Appl. Phys. A 117, 295–300

Appl. Phys. A (2014) 117:295–300
DOI 10.1007/s00339-013-8219-9
Laser-induced periodic surface structures on polymers
for formation of gold nanowires and activation of human cells
R.-A. Barb • C. Hrelescu • L. Dong • J. Heitz • J. Siegel
P. Slepicka • V. Vosmanska • V. Svorcik • B. Magnus •
R. Marksteiner • M. Schernthaner • K. Groschner
•
Received: 31 October 2013 / Accepted: 19 December 2013 / Published online: 19 January 2014
Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Frequently observed coherent structures in lasersurface processing are ripples, also denoted as laser-induced
periodic surface structures (LIPSS). For polyethylene terephthalate (PET) and polystyrene (PS), LIPSS can be induced
by irradiation with linearly polarized ns-pulsed UV laser
light. Under an angle of incidence of h, their lateral period is
close to the laser wavelength k divided by (neff - sinh). Here,
neff is the effective refractive index which is 1.32 and 1.23 for
PET and PS, respectively. We describe potential applications
of LIPSS for alignment and activation of human cells cultivated on polymer substrates, as well as for formation of
separated gold nanowires which show pronounced surface
plasmon resonances, e.g., at 775 nm for PET.
1 Introduction
The interference of an incident laser beam with first order
diffracted light running parallel to a surface can induce the
formation of ripple structures [1]. These are spatially periodic
R.-A. Barb C. Hrelescu L. Dong J. Heitz (&)
Institute of Applied Physics, Johannes Kepler University Linz,
4040 Linz, Austria
e-mail: [email protected]
URL: http://www.jku.at/applphys
J. Siegel P. Slepicka V. Vosmanska V. Svorcik
Institute of Chemical Technology, 166 28 Prague,
Czech Republic
B. Magnus R. Marksteiner
Innovacell Biotechnologie AG, 6020 Innsbruck, Austria
M. Schernthaner K. Groschner
Institute of Biophysics, Medical University Graz,
8010 Graz, Austria
structures, also denoted as laser-induced periodic surface
structures (LIPSS). Ripple formation was first observed by
Birnbaum [2] after ruby-laser irradiation of various semiconductor surfaces. Further investigations have shown that
ripple formation is a general phenomenon, observed practically always on solid or liquid surfaces after laser irradiation
with polarized light within certain ranges of laser parameters.
The precise physical mechanism of ripple formation depends
on the material used and the irradiation parameters and is the
topic of an ongoing research effort [3–6].
Laser pulse lengths in the order of some ns, fluences well
below the ablation threshold, and a large number of laser
pulses have to be applied to induce LIPSS formation on
polyethylene terephthalate (PET) or polystyrene (PS) surfaces. The formed nanostructures depend on the laser wavelength, k, as well as the angle of incidence, h, of the laser beam
[7–10]. For s-polarization, the lateral periodicity K is given by
K ¼
neff
k
sin h
ð1Þ
where neff is the effective refractive index, which lies
between the index of air (&1) and the index of the polymer. The direction of the ripples in case of polymers is
parallel to the polarization direction. The structure periodicity K can be varied by means of different irradiation
parameters, but not independently from the structure
height, h. The aspect ratio of the ripples (i.e., h divided by
half of K) is typically around 0.5.
In previous studies, it has been demonstrated that laserinduced oriented periodic structures on polymer surfaces
can be employed for alignment of mammalian cells cultivated thereon, at least if the periodicity K was larger than
300 nm [8, 11, 12]. Proliferative nuclear signaling in biological cells is profoundly activated by the nanotopography
of the cell culture substrate, even if the LIPSS structures
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Fig. 1 AFM images of LIPSS structures on a, b PET and c, d PS after irradiation with 6,000 KrF* laser pulses under an angle of incidence of
a, c 30° or b, d 45°, respectively
are too small to induce alignment [12]. Another application
of LIPSS on PET is the self-organized formation of separated gold nanowires [13], which can be easily and fast
fabricated on large areas. In this publication, we report for
the first time that these separated gold nanowires arrays
exhibit localized surface plasmon resonances (SPR) in the
NIR spectral region, overlapping with the so-called biological window. This together with the alignment and
activation of human cells on such LIPSS structures render
our nanowires as very promising candidates for biosensing
applications.
2 Experimental
The experiments were performed on flat PS or PET foils
with a thickness of 25 or 50 lm, purchased from
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Goodfellow Ltd. Ripple structures were produced using a
KrF* excimer laser (LPX 300, Lambda Physik) at
wavelength of k = 248 nm with pulse length of 20 ns.
The repetition rate was set to 10 Hz. The light was linearly polarized by an a-BBO polarizer (Melles Griot). A
pair of fused silica lenses imaged the output of the
polarizer onto the samples, mounted on a rotatable
sample holder. We used 6,000 laser pulses with a fluence
of 10.5 and 12.5 mJ/cm2 for PET and PS, respectively.
The laser beam energy was adjusted by means of an
attenuator with a dielectric coating. The pulse energy was
measured with a pyroelectric detector (Ophir Optronics)
after the last lens. Atomic force microscope (AFM)
images were obtained with a Digital Instruments CP II
set-up operated in the tapping mode. The average
roughness Ra (calculated with the AFM software) represents the average deviations from the center plane of the
Laser-induced periodic surface on polymers
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Fig. 2 a Phase contrast microscopy image (1009 magnification) of aligned human myoblasts on LIPSS structures on PS, b AFM image of the
corresponding ripple structure
AFM image and gives a lower estimate for half of the
ripple height h.
Primary human skeletal myoblasts were isolated from
muscle biopsies. The myoblast cells and all tissue material
used here originated from clinical studies with declarations
of consent for the use of rest materials for research purposes. The myoblasts were grown in an incubator with an
appropriate culture medium. Cells were harvested by
trypsination and seeded onto PS foils, which were placed in
a six well cell culture microplate. Typically, 500,000 cells
were seeded per well. Prior to cell seeding the foils were
rinsed twice with 70 % ethanol solution. After 24 h in
culture, the cells in the well plates were investigated by a
phase contrast microscope in inverted configuration (1009
magnification), which was equipped with a CCD camera.
HMEC-1 is a well-established immortalized cell line of
human microvascular endothelial cells (HMEC) [14]. For
our experiments, HMEC-1 cells were cultured as described
in reference [12]. For immunostainings, the cells were
seeded on the different substrates and fixed and immunostained with an anti-b-catenin antibody (1:150, BD Biosciences) or an anti-VE-cadherin antibody (1:150, BD
Biosciences) after 48 h of culture. Fluorescence microscopy of the samples was performed on a Leica TCS SP5
confocal microscope.
Gold layers of 15 nm thickness were deposited with a
rate of 0.3 nm/s on the polymer samples with ripples by
thermal vacuum evaporation (Pfeiffer Vacuum Classic
500). For the evaporation, the samples were fixed on an
inclined sample holder to achieve directional evaporation
under an angle of 70° to the sample normal. The sample
position was adjusted such that the ripple orientation was
horizontal (i.e., perpendicular to the evaporation direction).
The deposited gold thickness was derived from AFM
measurements of flat gold films, as control samples.
Scanning electron microscope (SEM) images of the polymer samples with gold-wires were performed with the
Zeiss-SEM Supra 55VP. The samples were imaged without
additional conductive coating, since the gold nanowires
provide sufficient conductivity. The polarization dependent
VIS–NIR transmission spectra were measured with a
homebuilt transmission setup [15]. The normal transmission for normal incident white light was recorded with a
BWspec spectrometer for s- and p- incident polarizations,
defined relative to the gold-nanowire orientations.
3 Results and discussion
Figure 1 shows AFM images of ripples on PET and PS
surfaces, measured in tapping mode with a scan rate of
1 Hz. The PET ripples in Fig. 1a at h = 30° have a periodicity K = 310 nm and an average roughness
Ra = 32 nm. The corresponding values in Fig. 1b at
h = 45° are K = 395 nm and Ra = 37 nm. From formula
(1), we can estimate that the effective refraction index of
PET is neff(PET) & 1.32. The PS ripples in Fig. 1c at
h = 30° have K = 345 nm and Ra = 38 nm and in Fig. 1d
at h = 45° have K = 465 nm and Ra = 42 nm, which
result in an effective index of refraction for PS of
neff(PS) & 1.23. In all four sub-figures the coverage of the
surface with ripples appears quite uniform, although the
ripples do not follow strictly one orientation, but exhibit
some waves, line breaks, and Y-bifurcations. At 248 nm,
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Fig. 3 Fluorescence microscopy images of a, c fluorescence-labeled b-catenin in HMEC-1 cells compared to images of b, d fluorescencelabeled VE-cadherin on a, c flat PET and b, d PET with LIPSS structures (similar to those shown in Fig. 1a)
the refractive index of PS is n(PS) = 1.8 [16]. From
reflectivity
measurements,
we
concluded
that
n(PET) [ n(PS). This is consistent with our observed neff
values.
Figure 2a shows a phase contrast microscope image of
myoblasts grown on a PS substrate with ripples, similar to
those presented in Fig. 2b. As we have reported before [8],
the main alignment axis is most probably identical with
orientation of the underlying ripple structure within an
uncertainty of about ±10° even though the lateral dimensions of the cells are much larger than the ripples as well as
the height of the cells is much larger than the ripple height.
We have proposed that alignment of myoblasts is based on
organization of the fibrils of the cell cytoskeleton especially in protrusions of the cell. We have chosen PS here,
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since most plastic consumables used in cell culture are
made from this material. We have recently shown that
commercial Petri dishes made from PS can be covered by
laser-induced ripples leading to cell alignment [17]. Myoblasts are progenitor cells for muscle regeneration and a
parallel alignment is an important step required for myoblast fusion into multi-nucleic mature muscle cells,
which form hierarchically organized myotubes and muscle
fibers [18].
When the oriented topographic features on a polymer
surface are too small, the cells can not align along these
structures any more. For ripples, cell alignment is observed
only when the periodicity is above a critical periodicity
threshold (about 300 nm), which is cell type specific [8].
Nevertheless, we observed a strong activation of cells
Laser-induced periodic surface on polymers
299
Fig. 4 a SEM image of gold nanowires on LIPSS structures on PET
(similar to those in Fig. 1a) and b corresponding transmission spectra
obtained under normal incidence for s-polarization (black) and
p-polarization (red), respectively. Orientation of polarizations relative
to the wires is indicated by the arrows in a
grown on laser-induced ripples even or especially without
alignment [12]. This is exemplarily shown in Fig. 3 for
HMEC-1 cells grown on PET surface with laser-induced
ripples (similar to those of Fig. 1a) in comparison with
cells grown on flat PET surface. In HMEC-1 cells grown
on flat PET (Fig. 3a), the transcriptional regulator b-catenin (red-stained) is mainly located in adhesion contacts of
the plasma membrane as well as at low levels in the
cytoplasm. In contrast, b-catenin is preferentially localized
in the cell nucleus for HMEC-1 grown on PET with ripples
(Fig. 3b). This translocation is of high relevance, because
b-catenin, as a crucial component of essential signaling
pathways determining cell fate, controls quiescence of cells
or induces target gene transcription leading to proliferation
and differentiation [19]. In the first case, b-catenin forms a
key component of the cell–cell junction complex associated with VE-cadherin in endothelial cells. While in the
latter case, b-catenin is connected to transcription factors in
the nucleus regulating gene expression. As control, we
show in Fig. 3c, d that the cell membrane as well as VEcadherin localization remains intact in both situations.
Nonetheless, changes in membrane architecture induced by
the LIPSS were observed by electron microscopy [20].
Another promising application of laser-induced ripples
is the formation of separated gold nanowires due to shadowing effects during evaporation under an inclined angle
[13]. In Fig. 4a, we show a SEM image of the gold
nanowires on a PET surface, structured before gold deposition with laser-induced ripples. Figure 4b shows the
corresponding normal transmission spectra for s- and pincident polarizations, respectively. The spectra depend
strongly on the incident light polarization. For s-polarization (perpendicular to the wire axis) a minimum in transmission is observed at 775 nm, which can be attributed to
the excitations of SPR, namely to the dipolar plasmon
resonances of the nanowires [21]. For the opposite polarization, where the polarization vector of the light is parallel
to the direction of the laser-induced ripples and the gold
nanowires, there is no such pronounced resonance visible
in this spectral range, since the transmission spectra in this
case depend mainly on the grating constant. Although
similar results were reported for silver and gold nanowire
arrays prepared by e-beam lithography [21], the here presented nanowires exhibit several advantages for biosensing. The plasmon resonances are overlapping with the socalled biological window and a large area fabrication of
such wires is not only cheaper and faster than the e-beam
lithography, but also can be integrated directly on relatively large areas in plastic devices used in cell culture.
Additionally, we have strong indications that a preferential
spatial confinement of focal adhesions in the cell membrane to the ripple ridges and/or the nanowires may be
achieved [20].
In summary, our experiments demonstrated that laser
induced periodic surface structures (LIPSS) created by
nanosecond UV laser can be used for nanopatterning of
relative large areas on synthetic polymers commonly used
as cell culture substrates. The polymer structures can be
transformed into gold nanowires by means of evaporation
under an inclined angle. We see a large potential for both
types of structures, especially in the field of tissue engineering and biosensors. In the latter case we think especially of surface plasmon resonance sensors directly
integrated into biotechnological devices.
Acknowledgments The support by the Austrian Research Promotion Agency FFG by project 838955 CellStretch (J. H., R.-A. B., B.
M., R. M.) and the support by the Grant Agency of the CR under the
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project 13-06609S (P. S., V.S.) are gratefully acknowledged. We also
want to thank H. Piglmayer-Brezina for gold deposition and SEM
characterization.
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