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Antibacterial properties of angle-dependent nanopatterns on polystyrene
Reactive and Functional Polymers 136 (2019) 173–180
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Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react
Antibacterial properties of angle-dependent nanopatterns on polystyrene a
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O. Neděla , P. Slepička , N. Slepickova Kasalkova , P. Sajdl , Z. Kolská , S. Rimpelová , V. Švorčíka a
Department of Solid State Engineering, University of Chemistry and Technology, 166 28 Prague, Czech Republic Department of Power Engineering, University of Chemistry and Technology, 166 28 Prague, Czech Republic Faculty of Science, J.E. Purkyne University, 400 96 Usti nad Labem, Czech Republic d Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Polystyrene Excimer laser treatment Atomic force microscopy Scanning electron microscopy Periodic nanostructures Antibacterial properties
Surface modification of polystyrene by a KrF pulse excimer UV laser under the angles of incidence of the laser beam of 0, 22.5 and 45° was proposed in this paper. The influence of the angle of incidence, laser fluence, number of pulses and a deposition of a thin gold layer on the physical and chemical properties of the surface was studied closely. Changes in the surface morphology, the formation of the periodic surface structures as well as the dependence of their dimension on the angle of incidence of the laser beam were determined by Atomic force microscopy and Scanning electron microscopy. Changes in the surface chemistry were studied by X-ray Photoelectron spectroscopy, which revealed a significant increase of oxygen concentration for all laser treated samples and also by electrokinetic analysis to further clarify the surface chemistry and charge changes occurring during the laser treatment. An optimal conditions for antibacterial surface construction was found on the basis of combination of silver nanolayer and PS treated under angle of laser beam incidence 45°. The potential application of laser-treated polystyrene foils can be found mainly in the biotechnological industry, as well as in various other fields dealing with surface modification and micro-patterning of solids.
1. Introduction Polymers are widely utilized materials, popular in industries such as electronics, construction, medicine, and others. Since the vast majority of polymers are chemically stable and thus have an inert surface with low surface energy, some form of surface treatment or modifications are necessary for most industrial applications. As it is desirable to modify the surface by introducing reactive, functional groups (oxidization) into it while keeping the bulk of the polymer material intact, a specific treatment method is required. There are various ways of modifying the surface of polymer substrates, each with their own pros and cons, such as, for example, chemical treatment, treatment based on physical principles, plasma treatment or laser treatment [1–3]. In this work, we focused on surface treatment of polystyrene (PS) by pulse excimer UV laser. PS, one of the many polymers with aromatic rings, is a popular thermoplastic material utilized in many industrial, technological and medical applications, thanks to its good physical and chemical properties [4–7]. Due to the benzene rings contained in PS molecules, the polymer exhibits good adsorption in the ultraviolet range of radiation wavelengths. Since UV light excimer laser treatment has a very low penetration ⁎
depth which ranges from fractions of a micrometer to several tens of micrometers [8], the chemical and physical changes occur only in the top few surface layers, and the bulk remains unaltered [9,10]. This leads to the popularity of surface modification of various substrates by excimer lasers in microprocessing of organic and inorganic substrates [11,12], metallization of a polymer materials - a process often utilized in microelectronics, as polymer surface in its pristine state exhibits next to no adhesion to the deposited metal [13,14], or in biochemical applications and tissue engineering, as the laser-treated surface exhibits a better adhesion to living cells [15]. UV excimer laser radiation can furthermore be utilized for etching not only of polymeric materials but also of biological ones [16]. When a laser beam hits the surface of a polymer, it can be either reflected or be scattered or absorbed by the bulk, depending on the chemical structure of the material. Presence of double bonds between two carbon atoms and/or carbon atom and a heteroatom shifts the absorption peak to higher wavelengths compared to single bonds. Furthermore, since crystallites scatter incident light and thus increase its path through the bulk of the material, semicrystalline polymers exhibit absorption of higher wavelengths than amorphous ones [17]. Depending on laser fluence and to a lesser degree on the number of
Corresponding author. E-mail address: [email protected] (P. Slepička).
https://doi.org/10.1016/j.reactfunctpolym.2019.01.007 Received 12 September 2018; Received in revised form 29 December 2018; Accepted 13 January 2019 Available online 14 January 2019 1381-5148/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. A dependence of surface roughness on laser fluence and the angle of incidence for 4000 (A), 6000 (B) and 8000 pulses (C). The Au shows the structures with gold nanolayer.
high importance, a very interesting papers focused on the preparation and characterization have been published by Bicmarck et al. [28,29], the anti-bacterial properties be in spot of research [30]. Even though the construction of ripple pattern of several polymer substrates was studied in papers before, e.g. [25–27,31–33], we have focused in this work on changes of input laser properties, mainly the angle of laser beam incidence, so that the ideal dot structure could be constructed on the PS surface, which was not published before by simple excimer exposure. Also the combination of certain input parameters leading not only to optimal narrow pattern, but to combination of ripple and wrinkle-like structure, which could be optimal candidate for cell filopodia attachment was proposed since the polystyrene acts as a standard for cell culture growth in tissue engineering. The polystyrene foils were treated under the angles of incidence (0, 22.5 and 45°) by KrF excimer laser for the duration (4000, 6000 and 8000) pulses with laser fluence (6–14 mJ/cm2, a step of 1 mJ/cm2). Surface chemistry of selected samples was then studied using XPS analysis and electrokinetic analysis. A thin (~100 nm) layer of gold was furthermore deposited on all the laser-treated samples, and the gold-coated samples were studied using the FIB-SEM technology. Surface morphology of the laser-treated samples both with and without a deposited gold layer was examined by atomic force microscopy. Also antibacterial properties of selected samples was tested, the gram-positive S. epidermidis bacteria was selected.
pulses, an UV excimer pulse laser treatment of polymer foils can result in ablation of the material, chemical modification of the surface layer and changes in the surface morphology [18]. Laser treatment by laser fluence below the ablation threshold for a given material changes the physical and chemical properties of the surface and can cause various surface structures to form. Using fluencies above the ablation threshold leads to material loss from the surface layer and can reveal quasiperiodic microstructures [19]. Treating a surface of a solid substrate by laser fluence well below the ablation threshold can, under the right conditions, lead to a formation of various surface structures, which may or may not be periodic. On polymers, the most frequently observed type of periodic surface structures are ripples with low spatial frequency [20]. These ripples have a period roughly comparable to the wavelength of the incident laser radiation, and their dimensions depend on many factors, such as chemical composition of the polymer substrate, wavelength of the laser radiation, angle of incidence, duration of laser treatment, atmosphere in the treatment chamber, etc. [21–23]. An equation [24] predicting the period of such structures based on the wavelength of laser light λ, the modified refraction index n and the angle of incidence of the laser beam θ, has been suggested and experimentally confirmed for several kinds of polymers.
⋀=
λ n ± sin θ
(1) 2. Materials and methods
The above mentioned low spatial frequency ripples have been successfully created on polymer substrates such as polyethyleneterephthalate [25], polyethylenenapthalate [26], PS [27] and many other polymers in the past and their application in various industries is a focus of ongoing research. Also the porous polymers are of
Biaxially oriented polystyrene foils (PS, 1.05 g cm−3, thickness of 0.05 mm, Tm ~ 240 °C, Tg ~ 100 °C, supplied by Goodfellow Ltd., UK) were used. 174
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Fig. 2. Images obtained by AFM showing the differences in surface morphology of PS treated under different conditions: 0°, 7 mJ/cm2, 8000 pulses (A), 0°, 10 mJ/ cm2, 8000 pulses (B), 0°, 14 mJ/cm2, 8000 pulses (C), 22.5°, 7 mJ/cm2, 8000 pulses (D), 22.5°, 10 mJ/cm2, 8000 pulses (E), 22.5°, 14 mJ/cm2, 8000 pulses (F), 45°, 7 mJ/cm2, 8000 pulses (G), 45°, 10 mJ/cm2, 8000 pulses (H) and 45°, 14 mJ/cm2, 8000 pulses (I).
spectra. XPS analysis was performed at the pressure of 2·10−8 Pa. Exposed and analyzed area had dimension of 2 × 3 mm2. X-ray source was monochromatic at 1486.7 eV with step size of 0.05 eV. The spectra evaluation was carried out by CasaXPS programme. Electrokinetic potential (zeta potential) of all samples was determined by SurPASS Instrument (Anton Paar) in an adjustable gap cell in contact with the electrolyte (0.001 mol/dm3 KCl in water) at constant pH = 7.0. For each measurement the two substrates of the same dimension (2 × 1 cm2) were kept on the sample holders and put into the measurement cell. The distance between the samples was 100 μm. Each sample was measured four times with the relative error of measurement not exceeding 5%. For zeta potential determination the streaming current method and Helmholtz–Smoluchowski equation were used. FIB (focused ion beam) cuts were prepared with an adapted scanning electron microscope (FIB-SEM, LYRA3 GMU, Tescan, CzechRepublic). The FIB cuts were made with a Ga ion beam. The polishing procedure was performed to clean and flatten the investigated surfaces. The FIB-SEM images were taken under an angle of 54.8°. The influence of the investigation angle on the measurement was automatically corrected by the SEM software. Surface roughness and the dimensions of the ripple-like structures were measured by atomic force microscopy (AFM) in a tapping mode. The AFM images were taken under ambient conditions on a Digital Instruments CP II set-up. Surface roughness and the dimensions of the
Samples were treated by a KrF excimer pulse UV laser (Lambda Physik Compex Pro 50, wavelength of 248 nm, frequency of 10 Hz) for the duration of 4000, 6000 and 8000 pulses under the angles of incidence of 0, 22.5 and 45°, with laser fluence in the range of 6–14 mJ/ cm2 with a step of 1 mJ/cm2. The modification was performed under standard laboratory conditions, ambient atmosphere and room temperature. The gold layer was deposited from a gold target (99.999%) by means of diode sputtering technique (BAL-TEC SCD 050 equipment). Typical sputtering conditions were: room temperature, time 300 s, total argon pressure of about 5 Pa, electrode distance of 50 mm and current of 40 mA. For the measurement of the gold layer thickness we deposited gold under the same conditions on a Si(100) substrate. The gold thickness was determined from scratches measured with atomic force microscopy. Typically five measurements on three scratches each was accomplished on each sample. The thickness of the gold layer sputtered under above described conditions was about 100 nm. For antimicrobial tests, the silver nanostructures were sputtered on the patterned surface, the sputtering conditions were: room temperature, time 50 s, total argon pressure of about 5 Pa, electrode distance of 50 mm and current of 20 mA. Concentrations of C(1 s) and O(1 s) atoms in the treated surface layer were measured by X-ray Photoelectron spectroscopy (XPS). Omicron Nanotechnology ESCAProbe P spectrometer (Omicron Nanotechnology GmbH, DE) was used to measure photoelectron 175
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Fig. 4. SEM (5 μm, on the left) and FIB-SEM (2 μm, on the right) images showing the morphology of the thin gold layer sputtered on the laser-treated PS surface and the thickness of the gold layer in between the ripples in an area cut out by a Ga ion beam. Both samples were treated by laser fluence of 10 mJ/cm2 under the angle of incidence 0° (A, B); 22.5° (C, D) and 45° (E, F) for the duration of 4000 pulses.
(0.9% NaCl) together with the tested samples (0.5 cm2). Subsequently, the samples were gently mixed and incubated statically at 24 °C for 2 and 24 h. Then, the samples were gently mixed again and 25 μL drops of each sample (5 times) were pipetted onto PCA (S. epidermidis) agar plates. The plates were cultured at 37 °C. After that, the number of CFU was counted and compared to the number of CFU on control plates (bacteria incubated only in the physiological solution). The samples were tested in duplicates. The experiment was conducted under sterile conditions.
3. Results and discussion 3.1. Surface morphology - atomic force microscopy In general, increasing laser fluence and the number of pulses leads to an increase in surface roughness for all angles of incidence. In Fig. 1 showing the dependence of surface roughness on laser fluence and the angle of incidence, there are 3 distinct areas of the plot. The first is an area of constant roughness in the 6–8 mJ/cm2 range, in which laser fluence is too low to allow the formation of periodic surface structures. Increasing the number of pulses leads to a decrease in the fluence range of the first area, as ripples form at lower values of laser fluence at longer treatment times. After the first area, there's a sharp increase in roughness leading to the second area, also more-or-less constant, in the 8–12 mJ/cm2 range, where laser fluence is high enough for the formation of periodic surface structures. Increasing laser fluence further causes a steep and mostly linear increase in surface roughness, caused by the
Fig. 3. A dependence of the dimensions of the periodic surface structures on laser fluence and the angle of incidence for 4000 (A), 6000 (B) and 8000 pulses (C). The Au shows the structures with gold nanolayer.
surface structures were determined. All scans were acquired at a line scanning rate of 1 Hz. The antimicrobial properties of PS sputtered with Ag were tested using a Gram-positive bacteria S. epidermidis (DBM3179). Bacteria were inoculated from agar plates into Luria–Bertani (LB) medium and cultivated overnight at 37 °C in an orbital shaker. Optical densities of the overnight cultures were measured at 600 nm. Then, 4·104 CFU of S. epidermidis were inoculated per 1 mL of sterile physiological solution 176
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Fig. 5. A dependence of the atomic oxygen content of the modified surface on laser fluence and the angle of incidence. All samples were treated for the duration of 6000 pulses.
periodic structure being disrupted and losing its homogeneity due to excess of energy. Fusing of the individual ripples occurs in this highest range of laser fluence, which leads to formation of various irregular shapes, which are much larger in their dimensions (width and height) than the original periodic structures. While the surface structures in the third area of the plot lose both their homogeneity and periodicity, they still retain an orientation along the main axis of the polarization of the incident laser beam. Increasing the angle of incidence leads to the respective areas being more distinctive and sharply separated. Surface structures first appear at the fluence of 7 mJ/cm2 in case of samples treated for the duration of 8000 pulses (Fig. 2A,D,G). This energy is insufficient for the formation of ripples and leads only to a semi-periodic globular structure, which transforms into ripples at higher values of laser fluence. Ripple structure forms in the 8–12 mJ/ cm2 range, increasing the number of pulses and the angle of incidence preserves the ripple pattern, but its homogeneity suffers (more noticeably with the increase of the angle of incidence than with the number of pulses). The ripple pattern originates from the superposition of the incoming wave and the refracted beam which spreads over the surface [19,22,24]. The interference of the incoming and refracted beam leads to the inhomogeneous energy and consequently heat distribution on the surface, thus leads to the polymer mass transfer on the surface. As a consequence due to mass diffusion and redistribution the periodic pattern appears on the surface. The excimer laser beam is also responsible for the change of effective (or modified) refractive index (n in Eq. (1)), which is one of the material constant responsible for light wave propagation. The modified refractive index is based on the changes of surface thin layer of modified material. Even the number of laser pulses increase the integral energy dose, the main influence on the wave propagation has the refractive index of the polymer. Since the angle of incidence of laser beam leads to the change of effective refractive index of the material, this change is the leading factor for the pattern homogeneity. The period of the ripples depends on the angle of incidence (Fig. 3) according to Eq. (1), however, due to the mechanism of the formation
Fig. 6. XPS spectra of PS foils treated with 6000 pulses and laser fluence 6 and 14 mJ/cm2 under the angles of incidence of 22.5 and 45°. XPS spectrum of pristine PS was added for comparison.
of the periodic surface structures. Increasing of the laser fluence further causes the homogenous ripple pattern to disintegrate – individual ripples fuse together, and the surface loses its periodicity (Fig. 2C,F,I). The dimensions of the resulting non-periodic structures are slightly more dependent on the number of pulses than on the angle of incidence - an increase in the number of pulses causes thicker structures to form. It is obvious that for the disintegrated pattern the wave propagation and interference is disrupted. The effective refractive index is no longer of great significance, since the disintegrated surface inhibits the wave propagation. From this point of view, the increased number of laser pulses complemented with higher integral energy dose becomes a major factor, which influences the pattern formation and dimensions. 3.2. Surface morphology - scanning electron microscopy For better description of surface morphology induced by KrF laser under different conditions we introduced images obtained by FIB-SEM microscopy for samples treated with only 4000 pulses and 10 mJ/cm2, the variable was the angle of incidence of the laser beam. The FIB-SEM and SEM images show good homogeneity of the deposited gold layer, 177
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surface morphology was determined. A detailed study on the PS treated under angle of 0° [27] was extended substantially. The detailed parameters for dot pattern, the ripple pattern and a combination of these types of surface structures were determined. The information provided in this section may be of great benefit for consequent sensor construction (SERS enhancement) and also for study of cell filopodia attachment which is of great importance in cytocompatibility studies. The following paragraphs will focus on surface chemistry determination, which, in combination with surface morphology, may be of great benefit for biosensor construction. 3.3. Surface chemistry Since the zeta potential and oxygen concentration in the surface layer of samples treated under the angle of incidence of 0° were determined previously [27], we have focused on the influence of the angle of incidence of the laser beam on these parameters. Surface chemistry of samples treated by laser fluence of 6–14 mJ/cm2 with a step of 2 mJ/ cm2 under the angles of incidence of 22.5 and 45° for the duration of 6000 pulses was studied using the XPS elemental composition analysis and by electrokinetic analysis (zeta potential determination). While the styrene molecule contains no oxygen atoms, a trace value of around 1 at. % of oxygen is usually found in pristine PS, due to adsorption from air. The oxygen content in the surface layer of PS rises dramatically after the laser treatment for all values of laser fluence and angles of incidence (Fig. 5). The highest content of oxygen (26.6 at. %) on samples treated under the angle of incidence of 22.5° was found on a sample irradiated by laser fluence of 6 mJ/cm2. Similar amount of oxygen was found for a sample treated by laser fluence of 8 mJ/cm2. Increasing the laser fluence further leads to a decrease in oxygen in the surface layer, resulting in an oxygen content of roughly 22.9% on samples treated by laser fluence of 10 and 12 mJ/cm2 and finally 17.7% on sample treated by laser fluence of 14 mJ/cm2. Samples treated under the angle of incidence of 45° show a similar trend of decreasing oxygen content with increasing laser fluence. The amount of oxygen in these samples is generally lower than in those treated under the angle of incidence of 22.5°. The highest oxygen content (25.7 at. %) was once again found in a sample treated by laser fluence of 6 mJ/cm2. Further increase of laser fluence leads to an almost linear decrease in the amount of oxygen, resulting in the lowest value (12.8 at. %) being measured in a sample treated by laser fluence of 14 mJ/cm2. On the samples treated with 6 mJ/cm2 a small amount of nitrogen was detected for both samples treated under angle of laser beam incidence 22.5° (1.0 at. % of nitrogen) and 45° (1.4 at. % of
Fig. 7. A dependence of the zeta potential of the modified surface on laser fluence and the angle of incidence. All samples were treated for the duration of 6000 pulses.
which also preserves the original morphology of the laser-treated PS substrate (Fig. 4). Both the extruded ripples as well as the flat surface between them are covered uniformly by the gold layer. Cutting through the gold-sputtered sample with a Ga ion beam confirms that the gold layer retains its 100 nm thickness not only on top of the individual ripples, but also in the gap between them (Fig. 4). It is evident, that for lower number of laser pulses the most homogeneous structures are constructed under the angles of incidence of the laser beam of 0 and 22.5°. The periodicity of ripples increases with the angle of incidence of the laser beam according to Eq. (1), and under the angle of incidence of 45°, the distortion of the surface structures becomes very apparent and the ripple periodicity disappears. Summarized, the influence of number of laser pulses with combination of laser fluence and angle of laser beam incidence on the PS
Fig. 8. The amount of S. epidermidis bacteria in dependence on surface modification of PS foil. The following samples are introduced: control sample, laser exposed samples with the fluence 10 mJcm−2, 6000 pulses and angle of laser beam incidence 0°, 22.5; and 45°, and also the same set of laser treated samples deposited with thin Ag nanolayer (50 s).
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Fig. 9. The photographs of S. epidermidis colony forming units on the same samples described in Fig. 8.
nitrogen). Due to the increase of laser fluence up to 14 mJ/cm2 the diminishing of nitrogen on the ripple pattern was observed (see XPS spectra, Fig. 6).
antibacterial properties, (ii) what is the influence of silver nanostructure deposited on excimer treated polystyrene. Mainly the first studied aspect could be of great importance, since only simple change of surface morphology may significantly influence the antibacterial properties of polystyrene. As it is obvious from Figs. 8 and 9, the laser exposure itself has almost no effect on inhibition of bacterial growth of S. epidermidis. Even under different angles of laser beam incidence, where the pattern dimensions are significantly altered, it has almost no influence of antibacterial properties. However, significantly different situation occurs, when a silver nanostructure is deposited on prepared ripple nanopattern. Accordingly to our assumption, the silver layer had a significant effect on growth of Gram-positive bacteria of S. epidermidis. It is evident, that the silver deposition itself significantly decreases the number of CFU on the nanopatterned surface. But surprisingly, if silver nanostructure is deposited on polystyrene pattern, the polystyrene exposure under different angles of laser beam incidence represent the key factor of antimicrobial improvement. As it is obvious from graph in Fig. 8, the higher the angle of laser beam incidence the stronger antimicrobial effect of prepared nanostructure is observed. The exposure of polystyrene under higher laser beam incidence increases the effective surface area of the sample. The changed surface morphology and effective surface area significantly influence both number of metal clusters and their shape. The increased height and width of the pattern in combination with the partially disrupted periodic structure leads to more inhomogeneous metal layer. The metal layer discontinuity with an increased number of metal island increases the antimicrobial activity of the polystyrene pattern. The lowest numbers of CFUs were observed on polystyrene sample deposited with 50s of silver which was previously exposed with 10 mJcm−2 and 6000 pulses under angle 45°.
3.4. Zeta potential measurements Zeta potential values differ significantly between samples treated under the angle of incidence of 22.5° and those treated under the angle of incidence of 45° (Fig. 7). Samples treated under the angle of incidence of 22.5° show no clear trend of dependence of zeta potential on laser fluence. The lowest (the most negative) value of zeta potential (−71.8 mV) was measured on a sample treated by laser fluence of 8 mJ/cm2 while the highest one (−61.9 mV) was measured on a sample treated by laser fluence of 12 mJ/cm2. This trend of zeta potential is somewhat similar to the curve of the oxygen content for samples treated by the same laser fluence (see Fig. 5), and there is no smooth trend in either of those. The samples treated under the angle of incidence of 45° are in better agreement with the declining curve of oxygen content of samples treated under the same angle of incidence (see Fig. 5), with the exception of the sample treated by laser fluence of 8 mJ/cm2. Sample treated by laser fluence of 10 mJ/cm2 and 45° shows a zeta potential value roughly equal to the highest zeta potential value measured on the batch of samples treated under the angle of incidence of 22.5°. Further increase in laser fluence leads to a linear change of zeta potential, with the sample treated by laser fluence of 12 mJ/cm2 having a zeta potential value close to that of pristine PS. The changes of zeta potential should be in accordance with the change in oxygen content, however, zeta potential is affected not only by the chemistry of the surface but also by its morphology [34]. We can therefore attribute the differences between the curves of oxygen content (Fig. 5) and zeta potential (Fig. 7) to the changes in surface roughness of the laser-treated samples.
4. Conclusions 3.5. Antibacterial properties UV excimer laser treatment causes significant changes in the chemistry and morphology of the surface of PS in the 6–14 mJ/cm2 fluence range. At first, globular structures appear, which then quickly transform into ripples as laser fluence increases. These ripples are stable in a relatively wide range of fluencies (8–12 mJ/cm2), although their homogeneity suffers as laser fluence increases. Surface roughness is relatively constant in this range of fluence values. The number of pulses has a negligible effect on the dimensions of the ripples, unlike the angle
Gram-positive (S. epidermidis) bacterial strain was used to test the antibacterial properties of the prepared samples. This bacterial strain was used since it is very often involved in infections connected with a biofilm formation. For the antibacterial tests, the excimer laser modified PS was tested as the first set of samples. We have focused on two main aspects, (i) does the different surface morphology induced by the treatment under different angle of laser beam incidence influence the 179
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of incidence of the laser beam, which determines their period. The modified refraction index of PS also exhibits a dependence on the angle of incidence, which will be a subject of our further study. Values of laser fluence above 12 mJ/cm2 cause the ripple pattern to disappear as individual ripples are disrupted and fuse together and the roughness of the surface increases dramatically. Laser treatment also causes a dramatic increase of the oxygen content in the surface layer, which decreases with both an increase of the angle of incidence and laser fluence. The values of zeta-potential were lower for samples treated under the angle of incidence of 45° compared to those treated under the angle of incidence of 22.5°, with the exception of the sample treated by laser fluence of 8 and 14 mJ/cm2. The higher the angle of laser beam incidence the stronger antimicrobial effect of prepared nanostructure is observed. The lowest numbers of CFUs of S. epidermidis were observed on polystyrene sample deposited with 50 s of silver which was previously exposed with 10 mJcm−2 and 6000 pulses under angle 45°.
Diss Universitat Hannover, 1992. [13] D.A. Wesner, H. Horn, E.W. Kreutz, Pretreatment by laser radiation and its effect on adhesion in the metallization of poly(butylene terephthalate), J. Adhes. Sci. Technol. 9 (1997) 1229–1242. [14] H. Horn, S. Beil, D.A. Wesner, R. Weichenhain, E.W. Kreutz, Excimer laser pretreatment and metallization of polymers, Nucl. Instrum. Methods Phys. Res., Sect. B 151 (1999) 279–284. [15] L.J. Lee, Polymer nanoengineering for biomedical applications, Ann. Biomed. Eng. 34 (2006) 75–88. [16] B.J. Garrison, R. Srinivasan, Laser ablation of organic polymers: Microscopic models for photochemical and thermal processes, Appl. Phys. Lett. 57 (8) (1985) 2909–2914. [17] E. Sancaktar, H. Lu, The effects of excimer laser irradiation at 248 nm on the surface mass loss and thermal properties of PS, ABS, PA6, and PC polymers, J. Appl. Polym. Sci. 99 (2006) 1024–1037. [18] W. Kesting, D. Knittel, T. Bahaners, E. Schollmer, Pulse- and time-dependent observation of UV-laser-induced structures on polymer surfaces, Appl. Surf. Sci. 54 (1992) 330–335. [19] E. Arenholz, J. Heitz, M. Wagner, D. Bauerle, H. Hibst, A. Hagemeyer, Laser-induced surface modification and structure formation of polymers, Appl. Surf. Sci. 69 (1993) 16–19. [20] M. Bolle, S. Lazare, Large scale excimer laser production of submicron periodic structures on polymer surfaces, Appl. Surf. Sci. 69 (1993) 31–37. [21] J.E. Sipe, J.F. Young, J.S. Preston, H.M. van Driel, Laser-induced periodic surface structure. I. Theory, Phys. Rev. B 27 (1983) 1141–1154. [22] J. Bonse, J. Krüger, Pulse number dependence of laser-induced periodic surface structures for femtosecond laser irradiation of silicon, J. Appl. Phys. 108 (2010) 034903. [23] A. Borowiec, H.K. Haugen, Ubwavelength ripple formation on the surfaces of compound semiconductors irradiated with femtosecond laser pulses, Appl. Phys. Lett. 82 (2003) 4462–4464. [24] M. Csete, Z. Bor, Laser-induced periodic surface structure formation on polyethylene-terephthalate, Appl. Surf. Sci. 133 (1998) 5–16. [25] R. Krajcar, J. Siegel, P. Slepička, P. Fitl, V. Švorčík, Silver nanowires prepared on PET structured by laser irradiation, Mater. Lett. 117 (2014) 184–187. [26] P. Slepička, O. Neděla, P. Sajdl, Z. Kolská, V. Švorčík, Polyethylene naphthalate as an excellent candidate for ripplenanopatterning, Appl. Surf. Sci. 285 (2013) 885–892. [27] P. Slepička, O. Neděla, J. Siegel, R. Krajcar, Z. Kolská, V. Švorčík, Ripple polystyrene nano-pattern induced by KrF laser, Express Polym Lett 8 (2014) 459–466. [28] R.Wu, A. Menner, A. Bismarck, Macroporous polymers made from medium internal phase emulsion templates: effect of emulsion formulation on the pore structure of polyMIPEs, Polymer 54 (2013) 5511–5517. [29] Q. Jiang, A. Menner, A. Bismarck, Robust macroporous polymers: using polyurethane diacrylate as property defining crosslinker, Polymer 97 (2016) 598–603. [30] K.-Y. Lee, A. Bismarck, Bacterial Nanocellulose From Biotechnology to BioEconomy, Chapter 6 - Bacterial NanoCellulose as Reinforcement for Polymer Matrices, (2016), pp. 109–122. [31] E. Rebollar, M. Sanz, S. Pérez, M. Hernández, I. Martín-Fabiani, D.R. Rueda, T.A. Ezquerra, C. Domingo, M. Castillejo, Gold coatings on polymer laser induced periodic surface structures: assessment as substrates for surface-enhanced Raman scattering, Phys. Chem. Chem. Phys. 14 (2012) 15699–15705. [32] E. Rebollar, S. Perez, M. Hernandez, C. Domingo, M. Tiberio, T.A. Ezquerra, J.P. Garcı'a-Ruiz, M. Castillejo, Physicochemical modifications accompanying UV laser induced surface structures on poly(ethylene terephthalate) and their effect on adhesion of mesenchymal cells, Phys. Chem. Chem. Phys. 16 (2014) 17551–17559. [33] E. Rebollar, M. Castillejo, T.A. Ezquerra, Laser induced periodic surface structures on polymer films: from fundamentals to applications, Eur. Polym. J. 73 (2015) 162–174. [34] Z. Kolská, A. Řezníčková, V. Švorčík, Surface characterization of polymer foils, ePolymers 83 (2012) 960–972.
Data availability statement The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. Acknowledgements This work was supported by the GACR under project 17-10907S. References [1] M. Ozdemir, H. Sadikoglu, Polymer a new and emerging technology: laser-induced surface modification of polymers, Trends Food Sci. Technol. 9 (1998) 159–167. [2] A. Buchman, H. Dodiuk, M. Rotel, J. Zahavi, Laser-induced adhesion enhancement of polymer composites and metal alloys, J. Adhesion Sci. Technol. 18 (1994) 1211–1224. [3] M. Strobel, M.J. Walzak, J.M. Hill, A. Lin, E. Karbashewski, C.S. Lyons, A comparison of gas-phase methods of modifying polymer surfaces, J. Adhesion Sci. Technol. 9 (1995) 365–383. [4] W. Brostow, H.E. Hagg Lobland, M. Narkis, Sliding wear, viscoelasticity, and brittleness of polymers, J. Mater. Res. 21 (2006) 2422. [5] M. Malmsten (Ed.), Biopolymers at Interfaces, Mercel Dekker, New York, 2003. [6] P. Slepička, N. Slepičková Kasálková, J. Siegel, Z. Kolská, L. Bačáková, V. Švorčík, Nano-structured and functionalized surfaces for cytocompatibility improvement and bactericidal action, Biotechnol. Adv. 33 (2015) 1120–1129. [7] V. Švorčík, V. Rybka, V. Hnatowicz, L. Bačáková, V. Lisá, Surface properties and biocompatibility of ion implanted polymers, J. Mater. Chem. 5 (1995) 27–30. [8] S. Kueper, M. Stuke, Femtosecond UV excimer laser ablation, Appl. Phys. B Lasers Opt. 44 (1987) 199–402. [9] J.M. Goddard, J.H. Hotchkiss, Polymer surface modification for the attachment of bioactive compounds, Prog. Polym. Sci. 32 (2007) 698–725. [10] W.L. Chan, E. Chason, Making waves: kinetic processes controlling surface evolution during low energy ion sputtering, J. Appl. Phys. 101 (2007) 121301. [11] D.J. Ehrlich, J.Y. Tsao (Eds.), Laser-Microfabrication, Thin Film Processes and Lithography, Academic Press, San Diego, 1989. [12] O. Gedrat, Strukturierung technischer Keramik mit Excimer-Laserstrahlung, Dr Ing.
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Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react
Antibacterial properties of angle-dependent nanopatterns on polystyrene a
a,⁎
a
b
c
T
d
O. Neděla , P. Slepička , N. Slepickova Kasalkova , P. Sajdl , Z. Kolská , S. Rimpelová , V. Švorčíka a
Department of Solid State Engineering, University of Chemistry and Technology, 166 28 Prague, Czech Republic Department of Power Engineering, University of Chemistry and Technology, 166 28 Prague, Czech Republic Faculty of Science, J.E. Purkyne University, 400 96 Usti nad Labem, Czech Republic d Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Polystyrene Excimer laser treatment Atomic force microscopy Scanning electron microscopy Periodic nanostructures Antibacterial properties
Surface modification of polystyrene by a KrF pulse excimer UV laser under the angles of incidence of the laser beam of 0, 22.5 and 45° was proposed in this paper. The influence of the angle of incidence, laser fluence, number of pulses and a deposition of a thin gold layer on the physical and chemical properties of the surface was studied closely. Changes in the surface morphology, the formation of the periodic surface structures as well as the dependence of their dimension on the angle of incidence of the laser beam were determined by Atomic force microscopy and Scanning electron microscopy. Changes in the surface chemistry were studied by X-ray Photoelectron spectroscopy, which revealed a significant increase of oxygen concentration for all laser treated samples and also by electrokinetic analysis to further clarify the surface chemistry and charge changes occurring during the laser treatment. An optimal conditions for antibacterial surface construction was found on the basis of combination of silver nanolayer and PS treated under angle of laser beam incidence 45°. The potential application of laser-treated polystyrene foils can be found mainly in the biotechnological industry, as well as in various other fields dealing with surface modification and micro-patterning of solids.
1. Introduction Polymers are widely utilized materials, popular in industries such as electronics, construction, medicine, and others. Since the vast majority of polymers are chemically stable and thus have an inert surface with low surface energy, some form of surface treatment or modifications are necessary for most industrial applications. As it is desirable to modify the surface by introducing reactive, functional groups (oxidization) into it while keeping the bulk of the polymer material intact, a specific treatment method is required. There are various ways of modifying the surface of polymer substrates, each with their own pros and cons, such as, for example, chemical treatment, treatment based on physical principles, plasma treatment or laser treatment [1–3]. In this work, we focused on surface treatment of polystyrene (PS) by pulse excimer UV laser. PS, one of the many polymers with aromatic rings, is a popular thermoplastic material utilized in many industrial, technological and medical applications, thanks to its good physical and chemical properties [4–7]. Due to the benzene rings contained in PS molecules, the polymer exhibits good adsorption in the ultraviolet range of radiation wavelengths. Since UV light excimer laser treatment has a very low penetration ⁎
depth which ranges from fractions of a micrometer to several tens of micrometers [8], the chemical and physical changes occur only in the top few surface layers, and the bulk remains unaltered [9,10]. This leads to the popularity of surface modification of various substrates by excimer lasers in microprocessing of organic and inorganic substrates [11,12], metallization of a polymer materials - a process often utilized in microelectronics, as polymer surface in its pristine state exhibits next to no adhesion to the deposited metal [13,14], or in biochemical applications and tissue engineering, as the laser-treated surface exhibits a better adhesion to living cells [15]. UV excimer laser radiation can furthermore be utilized for etching not only of polymeric materials but also of biological ones [16]. When a laser beam hits the surface of a polymer, it can be either reflected or be scattered or absorbed by the bulk, depending on the chemical structure of the material. Presence of double bonds between two carbon atoms and/or carbon atom and a heteroatom shifts the absorption peak to higher wavelengths compared to single bonds. Furthermore, since crystallites scatter incident light and thus increase its path through the bulk of the material, semicrystalline polymers exhibit absorption of higher wavelengths than amorphous ones [17]. Depending on laser fluence and to a lesser degree on the number of
Corresponding author. E-mail address: [email protected] (P. Slepička).
https://doi.org/10.1016/j.reactfunctpolym.2019.01.007 Received 12 September 2018; Received in revised form 29 December 2018; Accepted 13 January 2019 Available online 14 January 2019 1381-5148/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. A dependence of surface roughness on laser fluence and the angle of incidence for 4000 (A), 6000 (B) and 8000 pulses (C). The Au shows the structures with gold nanolayer.
high importance, a very interesting papers focused on the preparation and characterization have been published by Bicmarck et al. [28,29], the anti-bacterial properties be in spot of research [30]. Even though the construction of ripple pattern of several polymer substrates was studied in papers before, e.g. [25–27,31–33], we have focused in this work on changes of input laser properties, mainly the angle of laser beam incidence, so that the ideal dot structure could be constructed on the PS surface, which was not published before by simple excimer exposure. Also the combination of certain input parameters leading not only to optimal narrow pattern, but to combination of ripple and wrinkle-like structure, which could be optimal candidate for cell filopodia attachment was proposed since the polystyrene acts as a standard for cell culture growth in tissue engineering. The polystyrene foils were treated under the angles of incidence (0, 22.5 and 45°) by KrF excimer laser for the duration (4000, 6000 and 8000) pulses with laser fluence (6–14 mJ/cm2, a step of 1 mJ/cm2). Surface chemistry of selected samples was then studied using XPS analysis and electrokinetic analysis. A thin (~100 nm) layer of gold was furthermore deposited on all the laser-treated samples, and the gold-coated samples were studied using the FIB-SEM technology. Surface morphology of the laser-treated samples both with and without a deposited gold layer was examined by atomic force microscopy. Also antibacterial properties of selected samples was tested, the gram-positive S. epidermidis bacteria was selected.
pulses, an UV excimer pulse laser treatment of polymer foils can result in ablation of the material, chemical modification of the surface layer and changes in the surface morphology [18]. Laser treatment by laser fluence below the ablation threshold for a given material changes the physical and chemical properties of the surface and can cause various surface structures to form. Using fluencies above the ablation threshold leads to material loss from the surface layer and can reveal quasiperiodic microstructures [19]. Treating a surface of a solid substrate by laser fluence well below the ablation threshold can, under the right conditions, lead to a formation of various surface structures, which may or may not be periodic. On polymers, the most frequently observed type of periodic surface structures are ripples with low spatial frequency [20]. These ripples have a period roughly comparable to the wavelength of the incident laser radiation, and their dimensions depend on many factors, such as chemical composition of the polymer substrate, wavelength of the laser radiation, angle of incidence, duration of laser treatment, atmosphere in the treatment chamber, etc. [21–23]. An equation [24] predicting the period of such structures based on the wavelength of laser light λ, the modified refraction index n and the angle of incidence of the laser beam θ, has been suggested and experimentally confirmed for several kinds of polymers.
⋀=
λ n ± sin θ
(1) 2. Materials and methods
The above mentioned low spatial frequency ripples have been successfully created on polymer substrates such as polyethyleneterephthalate [25], polyethylenenapthalate [26], PS [27] and many other polymers in the past and their application in various industries is a focus of ongoing research. Also the porous polymers are of
Biaxially oriented polystyrene foils (PS, 1.05 g cm−3, thickness of 0.05 mm, Tm ~ 240 °C, Tg ~ 100 °C, supplied by Goodfellow Ltd., UK) were used. 174
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Fig. 2. Images obtained by AFM showing the differences in surface morphology of PS treated under different conditions: 0°, 7 mJ/cm2, 8000 pulses (A), 0°, 10 mJ/ cm2, 8000 pulses (B), 0°, 14 mJ/cm2, 8000 pulses (C), 22.5°, 7 mJ/cm2, 8000 pulses (D), 22.5°, 10 mJ/cm2, 8000 pulses (E), 22.5°, 14 mJ/cm2, 8000 pulses (F), 45°, 7 mJ/cm2, 8000 pulses (G), 45°, 10 mJ/cm2, 8000 pulses (H) and 45°, 14 mJ/cm2, 8000 pulses (I).
spectra. XPS analysis was performed at the pressure of 2·10−8 Pa. Exposed and analyzed area had dimension of 2 × 3 mm2. X-ray source was monochromatic at 1486.7 eV with step size of 0.05 eV. The spectra evaluation was carried out by CasaXPS programme. Electrokinetic potential (zeta potential) of all samples was determined by SurPASS Instrument (Anton Paar) in an adjustable gap cell in contact with the electrolyte (0.001 mol/dm3 KCl in water) at constant pH = 7.0. For each measurement the two substrates of the same dimension (2 × 1 cm2) were kept on the sample holders and put into the measurement cell. The distance between the samples was 100 μm. Each sample was measured four times with the relative error of measurement not exceeding 5%. For zeta potential determination the streaming current method and Helmholtz–Smoluchowski equation were used. FIB (focused ion beam) cuts were prepared with an adapted scanning electron microscope (FIB-SEM, LYRA3 GMU, Tescan, CzechRepublic). The FIB cuts were made with a Ga ion beam. The polishing procedure was performed to clean and flatten the investigated surfaces. The FIB-SEM images were taken under an angle of 54.8°. The influence of the investigation angle on the measurement was automatically corrected by the SEM software. Surface roughness and the dimensions of the ripple-like structures were measured by atomic force microscopy (AFM) in a tapping mode. The AFM images were taken under ambient conditions on a Digital Instruments CP II set-up. Surface roughness and the dimensions of the
Samples were treated by a KrF excimer pulse UV laser (Lambda Physik Compex Pro 50, wavelength of 248 nm, frequency of 10 Hz) for the duration of 4000, 6000 and 8000 pulses under the angles of incidence of 0, 22.5 and 45°, with laser fluence in the range of 6–14 mJ/ cm2 with a step of 1 mJ/cm2. The modification was performed under standard laboratory conditions, ambient atmosphere and room temperature. The gold layer was deposited from a gold target (99.999%) by means of diode sputtering technique (BAL-TEC SCD 050 equipment). Typical sputtering conditions were: room temperature, time 300 s, total argon pressure of about 5 Pa, electrode distance of 50 mm and current of 40 mA. For the measurement of the gold layer thickness we deposited gold under the same conditions on a Si(100) substrate. The gold thickness was determined from scratches measured with atomic force microscopy. Typically five measurements on three scratches each was accomplished on each sample. The thickness of the gold layer sputtered under above described conditions was about 100 nm. For antimicrobial tests, the silver nanostructures were sputtered on the patterned surface, the sputtering conditions were: room temperature, time 50 s, total argon pressure of about 5 Pa, electrode distance of 50 mm and current of 20 mA. Concentrations of C(1 s) and O(1 s) atoms in the treated surface layer were measured by X-ray Photoelectron spectroscopy (XPS). Omicron Nanotechnology ESCAProbe P spectrometer (Omicron Nanotechnology GmbH, DE) was used to measure photoelectron 175
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Fig. 4. SEM (5 μm, on the left) and FIB-SEM (2 μm, on the right) images showing the morphology of the thin gold layer sputtered on the laser-treated PS surface and the thickness of the gold layer in between the ripples in an area cut out by a Ga ion beam. Both samples were treated by laser fluence of 10 mJ/cm2 under the angle of incidence 0° (A, B); 22.5° (C, D) and 45° (E, F) for the duration of 4000 pulses.
(0.9% NaCl) together with the tested samples (0.5 cm2). Subsequently, the samples were gently mixed and incubated statically at 24 °C for 2 and 24 h. Then, the samples were gently mixed again and 25 μL drops of each sample (5 times) were pipetted onto PCA (S. epidermidis) agar plates. The plates were cultured at 37 °C. After that, the number of CFU was counted and compared to the number of CFU on control plates (bacteria incubated only in the physiological solution). The samples were tested in duplicates. The experiment was conducted under sterile conditions.
3. Results and discussion 3.1. Surface morphology - atomic force microscopy In general, increasing laser fluence and the number of pulses leads to an increase in surface roughness for all angles of incidence. In Fig. 1 showing the dependence of surface roughness on laser fluence and the angle of incidence, there are 3 distinct areas of the plot. The first is an area of constant roughness in the 6–8 mJ/cm2 range, in which laser fluence is too low to allow the formation of periodic surface structures. Increasing the number of pulses leads to a decrease in the fluence range of the first area, as ripples form at lower values of laser fluence at longer treatment times. After the first area, there's a sharp increase in roughness leading to the second area, also more-or-less constant, in the 8–12 mJ/cm2 range, where laser fluence is high enough for the formation of periodic surface structures. Increasing laser fluence further causes a steep and mostly linear increase in surface roughness, caused by the
Fig. 3. A dependence of the dimensions of the periodic surface structures on laser fluence and the angle of incidence for 4000 (A), 6000 (B) and 8000 pulses (C). The Au shows the structures with gold nanolayer.
surface structures were determined. All scans were acquired at a line scanning rate of 1 Hz. The antimicrobial properties of PS sputtered with Ag were tested using a Gram-positive bacteria S. epidermidis (DBM3179). Bacteria were inoculated from agar plates into Luria–Bertani (LB) medium and cultivated overnight at 37 °C in an orbital shaker. Optical densities of the overnight cultures were measured at 600 nm. Then, 4·104 CFU of S. epidermidis were inoculated per 1 mL of sterile physiological solution 176
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Fig. 5. A dependence of the atomic oxygen content of the modified surface on laser fluence and the angle of incidence. All samples were treated for the duration of 6000 pulses.
periodic structure being disrupted and losing its homogeneity due to excess of energy. Fusing of the individual ripples occurs in this highest range of laser fluence, which leads to formation of various irregular shapes, which are much larger in their dimensions (width and height) than the original periodic structures. While the surface structures in the third area of the plot lose both their homogeneity and periodicity, they still retain an orientation along the main axis of the polarization of the incident laser beam. Increasing the angle of incidence leads to the respective areas being more distinctive and sharply separated. Surface structures first appear at the fluence of 7 mJ/cm2 in case of samples treated for the duration of 8000 pulses (Fig. 2A,D,G). This energy is insufficient for the formation of ripples and leads only to a semi-periodic globular structure, which transforms into ripples at higher values of laser fluence. Ripple structure forms in the 8–12 mJ/ cm2 range, increasing the number of pulses and the angle of incidence preserves the ripple pattern, but its homogeneity suffers (more noticeably with the increase of the angle of incidence than with the number of pulses). The ripple pattern originates from the superposition of the incoming wave and the refracted beam which spreads over the surface [19,22,24]. The interference of the incoming and refracted beam leads to the inhomogeneous energy and consequently heat distribution on the surface, thus leads to the polymer mass transfer on the surface. As a consequence due to mass diffusion and redistribution the periodic pattern appears on the surface. The excimer laser beam is also responsible for the change of effective (or modified) refractive index (n in Eq. (1)), which is one of the material constant responsible for light wave propagation. The modified refractive index is based on the changes of surface thin layer of modified material. Even the number of laser pulses increase the integral energy dose, the main influence on the wave propagation has the refractive index of the polymer. Since the angle of incidence of laser beam leads to the change of effective refractive index of the material, this change is the leading factor for the pattern homogeneity. The period of the ripples depends on the angle of incidence (Fig. 3) according to Eq. (1), however, due to the mechanism of the formation
Fig. 6. XPS spectra of PS foils treated with 6000 pulses and laser fluence 6 and 14 mJ/cm2 under the angles of incidence of 22.5 and 45°. XPS spectrum of pristine PS was added for comparison.
of the periodic surface structures. Increasing of the laser fluence further causes the homogenous ripple pattern to disintegrate – individual ripples fuse together, and the surface loses its periodicity (Fig. 2C,F,I). The dimensions of the resulting non-periodic structures are slightly more dependent on the number of pulses than on the angle of incidence - an increase in the number of pulses causes thicker structures to form. It is obvious that for the disintegrated pattern the wave propagation and interference is disrupted. The effective refractive index is no longer of great significance, since the disintegrated surface inhibits the wave propagation. From this point of view, the increased number of laser pulses complemented with higher integral energy dose becomes a major factor, which influences the pattern formation and dimensions. 3.2. Surface morphology - scanning electron microscopy For better description of surface morphology induced by KrF laser under different conditions we introduced images obtained by FIB-SEM microscopy for samples treated with only 4000 pulses and 10 mJ/cm2, the variable was the angle of incidence of the laser beam. The FIB-SEM and SEM images show good homogeneity of the deposited gold layer, 177
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surface morphology was determined. A detailed study on the PS treated under angle of 0° [27] was extended substantially. The detailed parameters for dot pattern, the ripple pattern and a combination of these types of surface structures were determined. The information provided in this section may be of great benefit for consequent sensor construction (SERS enhancement) and also for study of cell filopodia attachment which is of great importance in cytocompatibility studies. The following paragraphs will focus on surface chemistry determination, which, in combination with surface morphology, may be of great benefit for biosensor construction. 3.3. Surface chemistry Since the zeta potential and oxygen concentration in the surface layer of samples treated under the angle of incidence of 0° were determined previously [27], we have focused on the influence of the angle of incidence of the laser beam on these parameters. Surface chemistry of samples treated by laser fluence of 6–14 mJ/cm2 with a step of 2 mJ/ cm2 under the angles of incidence of 22.5 and 45° for the duration of 6000 pulses was studied using the XPS elemental composition analysis and by electrokinetic analysis (zeta potential determination). While the styrene molecule contains no oxygen atoms, a trace value of around 1 at. % of oxygen is usually found in pristine PS, due to adsorption from air. The oxygen content in the surface layer of PS rises dramatically after the laser treatment for all values of laser fluence and angles of incidence (Fig. 5). The highest content of oxygen (26.6 at. %) on samples treated under the angle of incidence of 22.5° was found on a sample irradiated by laser fluence of 6 mJ/cm2. Similar amount of oxygen was found for a sample treated by laser fluence of 8 mJ/cm2. Increasing the laser fluence further leads to a decrease in oxygen in the surface layer, resulting in an oxygen content of roughly 22.9% on samples treated by laser fluence of 10 and 12 mJ/cm2 and finally 17.7% on sample treated by laser fluence of 14 mJ/cm2. Samples treated under the angle of incidence of 45° show a similar trend of decreasing oxygen content with increasing laser fluence. The amount of oxygen in these samples is generally lower than in those treated under the angle of incidence of 22.5°. The highest oxygen content (25.7 at. %) was once again found in a sample treated by laser fluence of 6 mJ/cm2. Further increase of laser fluence leads to an almost linear decrease in the amount of oxygen, resulting in the lowest value (12.8 at. %) being measured in a sample treated by laser fluence of 14 mJ/cm2. On the samples treated with 6 mJ/cm2 a small amount of nitrogen was detected for both samples treated under angle of laser beam incidence 22.5° (1.0 at. % of nitrogen) and 45° (1.4 at. % of
Fig. 7. A dependence of the zeta potential of the modified surface on laser fluence and the angle of incidence. All samples were treated for the duration of 6000 pulses.
which also preserves the original morphology of the laser-treated PS substrate (Fig. 4). Both the extruded ripples as well as the flat surface between them are covered uniformly by the gold layer. Cutting through the gold-sputtered sample with a Ga ion beam confirms that the gold layer retains its 100 nm thickness not only on top of the individual ripples, but also in the gap between them (Fig. 4). It is evident, that for lower number of laser pulses the most homogeneous structures are constructed under the angles of incidence of the laser beam of 0 and 22.5°. The periodicity of ripples increases with the angle of incidence of the laser beam according to Eq. (1), and under the angle of incidence of 45°, the distortion of the surface structures becomes very apparent and the ripple periodicity disappears. Summarized, the influence of number of laser pulses with combination of laser fluence and angle of laser beam incidence on the PS
Fig. 8. The amount of S. epidermidis bacteria in dependence on surface modification of PS foil. The following samples are introduced: control sample, laser exposed samples with the fluence 10 mJcm−2, 6000 pulses and angle of laser beam incidence 0°, 22.5; and 45°, and also the same set of laser treated samples deposited with thin Ag nanolayer (50 s).
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Fig. 9. The photographs of S. epidermidis colony forming units on the same samples described in Fig. 8.
nitrogen). Due to the increase of laser fluence up to 14 mJ/cm2 the diminishing of nitrogen on the ripple pattern was observed (see XPS spectra, Fig. 6).
antibacterial properties, (ii) what is the influence of silver nanostructure deposited on excimer treated polystyrene. Mainly the first studied aspect could be of great importance, since only simple change of surface morphology may significantly influence the antibacterial properties of polystyrene. As it is obvious from Figs. 8 and 9, the laser exposure itself has almost no effect on inhibition of bacterial growth of S. epidermidis. Even under different angles of laser beam incidence, where the pattern dimensions are significantly altered, it has almost no influence of antibacterial properties. However, significantly different situation occurs, when a silver nanostructure is deposited on prepared ripple nanopattern. Accordingly to our assumption, the silver layer had a significant effect on growth of Gram-positive bacteria of S. epidermidis. It is evident, that the silver deposition itself significantly decreases the number of CFU on the nanopatterned surface. But surprisingly, if silver nanostructure is deposited on polystyrene pattern, the polystyrene exposure under different angles of laser beam incidence represent the key factor of antimicrobial improvement. As it is obvious from graph in Fig. 8, the higher the angle of laser beam incidence the stronger antimicrobial effect of prepared nanostructure is observed. The exposure of polystyrene under higher laser beam incidence increases the effective surface area of the sample. The changed surface morphology and effective surface area significantly influence both number of metal clusters and their shape. The increased height and width of the pattern in combination with the partially disrupted periodic structure leads to more inhomogeneous metal layer. The metal layer discontinuity with an increased number of metal island increases the antimicrobial activity of the polystyrene pattern. The lowest numbers of CFUs were observed on polystyrene sample deposited with 50s of silver which was previously exposed with 10 mJcm−2 and 6000 pulses under angle 45°.
3.4. Zeta potential measurements Zeta potential values differ significantly between samples treated under the angle of incidence of 22.5° and those treated under the angle of incidence of 45° (Fig. 7). Samples treated under the angle of incidence of 22.5° show no clear trend of dependence of zeta potential on laser fluence. The lowest (the most negative) value of zeta potential (−71.8 mV) was measured on a sample treated by laser fluence of 8 mJ/cm2 while the highest one (−61.9 mV) was measured on a sample treated by laser fluence of 12 mJ/cm2. This trend of zeta potential is somewhat similar to the curve of the oxygen content for samples treated by the same laser fluence (see Fig. 5), and there is no smooth trend in either of those. The samples treated under the angle of incidence of 45° are in better agreement with the declining curve of oxygen content of samples treated under the same angle of incidence (see Fig. 5), with the exception of the sample treated by laser fluence of 8 mJ/cm2. Sample treated by laser fluence of 10 mJ/cm2 and 45° shows a zeta potential value roughly equal to the highest zeta potential value measured on the batch of samples treated under the angle of incidence of 22.5°. Further increase in laser fluence leads to a linear change of zeta potential, with the sample treated by laser fluence of 12 mJ/cm2 having a zeta potential value close to that of pristine PS. The changes of zeta potential should be in accordance with the change in oxygen content, however, zeta potential is affected not only by the chemistry of the surface but also by its morphology [34]. We can therefore attribute the differences between the curves of oxygen content (Fig. 5) and zeta potential (Fig. 7) to the changes in surface roughness of the laser-treated samples.
4. Conclusions 3.5. Antibacterial properties UV excimer laser treatment causes significant changes in the chemistry and morphology of the surface of PS in the 6–14 mJ/cm2 fluence range. At first, globular structures appear, which then quickly transform into ripples as laser fluence increases. These ripples are stable in a relatively wide range of fluencies (8–12 mJ/cm2), although their homogeneity suffers as laser fluence increases. Surface roughness is relatively constant in this range of fluence values. The number of pulses has a negligible effect on the dimensions of the ripples, unlike the angle
Gram-positive (S. epidermidis) bacterial strain was used to test the antibacterial properties of the prepared samples. This bacterial strain was used since it is very often involved in infections connected with a biofilm formation. For the antibacterial tests, the excimer laser modified PS was tested as the first set of samples. We have focused on two main aspects, (i) does the different surface morphology induced by the treatment under different angle of laser beam incidence influence the 179
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of incidence of the laser beam, which determines their period. The modified refraction index of PS also exhibits a dependence on the angle of incidence, which will be a subject of our further study. Values of laser fluence above 12 mJ/cm2 cause the ripple pattern to disappear as individual ripples are disrupted and fuse together and the roughness of the surface increases dramatically. Laser treatment also causes a dramatic increase of the oxygen content in the surface layer, which decreases with both an increase of the angle of incidence and laser fluence. The values of zeta-potential were lower for samples treated under the angle of incidence of 45° compared to those treated under the angle of incidence of 22.5°, with the exception of the sample treated by laser fluence of 8 and 14 mJ/cm2. The higher the angle of laser beam incidence the stronger antimicrobial effect of prepared nanostructure is observed. The lowest numbers of CFUs of S. epidermidis were observed on polystyrene sample deposited with 50 s of silver which was previously exposed with 10 mJcm−2 and 6000 pulses under angle 45°.
Diss Universitat Hannover, 1992. [13] D.A. Wesner, H. Horn, E.W. Kreutz, Pretreatment by laser radiation and its effect on adhesion in the metallization of poly(butylene terephthalate), J. Adhes. Sci. Technol. 9 (1997) 1229–1242. [14] H. Horn, S. Beil, D.A. Wesner, R. Weichenhain, E.W. Kreutz, Excimer laser pretreatment and metallization of polymers, Nucl. Instrum. Methods Phys. Res., Sect. B 151 (1999) 279–284. [15] L.J. Lee, Polymer nanoengineering for biomedical applications, Ann. Biomed. Eng. 34 (2006) 75–88. [16] B.J. Garrison, R. Srinivasan, Laser ablation of organic polymers: Microscopic models for photochemical and thermal processes, Appl. Phys. Lett. 57 (8) (1985) 2909–2914. [17] E. Sancaktar, H. Lu, The effects of excimer laser irradiation at 248 nm on the surface mass loss and thermal properties of PS, ABS, PA6, and PC polymers, J. Appl. Polym. Sci. 99 (2006) 1024–1037. [18] W. Kesting, D. Knittel, T. Bahaners, E. Schollmer, Pulse- and time-dependent observation of UV-laser-induced structures on polymer surfaces, Appl. Surf. Sci. 54 (1992) 330–335. [19] E. Arenholz, J. Heitz, M. Wagner, D. Bauerle, H. Hibst, A. Hagemeyer, Laser-induced surface modification and structure formation of polymers, Appl. Surf. Sci. 69 (1993) 16–19. [20] M. Bolle, S. Lazare, Large scale excimer laser production of submicron periodic structures on polymer surfaces, Appl. Surf. Sci. 69 (1993) 31–37. [21] J.E. Sipe, J.F. Young, J.S. Preston, H.M. van Driel, Laser-induced periodic surface structure. I. Theory, Phys. Rev. B 27 (1983) 1141–1154. [22] J. Bonse, J. Krüger, Pulse number dependence of laser-induced periodic surface structures for femtosecond laser irradiation of silicon, J. Appl. Phys. 108 (2010) 034903. [23] A. Borowiec, H.K. Haugen, Ubwavelength ripple formation on the surfaces of compound semiconductors irradiated with femtosecond laser pulses, Appl. Phys. Lett. 82 (2003) 4462–4464. [24] M. Csete, Z. Bor, Laser-induced periodic surface structure formation on polyethylene-terephthalate, Appl. Surf. Sci. 133 (1998) 5–16. [25] R. Krajcar, J. Siegel, P. Slepička, P. Fitl, V. Švorčík, Silver nanowires prepared on PET structured by laser irradiation, Mater. Lett. 117 (2014) 184–187. [26] P. Slepička, O. Neděla, P. Sajdl, Z. Kolská, V. Švorčík, Polyethylene naphthalate as an excellent candidate for ripplenanopatterning, Appl. Surf. Sci. 285 (2013) 885–892. [27] P. Slepička, O. Neděla, J. Siegel, R. Krajcar, Z. Kolská, V. Švorčík, Ripple polystyrene nano-pattern induced by KrF laser, Express Polym Lett 8 (2014) 459–466. [28] R.Wu, A. Menner, A. Bismarck, Macroporous polymers made from medium internal phase emulsion templates: effect of emulsion formulation on the pore structure of polyMIPEs, Polymer 54 (2013) 5511–5517. [29] Q. Jiang, A. Menner, A. Bismarck, Robust macroporous polymers: using polyurethane diacrylate as property defining crosslinker, Polymer 97 (2016) 598–603. [30] K.-Y. Lee, A. Bismarck, Bacterial Nanocellulose From Biotechnology to BioEconomy, Chapter 6 - Bacterial NanoCellulose as Reinforcement for Polymer Matrices, (2016), pp. 109–122. [31] E. Rebollar, M. Sanz, S. Pérez, M. Hernández, I. Martín-Fabiani, D.R. Rueda, T.A. Ezquerra, C. Domingo, M. Castillejo, Gold coatings on polymer laser induced periodic surface structures: assessment as substrates for surface-enhanced Raman scattering, Phys. Chem. Chem. Phys. 14 (2012) 15699–15705. [32] E. Rebollar, S. Perez, M. Hernandez, C. Domingo, M. Tiberio, T.A. Ezquerra, J.P. Garcı'a-Ruiz, M. Castillejo, Physicochemical modifications accompanying UV laser induced surface structures on poly(ethylene terephthalate) and their effect on adhesion of mesenchymal cells, Phys. Chem. Chem. Phys. 16 (2014) 17551–17559. [33] E. Rebollar, M. Castillejo, T.A. Ezquerra, Laser induced periodic surface structures on polymer films: from fundamentals to applications, Eur. Polym. J. 73 (2015) 162–174. [34] Z. Kolská, A. Řezníčková, V. Švorčík, Surface characterization of polymer foils, ePolymers 83 (2012) 960–972.
Data availability statement The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. Acknowledgements This work was supported by the GACR under project 17-10907S. References [1] M. Ozdemir, H. Sadikoglu, Polymer a new and emerging technology: laser-induced surface modification of polymers, Trends Food Sci. Technol. 9 (1998) 159–167. [2] A. Buchman, H. Dodiuk, M. Rotel, J. Zahavi, Laser-induced adhesion enhancement of polymer composites and metal alloys, J. Adhesion Sci. Technol. 18 (1994) 1211–1224. [3] M. Strobel, M.J. Walzak, J.M. Hill, A. Lin, E. Karbashewski, C.S. Lyons, A comparison of gas-phase methods of modifying polymer surfaces, J. Adhesion Sci. Technol. 9 (1995) 365–383. [4] W. Brostow, H.E. Hagg Lobland, M. Narkis, Sliding wear, viscoelasticity, and brittleness of polymers, J. Mater. Res. 21 (2006) 2422. [5] M. Malmsten (Ed.), Biopolymers at Interfaces, Mercel Dekker, New York, 2003. [6] P. Slepička, N. Slepičková Kasálková, J. Siegel, Z. Kolská, L. Bačáková, V. Švorčík, Nano-structured and functionalized surfaces for cytocompatibility improvement and bactericidal action, Biotechnol. Adv. 33 (2015) 1120–1129. [7] V. Švorčík, V. Rybka, V. Hnatowicz, L. Bačáková, V. Lisá, Surface properties and biocompatibility of ion implanted polymers, J. Mater. Chem. 5 (1995) 27–30. [8] S. Kueper, M. Stuke, Femtosecond UV excimer laser ablation, Appl. Phys. B Lasers Opt. 44 (1987) 199–402. [9] J.M. Goddard, J.H. Hotchkiss, Polymer surface modification for the attachment of bioactive compounds, Prog. Polym. Sci. 32 (2007) 698–725. [10] W.L. Chan, E. Chason, Making waves: kinetic processes controlling surface evolution during low energy ion sputtering, J. Appl. Phys. 101 (2007) 121301. [11] D.J. Ehrlich, J.Y. Tsao (Eds.), Laser-Microfabrication, Thin Film Processes and Lithography, Academic Press, San Diego, 1989. [12] O. Gedrat, Strukturierung technischer Keramik mit Excimer-Laserstrahlung, Dr Ing.
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