180
9 Bioinspired Hierarchically Structured Polymer Interfaces …
X-100 were purchased from VWR. Anti-paxillin, anti-mouse IgG Alexa 488 and
Phalloidin-Rhodamin were purchased from Invitrongen, Life Technologies. Hoechst
33,258 and Fluorescein diacetate (FDA) were purchased from Sigma-Aldrich, and
propidium iodide (PI) was purchased from Carl Roth. Throughout the whole study,
Milli-Q water drawn from a Millipore Direct Q8 system (Millipore Advantage A10
system, Schwalbach, with Millimark Express 40 filter, Merck, Germany) with a
resistivity 18.2 M cm was used.
Preparation of Hierarchically Structured Surfaces. For fabrication of inverse rose
petal structures with concave curvature, the PDMS precursor mixed with the curing
agent (10:1) was poured on the untreated fresh rose petals and cured at 70 °C for 2 h.
After rose petals embedded into PDMS completely drying, they became crisp and
could be removed from the PDMS surface by slightly bending PDMS. To replicate
the rose petals (convexly curved microstructures), aqueous PVA solution (10 wt%)
was poured onto the fresh rose petals and was exposed to ambient air at 25 °C.
When the water was evaporated, the PVA film was peeled off affording inverse rose
petal structures. Then, the PVA mold was replicated with PDMS following the recipe
shown above.
X-ray Photoelectron Spectroscopy. The different steps of the surface modification were analyzed on cured PDMS thin films obtained by spin-coating on Si
wafers (Siegert Wafers, type/dopant: P/B, resistivity: 1–10 cm). The samples
were analyzed after each step of modification by XPS (S-Probe ESCA SSX-100 s,
Surface Science Instruments, USA) using Al Kα radiation of 200 W. The spectra
were analyzed using Casa XPS processing software.
Cell Capture and Staining. The PaTu 8988t cell line was obtained as a kind gift
from Dr. Jürgen Schnekenburger (Biomedical Technology Center of Medical Faculty
Münster, Germany). The MCF7 cell line was purchased from ATCC (ATCC HTB22). The SaOS-2 cell line was obtained as a kind gift from Dr. Ulrike Ritz (University
Medical Center of the Johannes Gutenberg University Mainz, Germany). The PDMS
substrates (1 cm × 1 cm) were placed into a 24-well cell culture plate and then 1 ml
of cell suspension (1 × 10
5 cells ml
−1 ) was pipetted into each well. After incubating at 37 °C and 5% CO 2 in a cell incubator, the substrates were gently rinsed
with PBS buffer. The captured cells were stained by Hoechst 33,258 and counted
by Image J software as follows. Firstly, the cells were fixed with 4% paraformaldehyde (PFA) in PBS buffer for 30 min, followed by washing three times with PBS
buffer. Secondly, the substrates were incubated with 0.5% Triton X-100 in PBS buffer
for 10 min to increase cellular permeability for intracellular staining, followed by
washing three times with PBS buffer. Thirdly, Hoechst 33,258 solution (1 μg ml
−1 )
was dropped onto the substrates for 15 min to stain the captured cells, followed by
washing three times with PBS buffer. Finally, we imaged the cells using a fluorescence microscope (Axiovert 135), and counted them by using Image J software. For
fluorescent imaging, all the PDMS substrates were placed upside down on the glass
slides. Finally, we imaged the cells using a fluorescence microscope (Axiovert 135,
Carl Zeiss, Oberkochen, Germany), and counted them by using Image J software.
9 Bioinspired Hierarchically Structured Polymer Interfaces …
X-100 were purchased from VWR. Anti-paxillin, anti-mouse IgG Alexa 488 and
Phalloidin-Rhodamin were purchased from Invitrongen, Life Technologies. Hoechst
33,258 and Fluorescein diacetate (FDA) were purchased from Sigma-Aldrich, and
propidium iodide (PI) was purchased from Carl Roth. Throughout the whole study,
Milli-Q water drawn from a Millipore Direct Q8 system (Millipore Advantage A10
system, Schwalbach, with Millimark Express 40 filter, Merck, Germany) with a
resistivity 18.2 M cm was used.
Preparation of Hierarchically Structured Surfaces. For fabrication of inverse rose
petal structures with concave curvature, the PDMS precursor mixed with the curing
agent (10:1) was poured on the untreated fresh rose petals and cured at 70 °C for 2 h.
After rose petals embedded into PDMS completely drying, they became crisp and
could be removed from the PDMS surface by slightly bending PDMS. To replicate
the rose petals (convexly curved microstructures), aqueous PVA solution (10 wt%)
was poured onto the fresh rose petals and was exposed to ambient air at 25 °C.
When the water was evaporated, the PVA film was peeled off affording inverse rose
petal structures. Then, the PVA mold was replicated with PDMS following the recipe
shown above.
X-ray Photoelectron Spectroscopy. The different steps of the surface modification were analyzed on cured PDMS thin films obtained by spin-coating on Si
wafers (Siegert Wafers, type/dopant: P/B, resistivity: 1–10 cm). The samples
were analyzed after each step of modification by XPS (S-Probe ESCA SSX-100 s,
Surface Science Instruments, USA) using Al Kα radiation of 200 W. The spectra
were analyzed using Casa XPS processing software.
Cell Capture and Staining. The PaTu 8988t cell line was obtained as a kind gift
from Dr. Jürgen Schnekenburger (Biomedical Technology Center of Medical Faculty
Münster, Germany). The MCF7 cell line was purchased from ATCC (ATCC HTB22). The SaOS-2 cell line was obtained as a kind gift from Dr. Ulrike Ritz (University
Medical Center of the Johannes Gutenberg University Mainz, Germany). The PDMS
substrates (1 cm × 1 cm) were placed into a 24-well cell culture plate and then 1 ml
of cell suspension (1 × 10
5 cells ml
−1 ) was pipetted into each well. After incubating at 37 °C and 5% CO 2 in a cell incubator, the substrates were gently rinsed
with PBS buffer. The captured cells were stained by Hoechst 33,258 and counted
by Image J software as follows. Firstly, the cells were fixed with 4% paraformaldehyde (PFA) in PBS buffer for 30 min, followed by washing three times with PBS
buffer. Secondly, the substrates were incubated with 0.5% Triton X-100 in PBS buffer
for 10 min to increase cellular permeability for intracellular staining, followed by
washing three times with PBS buffer. Thirdly, Hoechst 33,258 solution (1 μg ml
−1 )
was dropped onto the substrates for 15 min to stain the captured cells, followed by
washing three times with PBS buffer. Finally, we imaged the cells using a fluorescence microscope (Axiovert 135), and counted them by using Image J software. For
fluorescent imaging, all the PDMS substrates were placed upside down on the glass
slides. Finally, we imaged the cells using a fluorescence microscope (Axiovert 135,
Carl Zeiss, Oberkochen, Germany), and counted them by using Image J software.