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Laser-assisted modification of polystyrene surfaces for cell culture applications
Applied Surface Science 253 (2007) 9177–9184 www.elsevier.com/locate/apsusc
Laser-assisted modification of polystyrene surfaces for cell culture applications Wilhelm Pfleging a,*, Michael Bruns b, Alexander Welle c, Sandra Wilson d,e a
Institute for Materials Research I, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany Institute for Materials Research III, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany c Institute for Biological Surfaces, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany d Institute of Microstructure Technology, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany e School of Applied Sciences, Cranfield University, Cranfield, Beds. MK430AL, UK b
Received 25 April 2007; received in revised form 21 May 2007; accepted 21 May 2007 Available online 31 May 2007
Abstract Laser-assisted patterning and modification of polystyrene (PS) was investigated with respect to applications in micro-fluidics and cell culture. For this purpose the wettability, the adsorption of proteins and the adhesion of animal cells were investigated as function of laser- and processing parameters. The change of surface chemistry was characterized by X-ray photoelectron spectroscopy. The local formation of chemical structures suitable for improved cell adhesion was realized on PS surfaces by UV laser irradiation. Above and below the laser ablation threshold two different mechanisms affecting cell adhesion were detected. In the first case the debris deposited on and along laser irradiated areas was responsible for improved cell adhesion, while in the second case a photolytic activation of the polymer surface including a subsequent oxidization in oxygen or ambient air is leading to a highly localized alteration of protein adsorption from cell culture media and finally to increased cell adhesion. Laser modifications of PS using suitable exposure doses and an appropriate choice of the processing gas (helium or oxygen) enabled a highly localized control of wetting. The dynamic advancing contact angle could be adjusted between 28 and 1508. The hydrophilic and hydrophobic behaviour are caused by chemical and topographical surface changes. # 2007 Elsevier B.V. All rights reserved. Keywords: Polystyrene; Laser; Modification; Ablation; Wetting; Cell adhesion
1. Introduction While silicon based micro-devices such as sensors or actuators are well established commercial products in microsystem technology (MST), non-silicon, in particular polymer based, micro-fluidic systems have very recently been introduced to the market [1]. For this purpose techniques of rapid prototyping [2], rapid tooling and rapid manufacturing are increasingly used in MST. Large volume industrial manufacturing can be provided by replication of a micromachined master tool [3]. Typical replication methods are hot embossing [4] and injection molding [5]. They provide low-cost mass production of microstructured components with large aspect
* Corresponding author. Tel.: +49 7247 822889; fax: +49 7247 827288. E-mail address: [email protected] (W. Pfleging). 0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.05.047
ratios, structural details in the sub-micron range and a precision better than 2 mm in the final polymer product [6,7]. In MST UV-laser-assisted processes are of particular interest for applications in microfluidics, bio-analytics, bioreactors and micro-optics [8–12]. The current state of the art of UV-laser micro-processing of polymer materials with respect to laser ablation, micro-patterning and Laser-LIGA has been described elsewhere [13]. For the packaging of micro-structured polymers, laser transmission welding was successfully developed [14], and in current research even for channel structures with a width of 20 mm. UV-photon-induced surface modification of polymers for a functionalization of polymer-based micro-devices is a relatively new research field. For this purpose, laser radiation sources or UV-lamp systems may be applied [15–18]. The main advantage of laser-based technology is its high process flexibility. Three-dimensional structures may be modified and a variety of processing conditions (e.g. processing gases, liquids) can be applied. Excimer laser
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processing enables high local resolution via direct writing or direct optical imaging of complex structures or motorized masks. The process is in general initiated by direct bond breaking (e.g. separation of side chains or homolytic fissions) which leads to the formation of new bonds or radicals. As a consequence of this the formation or grafting of functional groups, such as amino-groups or carboxyl-groups is possible, which in turn leads, e.g. to a change of biocompatibility. This type of functionalization was studied in detail for polystyrene (PS) with respect to wettability and adhesion of animal cells.
(OCA 15 plus, software SCA20) from Dataphysics Instruments GmbH (Filderstadt, Germany). Each surface modification was repeated five times and measurements were averaged. X-ray photoelectron spectroscopy (XPS) measurements were carried out on an ESCALAB 5 spectrometer (Vacuum Generators) using non-monochromatized MgKa radiation at a pressure of <109 mbar. The binding energy scale was referenced to 285.0 eV for the main C1s (C–H bond) feature. The photoelectrons were detected at a take-off angle of 408 or 608 with respect to the normal of the sample surface. That means that the information depth of the measurements is equal or lower than 5 nm.
2. Experimental set-up 3. Results and discussion 2.1. Laser modification 3.1. Laser processing parameters Laser-induced modifications based on excimer laser radiation were performed with the following different laser micromachining systems: (1) Exitech PS2000 operates with a Lambda LPX 210i as radiation source at 193 nm (pulse length 20 ns); (2) Promaster (Optec s.a.) operates with an ATLEX-500-SI at 248 nm (pulse length 4–6 ns). It is expected that short laser pulses (ns range) reduce significantly thermal contributions to a laser process. A high beam homogeneity or ‘‘flat top’’ profile with intensity fluctuation better than 5% is necessary to successfully provide laser-assisted modification in polymers. Therefore, for the high power excimer laser radiation multi-lens arrays were used in order to homogenize the beam and to meet the process requirements. On the other hand, short pulse excimer generates a raw ‘‘flat-top’’ beam directly applicable without homogenizing devices for various micro-processing applications [13]. 2.2. Cell culture experiments Standard culture of L929 cells was performed as described in detail elsewhere [19]. L929 murine fibroblast cell culture medium was minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate, 0.1 mM nonessential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin and 10 vol.% horse serum (ATCC 30-2040). PC12-GFP cells were cultivated in RPMI 1640 media supplemented with 2 mM L-glutamine, 1.5 mg/ml sodiumbicarbonate, 4.5 mg/ml glucose, 10 mM HEPES, 1% non-essential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin, 10 vol.% horse serum (PAA, CatNo. B15-023), and 5 vol.% fetal calf serum (PAA, CatNo. A15-649). In both cases, prior to cell inoculation and during cell culture on modified polymeric substrates cell culture medium supplemented with 1 mg/ml Pluronic F-68 [20] was used. Light microscopy was performed during cell culture in phase contrast mode, after fixation and staining with crystal violet using bright-field illumination.
UV-laser assisted ablation and modification processes are mainly influenced by the laser fluence e. In principle three different process regimes with respect to the laser fluences are of interest: Firstly, below the ablation threshold et, secondly in the range of et and thirdly, significantly above et. Above the ablation threshold et three-dimensional shapes with a very small surface roughness of Ra = 50 nm can be realized [3,13]. In the range of et the surface roughness significantly increases and cone formation will be observed. Below the ablation threshold in general no significant change in topography can be determined. However, for a large laser pulse number ‘‘ablation’’ or formation of nanopores caused by polymer degradation can occur below the threshold et [13]. For PS the ablation rate R as function of laser fluence e was determined in order to locate the possible process regimes for modification and ablation (Fig. 1). For small laser fluences (<1 J/cm2) a single-photon absorption process describes the excimer laser ablation process well. Ablation rate increases with the logarithm of the laser fluence which can be depicted with Beer’s absorption law [13,21]: 1 e R ¼ ln ; (1) a et
2.3. Chemical analysis Contact angle measurements of modified and untreated polymer surfaces were used to determine the change of wettability with respect to de-ionized water. For this purpose the advancing contact angle was determined using a goniometer
Fig. 1. Ablation rate for polystyrene (PS) as function of laser fluences (ArFexcimer, wavelength 193 nm, pulse width 20 ns, laser repetition rate 10 Hz, number of laser pulses N = 100).
W. Pfleging et al. / Applied Surface Science 253 (2007) 9177–9184 Table 1 Ablation threshold eth and effective absorption coefficient a of PS for different excimer laser radiation wavelengths (248 nm, 193 nm) et (193 nm) PS
75.5 ml cm 9 ml cm2
2
a (193 nm) 1
16.7 mm 43 mm1
et (248 nm) 200 mJ/cm
2
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Table 2 Possible change of dynamic advancing angle (DQ) of PS for different laser fluences, laser wavelengths and processing gases (laser pulse number 400)
a (248 nm) 0.5 mm1
where et denotes the ablation threshold and a is called ‘‘effective absorption coefficient’’. It is obvious that the ablation rate shows two regions with linear laser absorption (Fig. 1). For laser fluences above 100 mJ/cm2 the theoretical ablation threshold et is equal to 75.5 mJ/cm2 corresponding to an effective absorption coefficient a of about 16.7 mm1 (Table 1). For a laser wavelength of 193 nm the effective absorption index a is significantly higher than 1 mm1, which is in opposite to the a values for 248 nm. That means that for 193 nm the laser energy is deposited within 60 nm, while for 248 nm the absorption length is of about 2 mm. This indicates that for laser ablation with 193 nm above the threshold et a pyrolytic process with good surface quality can be obtained, while for 248 nm a thermally driven process with debris and melt formation will dominate. The following discussion refers to the ArF-excimer laser wavelength (193 nm). A value of 80 mJ/cm2 for the ablation threshold of PS was measured elsewhere [22]. For laser fluences smaller than 100 mJ/cm2 a second ablation threshold of (9 1.8) mJ/ cm2 with an effective absorption coefficient of 43 mm1 was determined. During patterning of PS two mechanisms seem to be important. For high laser fluences the ablation of PS corresponds very well with the classical behaviour of polymer ablation and the ablation threshold given in the literature. By contrast, for very low laser fluences (<9 mJ/cm2) a reaction layer (e.g. with carboxyl groups) is build up (more details in Section 3.3). These ‘‘oxide layers’’ will be partially removed and rebuild for laser fluences above 9 mJ/cm2. That means that a laser dry etching process of the polymer surface above the fluences threshold of 9 mJ/cm2 is observed which dominates the ablation mechanism for laser fluences up to 100 mJ/cm2. 3.2. Control of wettability on polystyrene surfaces For the untreated polymer surfaces the dynamic advancing contact angles Q0 was measured as reference value. For PS Q0 is equal to 82.0 3.38. In the first step, large area modification in air using a conventional UV-Lamp (UVAPRINT, Dr. Ho¨nle A.G., Gra¨felfing, Germany) was performed to get a reference value. The UV-lamp radiates within a wavelength spectrum of l = 220– 420 nm and delivered an intensity of I = 2.4 mW/cm2. The dynamic advancing contact angle Q decreases with irradiation time and reached a saturation value after 120 min. The dynamic advancing contact angle Q reaches a value of 35.18 after 30 min and 23.18 after 120 min. Several laser modifications at 248 nm and 193 nm were performed below the ablation threshold, nearby the ablation threshold and above the ablation threshold and the wetting behaviour was observed as function of laser pulse number. Further, the influence of different processing gases, such
as helium, oxygen and air was investigated. Table 2 summarizes these investigations which are now described in detail. For the wavelength of 248 nm only a small change in wettability can be initiated and the slight increase of 10–208 of the contact angle is mainly caused by a change in surface topography. It was shown that with ArF-excimer laser radiation (193 nm) the wettability can be very well controlled via laser fluence, processing gas and laser pulse number. An enhancement of hydrophobic as well as hydrophilic properties is possible at 60 mJ/cm2 with oxygen as processing gas. After 40 laser pulses Q is reduced from 828 down to 138 and after 400 (200) pulses Q increases up to 1298 (1018). For laser fluences of 90 mJ/cm2, which are slightly above the ablation threshold, the hydrophilic effect is even more pronounced (Fig. 2). After 40 laser pulses, as well as after 100 and 300 laser pulses, the dynamic advancing contact angle Q decreases down to 1.78. After 400 laser pulses the topographic effect caused by an increased surface roughness dominates the chemical effect caused by an oxidization of the PS surface. Fig. 2 shows also that a change in processing gas can be used to switch between hydrophobic and hydrophilic surface properties, even after 40 laser pulses. For He as a processing gas and above 400 laser pulses a contact angle Q of 1508 is reached. Ex situ XPS measurements show, that for He and a laser fluence of 90 mJ/cm2 the surface composition of treated PS is similar to native PS carrying the natural contamination layer. We assume that the oxidization was completely suppressed and only the topographic change influenced the wettability. The topographic structure consists of columns with a diameter of 1–2 mm and a pitch distance of 2–5 mm (Fig. 2, right). For laser fluences at 5 mJ/cm2, which are below the threshold of laser dry etching of PS, no significant change of the dynamic advancing contact angle with respect to the value of the untreated PS-surface was measured. However, the dynamic receding contact angle is significantly reduced from 558 down to 188. The increase in the observed contact angle hysteresis indicates that the chemical structure at the surface is partly changed and inhomogeneous chemical groups leading to a hydrophilic behavior are formed.
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Fig. 2. Left: Dynamic advancing contact angle as function of laser pulse number for PS; right: SEM images of PS surface after laser treatment with 400 laser pulses in He- (top) and O2-atmosphere (bottom).
Ex situ XPS measurements of PS surfaces after laser treatment were performed in order to study the change of chemical binding structure and chemical composition as a function of laser fluence and processing gas. The highest amount of oxygen compounds at the surface is detected after laser treatment at 5 mJ/cm2 in He or O2 processing gas atmosphere (Fig. 3, right). The same behavior is observed for the laser exposure of PS through a quartz cover plate. This suggests that oxidization occurs also after laser treatment, when the sample is handled ex situ in air and indicates that material excitation and formed radicals are temporally stable after laser treatment. The formation of radicals can be due to breaking of phenyl rings or cracking of bonds of the main polymer chain or of side chains. XPS measurements show that in the case of He as a processing gas (Fig. 3) the line shape corresponds very well
with the line shape of the chemical group –COOH [23]. When oxygen is used as processing gas, the O1s line shape slightly changes for laser fluences of 90 mJ/cm2. The O1s line at low binding energy is an artefact and due to localized charge builtup. With He or O2 as processing gas and a laser fluence of 5 mJ/ cm2 the oxygen concentration increases up to 20 at.%. For 90 mJ/cm2, which is above the ablation threshold (75 mJ/cm2), the amount of oxygen-bonds is reduced to 3 at.% and 13 at.% for processing gas He and O2, respectively (Fig. 3). Above the ablation threshold oxidization occurs only in the presence of oxygen as processing gas. Apparently, no temporally stable material excitation is existent above the ablation threshold. The change of chemical bonding, which is observed in the O1s spectra (Fig. 3, left), and the change DQ of wettability between the use of 5 mJ/cm2 (DQ > 108) and 90 mJ/cm2 (DQ 808) are corresponding effects.
Fig. 3. O1s spectra from PS surfaces after modification in processing gas oxygen (left) or helium (right) for different laser fluences (laser pulse number 300).
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Fig. 4. Left: ArF-laser processing diagram for PS illustrates the threshold behavior for laser pulse number and laser fluence with respect to a successful L929 cell adhesion; right: optical photograph after L929 cell cultivation on laser modified PS Petri dishes (each square belongs to another laser pulse number) including optical microscope image for a closer view (laser fluence 5 mJ/cm2). Laser pulse numbers are denoted in each case.
3.3. Formation of carboxyl groups on PS for improved cell adhesion The adhesion of most animal cell lines on unmodified PS surfaces is generally poor. This is to be attributed to the strong adsorption of albumin from serum containing cell culture media. Since albumin does not present cell attractive peptide sequences the albumin covered surface is passivated with respect to cell adhesion. Due to the altered physico-chemical properties (wettability) of the UV irradiated surfaces also the competitive adsorption of plasma proteins is influenced. It was shown previously that albumin adsorption onto UV irradiated PS is hindered whereas the adsorption of cell attractive proteins is increased [19]. The adhesion of L929 cells was investigated on PS surfaces as function of processing gas, laser wavelength (193 nm, 248 nm), laser fluences and laser pulse number. For a significant change in dynamic advancing angle, as described in the preceding chapter, ArF-excimer laser fluences of larger or equal than 20 mJ/cm2 are necessary. In contrast to the change of wettability the type of processing gas (He, air or oxygen) has no detectable influence on the L929 cell adhesion. Furthermore, significant cell adhesion on PS surfaces can be obtained for laser fluences smaller or equal than 6 mJ/cm2. Here it is necessary to consider that the threshold for the dry etch process is in the range of 9 mJ/cm2. Above this threshold a subsequent removal of oxidized groups, assumably –COOH being mainly responsible for an improved L929 cell adhesion as described elsewhere [19], takes place. Not only the laser fluence has showed a threshold for cell adhesion, but also the laser pulse number. For a laser pulse number smaller than 100 pulses no cell adhesion was observed at 5 mJ/cm2. The threshold of the laser pulse number is very
sharp and was determined within an accuracy of 10 pulses. For 19 mJ/cm2 no cell adhesion was observed. The threshold of laser pulse number varies slightly with laser fluence as shown in Fig. 4. For a laser fluence of 6 mJ/cm2, 250 laser pulses are necessary for a subsequent cell adhesion. On PS surfaces the cell adhesion was also realized for pulse numbers of 1200. Table 3 summarizes the obtained results of laser-assisted surface modification of PS with respect to cell adhesion and wettability. For KrF-excimer laser radiation (wavelength 248 nm) no improved L929 cell adhesion can be established at low laser fluences. For 248 nm it was necessary to increase the laser fluences up to a critical value of about 200 mJ/cm2 (ablation threshold of PS for 248 nm, see Table 1) above which polymer ablation took place. Lee et al. have performed laser ablation with 248 nm on oxygen plasma treated PS and have observed, Table 3 Correlation between wettability and cell adhesion as function of laser fluences, laser pulse number and processing gases for the use of ArF-excimer laser Parameters for improved
Processing gas Laser fluence Laser pulse number Change of wettability Oxygen (at.%) Mechanisms
Cell adhesion
Hydrophilicity
Hydrophobicity
He, O2 2–6 mJ/cm2 100–1200
O2 90 mJ/cm2 40–300
He 90 mJ/cm2 400
0 > DQ > 108
DQ 808
DQ +708
20 Chemical
13 Topographical/ chemical
3 Topographical
Corresponding stoichiometry of oxygen obtained by XPS and specification of the dominant process mechanisms are given.
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Fig. 5. Optical microscope images of L929 cells cultured after debris formation during laser ablation of PS (laser wavelength 248 nm, laser fluence e = 2.2 J/cm2); left: cells grow beside of laser-irradiated areas (200 mm 200 mm) after a single laser-shot; right: cells grow in and outside of laser-generated grooves (grooves width 25 mm, groove-to-groove distance 200 mm).
that ablation leads to a removal of oxide layer and NG108-15 cell adhesion is avoided [24]. Zhu et al. reported for the use of frequency-quadrupled Nd:YAG laser (wavelength 266 nm) that the formation of nano-ripples or -grooves might be responsible for an improved cell adhesion [18]. However, we have demonstrated in the case of 248 nm and 193 nm that cell adhesion might be improved for laser fluences in the range and above the ablation threshold. We assume that for laser fluences above the ablation threshold two effects have to be considered: 1st chemical and topographical change caused by debris formation and 2nd ripple formation at the border area. Ripple formation might be caused by laser beam diffraction at the used mask geometry leading to small interference structures at the border of the ablated geometry. Also laser induced surface acoustic waves could induce a change of topography at nmscale. If grooves with a width of 25 mm were produced with 248 nm than cell adhesion in the grooves as well at the border area was observed (Fig. 5, right). KrF-excimer laser ablation of an area of 200 mm 200 mm led to a cell adhesion only at the border area outside of the generated structure profile (Fig. 5, left). From our investigation we can conclude that in the case of 193 nm cell adhesion above the ablation threshold can be
avoided if debris formation is suppressed. This can be used in order to combine laser patterning at high laser fluences and laser modification at low laser fluences without change of laser source and processing chamber. Modification of PS with ArF-exicmer laser radiation at low laser fluences enables a high lateral resolution control of cell adhesion. After modification of areas of 50 mm 50 mm clusters of PC-12 cells were adhered on the modified sites. If the modifications were confined to areas of 25 mm in diameter, it was possible to achieve single cell attachment (Fig. 6). The formation of a cellular network with neurites connecting single neuronal cells, which were attached on the polymer surface, was established. For this purpose tracks on PS with a line width of about 2 mm were laser-modified and the outgrowth of neurites along the exposed tracks was observed in cell culture after NGF induced cell differentiation. In Fig. 6 (right) the successful formation of those cell networks is illustrated. 3.4. Formation of amino groups on PS Carboxyl groups on PS were formed during or after laser exposure. It is assumed that radicals are formed by
Fig. 6. In situ microscope images of laser modified PS surfaces during PC-12 cell cultivation; left: A single-cell is attached to the modified area (diameter of modification 25 mm; no neurite paths were exposed); right: single cells and neurite paths in rectangular arrangement (width of modified tracks: 2 mm); laser wavelength for modification 193 nm and laser fluence 5 mJ/cm2.
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Fig. 7. Left: schematic view of irradiation of PS surface in presence of liquid 2-amino-ethanol; right: XPS spectra of N1s as function of laser pulse number (laser wavelength: 193 nm, laser fluences: 13 mJ/cm2, processing medium: 2-amino-ethanol).
laser-induced bond breaking. Oxygen was delivered from processing gas or from the ambient air. As a consequence of this, the following question was addressed: Is it possible to realize also the formation of other chemical groups at the polymer surface such as amino groups to enable other strategies of biomolecule immobilization? For this purpose other ambient conditions during laser processing would be absolutely necessary. First necessity is to reduce or to avoid the surface reaction with oxygen and second, the desired chemical group should be formed and exposed to the excited polymer surface. For this purpose the polymer was modified in presence of liquid 2-amino-ethanol as shown schematically in Fig. 7 (left). Ex situ XPS measurements after modification were established in order to verify a possible grafting of nitrogen containing compounds to the PS surface as function of laser pulse number (Fig. 7, right). A constant laser fluence of about 13 mJ/cm2 was used. The laser fluence at the interface of polymer and 2-amino-ethanol was surely smaller because of reflection losses and beam attenuation during propagation in the liquid phase. No change in topography was observed after modification. This means, that the effective laser fluence at the polymer surface was lower than the threshold value of 9 mJ/cm2. The N1s spectra show only one peak at binding energies between 399.3e V and 399.7e V. Unfortunately with XPS it is not doubtlessly possible to distinguish between primary amino group and some other nitrogen/carbon species [25]. A binding between oxygen and nitrogen is not supposed, because this should induce a shift of the N1s line to significant higher binding energies [26]. With increasing exposure dose the amount of nitrogen at the PS-surface increases (Fig. 7, right). Nevertheless, XPS measurements reveal that also carboxyl-groups were formed on the PS surface. It is assumed that the oxidization has occurred ex situ, very similar to the modification in He atmosphere.
4. Summary Laser processing at short wavelengths is an appropriate tool for a selective patterning of polymer surfaces. Here, patterning stands for chemical or topographic modification of surfaces on nanometer and micrometer scale. But patterning also encompasses ablation for the generation of two- and threedimensional shapes in polymer surfaces. It was demonstrated that these kinds of ‘‘patternings’’ could be combined with high lateral resolution, e.g. ablation and modification of polymer surfaces with respect to change of wettability and change of L929 and PC-12 cell adhesion (Table 3). The adhesion of cell clusters, single-cells as well as the formation of cell networks on polystyrene surfaces could be controlled with high accuracy. Different types of chemical groups (e.g. –COOH, –NH2) could be patterned onto polymer surfaces containing features with two or three dimensions. Besides the chemical effect also a change in topography on nm- and mm-scale can be used for a change of cell adhesion and wettability. Change of processing gas, O2 or He, can be used to induce complete wetting or to realize hydrophobic surfaces without changing the processing chamber. All these laser processes work without vacuum chambers and enable also the use of liquid media as possible reactant. Future investigations will integrate theses different aspects of modification and ablation for lab-on-chip applications and for the realization of bioreactors and polymer-based bio-medical devices, such as automated patch clamping systems. Acknowledgments We are grateful to our colleague M. Beiser for his technical assistance in SEM. We are indebted to Mrs. M. Marin for her excellent contributions to this research project. We also thank Mrs. V. Trouillet for her support in XPS and H. Besser for laser
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material processing. We gratefully acknowledge the financial support by the program NANOMIKRO of the Helmholtz association and the EU within the Sixth Framework Programme (‘‘Network of Excellence in Multi-Material Micro Manufacture (4M)’’ and ‘‘Network of Excellence in Microoptic (NEMO)’’). References [1] C.G. Khan Malek, Anal. Bioanal. Chem. 385 (2006) 1362. [2] M. Farsari, F. Claret-Tournier, S. Huang, J. Mater. Process. Technol. 107 (2000) 167. [3] W. Pfleging, T. Hanemann, M. Torge, W. Bernauer, J. Mech. Eng. Sci. 217 (2003) 53. [4] M. Heckele, W. Bacher, K.D. Mu¨ller, Microsystem Technol. 4 (1998) 122. [5] V. Piotter, N. Holstein, K. Plewa, R. Ruprecht, J. Hausselt, Microsystem Technol. 10 (2004) 537. [6] U. Wallrabe, H. Dittrich, G. Friedsam, T. Hanemann, J. Mohr, K. Mueller, V. Piotter, P. Ruther, T. Schaller, W. Zißler, SPIE 4408 (2001) 478. [7] T. Hanemann, M. Heckele, V. Piotter, Polym. News 25 (2000) 224. [8] S. Sinzinger, J. Jahns, Microoptics, Wiley-VCH, Weinheim, FRG, 1999. [9] W. Ehrfeld, V. Hessel, H. Lo¨we, Microreactors, Wiley-VCH, Weinheim, FRG, 2000. [10] E. Gottwald, S. Giselbrecht, C. Augspurger, N. Dambrowsky, R. Truckenmu¨ller, V. Piotter, T. Gietzelt, O. Wendt, W. Pfleging, A.
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Laser-assisted modification of polystyrene surfaces for cell culture applications Wilhelm Pfleging a,*, Michael Bruns b, Alexander Welle c, Sandra Wilson d,e a
Institute for Materials Research I, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany Institute for Materials Research III, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany c Institute for Biological Surfaces, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany d Institute of Microstructure Technology, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany e School of Applied Sciences, Cranfield University, Cranfield, Beds. MK430AL, UK b
Received 25 April 2007; received in revised form 21 May 2007; accepted 21 May 2007 Available online 31 May 2007
Abstract Laser-assisted patterning and modification of polystyrene (PS) was investigated with respect to applications in micro-fluidics and cell culture. For this purpose the wettability, the adsorption of proteins and the adhesion of animal cells were investigated as function of laser- and processing parameters. The change of surface chemistry was characterized by X-ray photoelectron spectroscopy. The local formation of chemical structures suitable for improved cell adhesion was realized on PS surfaces by UV laser irradiation. Above and below the laser ablation threshold two different mechanisms affecting cell adhesion were detected. In the first case the debris deposited on and along laser irradiated areas was responsible for improved cell adhesion, while in the second case a photolytic activation of the polymer surface including a subsequent oxidization in oxygen or ambient air is leading to a highly localized alteration of protein adsorption from cell culture media and finally to increased cell adhesion. Laser modifications of PS using suitable exposure doses and an appropriate choice of the processing gas (helium or oxygen) enabled a highly localized control of wetting. The dynamic advancing contact angle could be adjusted between 28 and 1508. The hydrophilic and hydrophobic behaviour are caused by chemical and topographical surface changes. # 2007 Elsevier B.V. All rights reserved. Keywords: Polystyrene; Laser; Modification; Ablation; Wetting; Cell adhesion
1. Introduction While silicon based micro-devices such as sensors or actuators are well established commercial products in microsystem technology (MST), non-silicon, in particular polymer based, micro-fluidic systems have very recently been introduced to the market [1]. For this purpose techniques of rapid prototyping [2], rapid tooling and rapid manufacturing are increasingly used in MST. Large volume industrial manufacturing can be provided by replication of a micromachined master tool [3]. Typical replication methods are hot embossing [4] and injection molding [5]. They provide low-cost mass production of microstructured components with large aspect
* Corresponding author. Tel.: +49 7247 822889; fax: +49 7247 827288. E-mail address: [email protected] (W. Pfleging). 0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.05.047
ratios, structural details in the sub-micron range and a precision better than 2 mm in the final polymer product [6,7]. In MST UV-laser-assisted processes are of particular interest for applications in microfluidics, bio-analytics, bioreactors and micro-optics [8–12]. The current state of the art of UV-laser micro-processing of polymer materials with respect to laser ablation, micro-patterning and Laser-LIGA has been described elsewhere [13]. For the packaging of micro-structured polymers, laser transmission welding was successfully developed [14], and in current research even for channel structures with a width of 20 mm. UV-photon-induced surface modification of polymers for a functionalization of polymer-based micro-devices is a relatively new research field. For this purpose, laser radiation sources or UV-lamp systems may be applied [15–18]. The main advantage of laser-based technology is its high process flexibility. Three-dimensional structures may be modified and a variety of processing conditions (e.g. processing gases, liquids) can be applied. Excimer laser
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processing enables high local resolution via direct writing or direct optical imaging of complex structures or motorized masks. The process is in general initiated by direct bond breaking (e.g. separation of side chains or homolytic fissions) which leads to the formation of new bonds or radicals. As a consequence of this the formation or grafting of functional groups, such as amino-groups or carboxyl-groups is possible, which in turn leads, e.g. to a change of biocompatibility. This type of functionalization was studied in detail for polystyrene (PS) with respect to wettability and adhesion of animal cells.
(OCA 15 plus, software SCA20) from Dataphysics Instruments GmbH (Filderstadt, Germany). Each surface modification was repeated five times and measurements were averaged. X-ray photoelectron spectroscopy (XPS) measurements were carried out on an ESCALAB 5 spectrometer (Vacuum Generators) using non-monochromatized MgKa radiation at a pressure of <109 mbar. The binding energy scale was referenced to 285.0 eV for the main C1s (C–H bond) feature. The photoelectrons were detected at a take-off angle of 408 or 608 with respect to the normal of the sample surface. That means that the information depth of the measurements is equal or lower than 5 nm.
2. Experimental set-up 3. Results and discussion 2.1. Laser modification 3.1. Laser processing parameters Laser-induced modifications based on excimer laser radiation were performed with the following different laser micromachining systems: (1) Exitech PS2000 operates with a Lambda LPX 210i as radiation source at 193 nm (pulse length 20 ns); (2) Promaster (Optec s.a.) operates with an ATLEX-500-SI at 248 nm (pulse length 4–6 ns). It is expected that short laser pulses (ns range) reduce significantly thermal contributions to a laser process. A high beam homogeneity or ‘‘flat top’’ profile with intensity fluctuation better than 5% is necessary to successfully provide laser-assisted modification in polymers. Therefore, for the high power excimer laser radiation multi-lens arrays were used in order to homogenize the beam and to meet the process requirements. On the other hand, short pulse excimer generates a raw ‘‘flat-top’’ beam directly applicable without homogenizing devices for various micro-processing applications [13]. 2.2. Cell culture experiments Standard culture of L929 cells was performed as described in detail elsewhere [19]. L929 murine fibroblast cell culture medium was minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate, 0.1 mM nonessential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin and 10 vol.% horse serum (ATCC 30-2040). PC12-GFP cells were cultivated in RPMI 1640 media supplemented with 2 mM L-glutamine, 1.5 mg/ml sodiumbicarbonate, 4.5 mg/ml glucose, 10 mM HEPES, 1% non-essential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin, 10 vol.% horse serum (PAA, CatNo. B15-023), and 5 vol.% fetal calf serum (PAA, CatNo. A15-649). In both cases, prior to cell inoculation and during cell culture on modified polymeric substrates cell culture medium supplemented with 1 mg/ml Pluronic F-68 [20] was used. Light microscopy was performed during cell culture in phase contrast mode, after fixation and staining with crystal violet using bright-field illumination.
UV-laser assisted ablation and modification processes are mainly influenced by the laser fluence e. In principle three different process regimes with respect to the laser fluences are of interest: Firstly, below the ablation threshold et, secondly in the range of et and thirdly, significantly above et. Above the ablation threshold et three-dimensional shapes with a very small surface roughness of Ra = 50 nm can be realized [3,13]. In the range of et the surface roughness significantly increases and cone formation will be observed. Below the ablation threshold in general no significant change in topography can be determined. However, for a large laser pulse number ‘‘ablation’’ or formation of nanopores caused by polymer degradation can occur below the threshold et [13]. For PS the ablation rate R as function of laser fluence e was determined in order to locate the possible process regimes for modification and ablation (Fig. 1). For small laser fluences (<1 J/cm2) a single-photon absorption process describes the excimer laser ablation process well. Ablation rate increases with the logarithm of the laser fluence which can be depicted with Beer’s absorption law [13,21]: 1 e R ¼ ln ; (1) a et
2.3. Chemical analysis Contact angle measurements of modified and untreated polymer surfaces were used to determine the change of wettability with respect to de-ionized water. For this purpose the advancing contact angle was determined using a goniometer
Fig. 1. Ablation rate for polystyrene (PS) as function of laser fluences (ArFexcimer, wavelength 193 nm, pulse width 20 ns, laser repetition rate 10 Hz, number of laser pulses N = 100).
W. Pfleging et al. / Applied Surface Science 253 (2007) 9177–9184 Table 1 Ablation threshold eth and effective absorption coefficient a of PS for different excimer laser radiation wavelengths (248 nm, 193 nm) et (193 nm) PS
75.5 ml cm 9 ml cm2
2
a (193 nm) 1
16.7 mm 43 mm1
et (248 nm) 200 mJ/cm
2
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Table 2 Possible change of dynamic advancing angle (DQ) of PS for different laser fluences, laser wavelengths and processing gases (laser pulse number 400)
a (248 nm) 0.5 mm1
where et denotes the ablation threshold and a is called ‘‘effective absorption coefficient’’. It is obvious that the ablation rate shows two regions with linear laser absorption (Fig. 1). For laser fluences above 100 mJ/cm2 the theoretical ablation threshold et is equal to 75.5 mJ/cm2 corresponding to an effective absorption coefficient a of about 16.7 mm1 (Table 1). For a laser wavelength of 193 nm the effective absorption index a is significantly higher than 1 mm1, which is in opposite to the a values for 248 nm. That means that for 193 nm the laser energy is deposited within 60 nm, while for 248 nm the absorption length is of about 2 mm. This indicates that for laser ablation with 193 nm above the threshold et a pyrolytic process with good surface quality can be obtained, while for 248 nm a thermally driven process with debris and melt formation will dominate. The following discussion refers to the ArF-excimer laser wavelength (193 nm). A value of 80 mJ/cm2 for the ablation threshold of PS was measured elsewhere [22]. For laser fluences smaller than 100 mJ/cm2 a second ablation threshold of (9 1.8) mJ/ cm2 with an effective absorption coefficient of 43 mm1 was determined. During patterning of PS two mechanisms seem to be important. For high laser fluences the ablation of PS corresponds very well with the classical behaviour of polymer ablation and the ablation threshold given in the literature. By contrast, for very low laser fluences (<9 mJ/cm2) a reaction layer (e.g. with carboxyl groups) is build up (more details in Section 3.3). These ‘‘oxide layers’’ will be partially removed and rebuild for laser fluences above 9 mJ/cm2. That means that a laser dry etching process of the polymer surface above the fluences threshold of 9 mJ/cm2 is observed which dominates the ablation mechanism for laser fluences up to 100 mJ/cm2. 3.2. Control of wettability on polystyrene surfaces For the untreated polymer surfaces the dynamic advancing contact angles Q0 was measured as reference value. For PS Q0 is equal to 82.0 3.38. In the first step, large area modification in air using a conventional UV-Lamp (UVAPRINT, Dr. Ho¨nle A.G., Gra¨felfing, Germany) was performed to get a reference value. The UV-lamp radiates within a wavelength spectrum of l = 220– 420 nm and delivered an intensity of I = 2.4 mW/cm2. The dynamic advancing contact angle Q decreases with irradiation time and reached a saturation value after 120 min. The dynamic advancing contact angle Q reaches a value of 35.18 after 30 min and 23.18 after 120 min. Several laser modifications at 248 nm and 193 nm were performed below the ablation threshold, nearby the ablation threshold and above the ablation threshold and the wetting behaviour was observed as function of laser pulse number. Further, the influence of different processing gases, such
as helium, oxygen and air was investigated. Table 2 summarizes these investigations which are now described in detail. For the wavelength of 248 nm only a small change in wettability can be initiated and the slight increase of 10–208 of the contact angle is mainly caused by a change in surface topography. It was shown that with ArF-excimer laser radiation (193 nm) the wettability can be very well controlled via laser fluence, processing gas and laser pulse number. An enhancement of hydrophobic as well as hydrophilic properties is possible at 60 mJ/cm2 with oxygen as processing gas. After 40 laser pulses Q is reduced from 828 down to 138 and after 400 (200) pulses Q increases up to 1298 (1018). For laser fluences of 90 mJ/cm2, which are slightly above the ablation threshold, the hydrophilic effect is even more pronounced (Fig. 2). After 40 laser pulses, as well as after 100 and 300 laser pulses, the dynamic advancing contact angle Q decreases down to 1.78. After 400 laser pulses the topographic effect caused by an increased surface roughness dominates the chemical effect caused by an oxidization of the PS surface. Fig. 2 shows also that a change in processing gas can be used to switch between hydrophobic and hydrophilic surface properties, even after 40 laser pulses. For He as a processing gas and above 400 laser pulses a contact angle Q of 1508 is reached. Ex situ XPS measurements show, that for He and a laser fluence of 90 mJ/cm2 the surface composition of treated PS is similar to native PS carrying the natural contamination layer. We assume that the oxidization was completely suppressed and only the topographic change influenced the wettability. The topographic structure consists of columns with a diameter of 1–2 mm and a pitch distance of 2–5 mm (Fig. 2, right). For laser fluences at 5 mJ/cm2, which are below the threshold of laser dry etching of PS, no significant change of the dynamic advancing contact angle with respect to the value of the untreated PS-surface was measured. However, the dynamic receding contact angle is significantly reduced from 558 down to 188. The increase in the observed contact angle hysteresis indicates that the chemical structure at the surface is partly changed and inhomogeneous chemical groups leading to a hydrophilic behavior are formed.
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Fig. 2. Left: Dynamic advancing contact angle as function of laser pulse number for PS; right: SEM images of PS surface after laser treatment with 400 laser pulses in He- (top) and O2-atmosphere (bottom).
Ex situ XPS measurements of PS surfaces after laser treatment were performed in order to study the change of chemical binding structure and chemical composition as a function of laser fluence and processing gas. The highest amount of oxygen compounds at the surface is detected after laser treatment at 5 mJ/cm2 in He or O2 processing gas atmosphere (Fig. 3, right). The same behavior is observed for the laser exposure of PS through a quartz cover plate. This suggests that oxidization occurs also after laser treatment, when the sample is handled ex situ in air and indicates that material excitation and formed radicals are temporally stable after laser treatment. The formation of radicals can be due to breaking of phenyl rings or cracking of bonds of the main polymer chain or of side chains. XPS measurements show that in the case of He as a processing gas (Fig. 3) the line shape corresponds very well
with the line shape of the chemical group –COOH [23]. When oxygen is used as processing gas, the O1s line shape slightly changes for laser fluences of 90 mJ/cm2. The O1s line at low binding energy is an artefact and due to localized charge builtup. With He or O2 as processing gas and a laser fluence of 5 mJ/ cm2 the oxygen concentration increases up to 20 at.%. For 90 mJ/cm2, which is above the ablation threshold (75 mJ/cm2), the amount of oxygen-bonds is reduced to 3 at.% and 13 at.% for processing gas He and O2, respectively (Fig. 3). Above the ablation threshold oxidization occurs only in the presence of oxygen as processing gas. Apparently, no temporally stable material excitation is existent above the ablation threshold. The change of chemical bonding, which is observed in the O1s spectra (Fig. 3, left), and the change DQ of wettability between the use of 5 mJ/cm2 (DQ > 108) and 90 mJ/cm2 (DQ 808) are corresponding effects.
Fig. 3. O1s spectra from PS surfaces after modification in processing gas oxygen (left) or helium (right) for different laser fluences (laser pulse number 300).
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Fig. 4. Left: ArF-laser processing diagram for PS illustrates the threshold behavior for laser pulse number and laser fluence with respect to a successful L929 cell adhesion; right: optical photograph after L929 cell cultivation on laser modified PS Petri dishes (each square belongs to another laser pulse number) including optical microscope image for a closer view (laser fluence 5 mJ/cm2). Laser pulse numbers are denoted in each case.
3.3. Formation of carboxyl groups on PS for improved cell adhesion The adhesion of most animal cell lines on unmodified PS surfaces is generally poor. This is to be attributed to the strong adsorption of albumin from serum containing cell culture media. Since albumin does not present cell attractive peptide sequences the albumin covered surface is passivated with respect to cell adhesion. Due to the altered physico-chemical properties (wettability) of the UV irradiated surfaces also the competitive adsorption of plasma proteins is influenced. It was shown previously that albumin adsorption onto UV irradiated PS is hindered whereas the adsorption of cell attractive proteins is increased [19]. The adhesion of L929 cells was investigated on PS surfaces as function of processing gas, laser wavelength (193 nm, 248 nm), laser fluences and laser pulse number. For a significant change in dynamic advancing angle, as described in the preceding chapter, ArF-excimer laser fluences of larger or equal than 20 mJ/cm2 are necessary. In contrast to the change of wettability the type of processing gas (He, air or oxygen) has no detectable influence on the L929 cell adhesion. Furthermore, significant cell adhesion on PS surfaces can be obtained for laser fluences smaller or equal than 6 mJ/cm2. Here it is necessary to consider that the threshold for the dry etch process is in the range of 9 mJ/cm2. Above this threshold a subsequent removal of oxidized groups, assumably –COOH being mainly responsible for an improved L929 cell adhesion as described elsewhere [19], takes place. Not only the laser fluence has showed a threshold for cell adhesion, but also the laser pulse number. For a laser pulse number smaller than 100 pulses no cell adhesion was observed at 5 mJ/cm2. The threshold of the laser pulse number is very
sharp and was determined within an accuracy of 10 pulses. For 19 mJ/cm2 no cell adhesion was observed. The threshold of laser pulse number varies slightly with laser fluence as shown in Fig. 4. For a laser fluence of 6 mJ/cm2, 250 laser pulses are necessary for a subsequent cell adhesion. On PS surfaces the cell adhesion was also realized for pulse numbers of 1200. Table 3 summarizes the obtained results of laser-assisted surface modification of PS with respect to cell adhesion and wettability. For KrF-excimer laser radiation (wavelength 248 nm) no improved L929 cell adhesion can be established at low laser fluences. For 248 nm it was necessary to increase the laser fluences up to a critical value of about 200 mJ/cm2 (ablation threshold of PS for 248 nm, see Table 1) above which polymer ablation took place. Lee et al. have performed laser ablation with 248 nm on oxygen plasma treated PS and have observed, Table 3 Correlation between wettability and cell adhesion as function of laser fluences, laser pulse number and processing gases for the use of ArF-excimer laser Parameters for improved
Processing gas Laser fluence Laser pulse number Change of wettability Oxygen (at.%) Mechanisms
Cell adhesion
Hydrophilicity
Hydrophobicity
He, O2 2–6 mJ/cm2 100–1200
O2 90 mJ/cm2 40–300
He 90 mJ/cm2 400
0 > DQ > 108
DQ 808
DQ +708
20 Chemical
13 Topographical/ chemical
3 Topographical
Corresponding stoichiometry of oxygen obtained by XPS and specification of the dominant process mechanisms are given.
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Fig. 5. Optical microscope images of L929 cells cultured after debris formation during laser ablation of PS (laser wavelength 248 nm, laser fluence e = 2.2 J/cm2); left: cells grow beside of laser-irradiated areas (200 mm 200 mm) after a single laser-shot; right: cells grow in and outside of laser-generated grooves (grooves width 25 mm, groove-to-groove distance 200 mm).
that ablation leads to a removal of oxide layer and NG108-15 cell adhesion is avoided [24]. Zhu et al. reported for the use of frequency-quadrupled Nd:YAG laser (wavelength 266 nm) that the formation of nano-ripples or -grooves might be responsible for an improved cell adhesion [18]. However, we have demonstrated in the case of 248 nm and 193 nm that cell adhesion might be improved for laser fluences in the range and above the ablation threshold. We assume that for laser fluences above the ablation threshold two effects have to be considered: 1st chemical and topographical change caused by debris formation and 2nd ripple formation at the border area. Ripple formation might be caused by laser beam diffraction at the used mask geometry leading to small interference structures at the border of the ablated geometry. Also laser induced surface acoustic waves could induce a change of topography at nmscale. If grooves with a width of 25 mm were produced with 248 nm than cell adhesion in the grooves as well at the border area was observed (Fig. 5, right). KrF-excimer laser ablation of an area of 200 mm 200 mm led to a cell adhesion only at the border area outside of the generated structure profile (Fig. 5, left). From our investigation we can conclude that in the case of 193 nm cell adhesion above the ablation threshold can be
avoided if debris formation is suppressed. This can be used in order to combine laser patterning at high laser fluences and laser modification at low laser fluences without change of laser source and processing chamber. Modification of PS with ArF-exicmer laser radiation at low laser fluences enables a high lateral resolution control of cell adhesion. After modification of areas of 50 mm 50 mm clusters of PC-12 cells were adhered on the modified sites. If the modifications were confined to areas of 25 mm in diameter, it was possible to achieve single cell attachment (Fig. 6). The formation of a cellular network with neurites connecting single neuronal cells, which were attached on the polymer surface, was established. For this purpose tracks on PS with a line width of about 2 mm were laser-modified and the outgrowth of neurites along the exposed tracks was observed in cell culture after NGF induced cell differentiation. In Fig. 6 (right) the successful formation of those cell networks is illustrated. 3.4. Formation of amino groups on PS Carboxyl groups on PS were formed during or after laser exposure. It is assumed that radicals are formed by
Fig. 6. In situ microscope images of laser modified PS surfaces during PC-12 cell cultivation; left: A single-cell is attached to the modified area (diameter of modification 25 mm; no neurite paths were exposed); right: single cells and neurite paths in rectangular arrangement (width of modified tracks: 2 mm); laser wavelength for modification 193 nm and laser fluence 5 mJ/cm2.
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Fig. 7. Left: schematic view of irradiation of PS surface in presence of liquid 2-amino-ethanol; right: XPS spectra of N1s as function of laser pulse number (laser wavelength: 193 nm, laser fluences: 13 mJ/cm2, processing medium: 2-amino-ethanol).
laser-induced bond breaking. Oxygen was delivered from processing gas or from the ambient air. As a consequence of this, the following question was addressed: Is it possible to realize also the formation of other chemical groups at the polymer surface such as amino groups to enable other strategies of biomolecule immobilization? For this purpose other ambient conditions during laser processing would be absolutely necessary. First necessity is to reduce or to avoid the surface reaction with oxygen and second, the desired chemical group should be formed and exposed to the excited polymer surface. For this purpose the polymer was modified in presence of liquid 2-amino-ethanol as shown schematically in Fig. 7 (left). Ex situ XPS measurements after modification were established in order to verify a possible grafting of nitrogen containing compounds to the PS surface as function of laser pulse number (Fig. 7, right). A constant laser fluence of about 13 mJ/cm2 was used. The laser fluence at the interface of polymer and 2-amino-ethanol was surely smaller because of reflection losses and beam attenuation during propagation in the liquid phase. No change in topography was observed after modification. This means, that the effective laser fluence at the polymer surface was lower than the threshold value of 9 mJ/cm2. The N1s spectra show only one peak at binding energies between 399.3e V and 399.7e V. Unfortunately with XPS it is not doubtlessly possible to distinguish between primary amino group and some other nitrogen/carbon species [25]. A binding between oxygen and nitrogen is not supposed, because this should induce a shift of the N1s line to significant higher binding energies [26]. With increasing exposure dose the amount of nitrogen at the PS-surface increases (Fig. 7, right). Nevertheless, XPS measurements reveal that also carboxyl-groups were formed on the PS surface. It is assumed that the oxidization has occurred ex situ, very similar to the modification in He atmosphere.
4. Summary Laser processing at short wavelengths is an appropriate tool for a selective patterning of polymer surfaces. Here, patterning stands for chemical or topographic modification of surfaces on nanometer and micrometer scale. But patterning also encompasses ablation for the generation of two- and threedimensional shapes in polymer surfaces. It was demonstrated that these kinds of ‘‘patternings’’ could be combined with high lateral resolution, e.g. ablation and modification of polymer surfaces with respect to change of wettability and change of L929 and PC-12 cell adhesion (Table 3). The adhesion of cell clusters, single-cells as well as the formation of cell networks on polystyrene surfaces could be controlled with high accuracy. Different types of chemical groups (e.g. –COOH, –NH2) could be patterned onto polymer surfaces containing features with two or three dimensions. Besides the chemical effect also a change in topography on nm- and mm-scale can be used for a change of cell adhesion and wettability. Change of processing gas, O2 or He, can be used to induce complete wetting or to realize hydrophobic surfaces without changing the processing chamber. All these laser processes work without vacuum chambers and enable also the use of liquid media as possible reactant. Future investigations will integrate theses different aspects of modification and ablation for lab-on-chip applications and for the realization of bioreactors and polymer-based bio-medical devices, such as automated patch clamping systems. Acknowledgments We are grateful to our colleague M. Beiser for his technical assistance in SEM. We are indebted to Mrs. M. Marin for her excellent contributions to this research project. We also thank Mrs. V. Trouillet for her support in XPS and H. Besser for laser
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material processing. We gratefully acknowledge the financial support by the program NANOMIKRO of the Helmholtz association and the EU within the Sixth Framework Programme (‘‘Network of Excellence in Multi-Material Micro Manufacture (4M)’’ and ‘‘Network of Excellence in Microoptic (NEMO)’’). References [1] C.G. Khan Malek, Anal. Bioanal. Chem. 385 (2006) 1362. [2] M. Farsari, F. Claret-Tournier, S. Huang, J. Mater. Process. Technol. 107 (2000) 167. [3] W. Pfleging, T. Hanemann, M. Torge, W. Bernauer, J. Mech. Eng. Sci. 217 (2003) 53. [4] M. Heckele, W. Bacher, K.D. Mu¨ller, Microsystem Technol. 4 (1998) 122. [5] V. Piotter, N. Holstein, K. Plewa, R. Ruprecht, J. Hausselt, Microsystem Technol. 10 (2004) 537. [6] U. Wallrabe, H. Dittrich, G. Friedsam, T. Hanemann, J. Mohr, K. Mueller, V. Piotter, P. Ruther, T. Schaller, W. Zißler, SPIE 4408 (2001) 478. [7] T. Hanemann, M. Heckele, V. Piotter, Polym. News 25 (2000) 224. [8] S. Sinzinger, J. Jahns, Microoptics, Wiley-VCH, Weinheim, FRG, 1999. [9] W. Ehrfeld, V. Hessel, H. Lo¨we, Microreactors, Wiley-VCH, Weinheim, FRG, 2000. [10] E. Gottwald, S. Giselbrecht, C. Augspurger, N. Dambrowsky, R. Truckenmu¨ller, V. Piotter, T. Gietzelt, O. Wendt, W. Pfleging, A.
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