. In this case, average cell density is defined as the cell density on the treated line for each individual day. This was calculated by dividing the total number of cells on the treated line over the whole area of the treated line. The results from Fig. 7 imply that lower scan speed promotes better cell adhesion to the oxygen radicaltreated surfaces. 3.2. Effects of oxygen flow rates
Fig. 2. Scanned line width profiles plotted from water contact angles measurements for different scanning speeds. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
minimal contact angle, θmin and treated line width, W are plotted in Fig. 3. W is defined here as the width at 10% of the maximum valley depth of the water contact angles’ profile. Contact angle for non-treated PDMS surface was approximately 112°. It is observed that as scanning speed increases from 0.5 to 6 mm/s, minimal contact angle increases from 72 to 79° while the treated line width decreases from 2.2 to 1.5 mm. This phenomenon could be ascribed to the shorter treatment time by the oxygen radicals on the PDMS surface as scanning speed increases, hence resulting in decreasing hydrophilicity and thus narrower line width and higher water contact angles. 3.1.2. Surface morphology of treated PDMS Since oxygen radicals are likely to etch polymer surface, surface morphology of treated PDMS was analyzed using AFM. Fig. 4 shows surface morphologies for 3 different scan speeds of 0.5, 3 and 6 mm/s. The surface roughness values were then plotted as shown in Fig. 5. Both root mean square (Rrms) and average roughness (Ra) values show decreasing phenomena with the increase of scanning speed. Such tendency can be ascribed to decreasing treatment time as scanning speed is increased. Ra and Rrms of non-treated PDMS surface were measured and found to be 2.2 nm and 2.9 nm, respectively.
Similarly to Section 3.1, effects of oxygen flow rates on wettability and surface morphology of treated PDMS and cell adhesion properties were also investigated. Fig. 8(a) shows the effects of oxygen flow rates on water contact angles of PDMS surfaces treated by SRMJ. As oxygen flow rate increases from 20 to 40 ccm, θmin shows gradual decrease from 83 and then remains almost constant at 69°. However, when oxygen flow rate increases further from 40 ccm to 110 ccm, the hydrophilization effect on the PDMS surface is saturated and resulted in almost constant contact angles. Similarly, with increasing oxygen flow rate from 20 to 60 ccm, line width, W increases and reaches constant value when flow rate is further increased from 80 to 110 ccm. Fig. 8(b) shows the results on the AFM images of PDMS surfaces treated by oxygen radicals using SRMJ. Both Ra and Rrms values show gradual increase in roughness values with the increase of oxygen flow rates. In Fig. 8(c), cell density shows monotonic increase as oxygen flow rates increase from 20 to 110 ccm. Since cell density increases
Fig. 3. Relationship between minimal contact angle, treated line width and scanning speeds of radical jet used. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
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Fig. 4. 3D profiles of treated PDMS surface morphology of different scan speeds taken by AFM. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
exponentially through the proliferation, high density of cell adhesion was observed on the treated surface for the long cultivation period of DIV 3. 4. Discussion It is commonly known that biological cells adhere easily to the surface with water contact angle of 60–70°. Thus, we conducted an additional experiment which adopted lowpressure oxygen-based ICP plasma treatment instead of using SRMJ, since it can achieve wide-range wettability control of polymer surface compared with radical treatment. As shown in Fig. 9, the relationship between HeLa cell attachment and wettability on PDMS surface is in good accordance with the reported values, namely, good cell adhesion occurs at approximately 60°. PDMS surfaces treated by low-pressure ICP and radical microjet may not show identical phenomena, for example, oxide layer thickness or surface roughness might be different. We still, however, believe the relationship between cell adhesion and hydrophilization effect for SRMJ is achieved in a similar way qualitatively. On the other hand, cell-adhesive protein, so-called cell attachment factor, such as fibronectin and collagen has a characteristic to combine with a receptor on the cell membrane. Thus, cell adhesion control by surface treatment is actually the result of controlling protein adhesion which works as extracellular matrix. The contact angle of approxi-
mately 60–70° is reported to be preferable for protein adsorption on solid surfaces. Subsequently, we further discuss about the chemical and physical change on PDMS surface by SMRJ treatment. It has been reported that hydrophilic change of PDMS surface by plasma- or UV-enhanced oxidation occurs as a result of successive substitution of methyl (\CH3) group on PDMS surface by −OH group [22]. As similar surface reactions are likely to occur in our samples, XPS analysis of Si 2p spectrum
Fig. 5. Relationship between surface roughness of treated PDMS and different scanning speeds used in SRMJ. Experimental conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
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2p3/2 levels due to the spin–orbit interaction, for spectra analysis, they were decomposed into the Si2p1/2 and Si2p3/2 spectra. Si2p3/2 spectra from PDMS films could be decomposed into three chemically shifted peaks, resulted from structures denoted as D[(CH3)2Si O2/2], T[(CH3)SiO3/2], and Q[SiO4/2] siloxy units. The binding-energy shifts for D, T, and Q were determined by spectra fitting of a mixed Gaussian–Lorentzian product function with the equally retained FWHM for each peak, and the binding-energy values obtained were 102.3, 102.9, and 103.8 eV, respectively. Also as shown in Fig. 10, the percentage composition of T-structure also shows an increase from 4.2% for the untreated sample to 13.8% after treatment by
Fig. 6. Cell proliferation profile of different scanning speeds for DIV 2 of cell culture process. Surface treatment conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
was conducted to investigate change in chemical state of Si near the top surface. Chemical states of Si on PDMS surface, Si2p photoelectron spectra were further analyzed in detail as shown in Fig. 10. Since the Si2p XPS spectra are composed of 2p1/2 and
Fig. 7. Average cell density over total proliferated area for DIV 1 and DIV 2 of cell culture process. Surface treatment conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
Fig. 8. (a) Relationship between minimal contact angle, θmin and treated line width, W for different oxygen flow rates; (b) root mean square (Rrms) value and average roughness (Ra) value taken by AFM for different oxygen flow rates measured; (c) average cell density over total proliferated area of HeLa cells cultured for 3 consecutive days (DIV 1, DIV 2, DIV 3) for each oxygen flow rate measured. Surface treatment conditions used were oxygen flow rate: 110 ccm; Ar flow rate: 180 ccm.
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Fig. 9. Attachment characteristics of HeLa cells on PDMS films treated in oxygen inductively coupled plasmas operated at 400 W and 6.5 × 10− 3 Pa. Fig. 11. A microphotograph of cells cultivated in line on treated PDMS surfaces using scan speed of 0.5 mm/s and oxygen flow rate of 90 ccm.
SRMJ. Thus, we confirm that surface oxidation had occurred on the PDMS surface after SRMJ treatment. Moreover, changes in surface morphology by SRMJ could also be considered as another factor which may affect cell attachment onto the PDMS film. Our results of AFM observation show that cells attach more easily when surface roughness values are higher. Although the result might be ascribed to stronger cell–surface interactions when surface is rough, further study is needed to clarify the cause-and-effect relationship between surface roughness and cell adhesion in nano-meter scale. Finally, based on our experimental results, we could demonstrate linear patterned cell cultivation of several hundred micro-meter width as shown in Fig. 11. Optimized surface treatment conditions of high oxygen flow rate of 90 ccm and
low scan speed of 0.5 mm/s were adopted. Such controlled cell cultivation will be useful for research and development of biomicrosystems in the near future. 5. Summary A scanning radical microjet has been developed and hydrophilic patterning and enhanced cell attachment on PDMS films have been successfully achieved. Effects of experimental parameters such as scanning speed and oxygen flow rate on hydrophilization were comprehensively investigated. By controlling these operation parameters, surface characteristics of PDMS could be modified accordingly, thus changing it from hydrophobic to hydrophilic nature; approximately 70° of water contact angle. HeLa cells could adhere much more easily on the treated PDMS surface at higher flow rate and lower scanning speed (longer treatment time on PDMS surface) due to higher \OH concentration which resulted in increasing hydrophilicity. In addition, cells adhered more easily to surfaces which indicated higher roughness values. Changes in surface morphology might also be another factor that affected cell–surface interactions, though the cause-and-effect relationship is still not clear. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References
Fig. 10. Si2p3/2 spectra of the PDMS samples for untreated (top) and oxygen plasma-treated (bottom) samples. XPS spectra were obtained using Mg Kα Xray source and the take-off angle of 35°.
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