Hydrogel film

Scanning
electron microscopy
(SEM), atomic force microscopy (AFM), rheology, and Fourier-transform
infrared spectroscopy (FTIR) were performed as previously described.^{15 (link),17 (link),18 (link),20 (link),22 (link)} For SEM, hydrogel samples were fixed in
glutaraldehyde, ethanol dehydrated, critical point dried, sputter
coated with gold/palladium, and imaged on a field emission SEM (LEO
1530VP). For AFM, the peptide solutions (0.01–0.05 wt %) were
spin-coated onto clean mica disks. Images were collected on a Dimension
Icon AFM (Bruker Instruments) in ScanAsyst mode. For rheology, 4 wt
% solution of peptide hydrogel was transferred onto an 8 mm parallel
plate geometry with a gap of 250 μm. Strain sweep (0–1000%
strain at 1 Hz) and shear recovery (1% strain at 1 Hz for 20 s, 100%
strain at 1 Hz for 60 s, and 1% strain at 1 Hz for 120 s) were performed
using an ARES-G2 rheometer (TA Instruments). For FTIR, the peptide
solutions (0.04 wt %) were pipetted onto a universal attenuated total
reflectance accessory and dried until a thin film of peptide was achieved.
Infrared spectrum was taken using a Spectrum 100 FTIR spectrometer
(PerkinElmer). For circular dichroism, we used an Olis Rapid Scanning
Monochromator to measure the ellipticity of a 0.004% peptide solution
from 190 to 250 nm in a 1 cm cuvette. The ellipticity (θ, measured
in millidegrees) was converted to molar residual ellipticity (θ)
according to the formula: (θ) = (θ·m)/(10·cln), where m is the
molecular weight of the peptide, c is the concentration
of the peptide solution in mg/mL, l is the path length
of the cuvette in centimeter, and n is the no. of
residues in the peptide sequence (similar to calculations described
before).^{25 (link)}

Hydrogel layers of thicknesses ranging between 30
and 330 nm have
been deposited in a custom-built initiated chemical vapor deposition
(iCVD) reactor. The deposition processes were run in a cylindrical
chamber (diameter 360 mm, height 55 mm), in which the pressure during
deposition is controlled by a Duo 5M rotary vane pump (Pfeiffer Vacuum,
Germany) and a throttle valve (MKS Instruments, USA). Single-sided
polished silicon wafers with a native oxide of 1.5–2 nm thickness
on top (Siegert Wafer, Germany) are used as substrates. The substrates
are positioned on the bottom of the reaction chamber, where the temperature
is set to 35 °C by an Accel 500 LC heater/chiller (Thermo Fisher
Scientific, USA). The deposited film thickness is monitored in situ by laser interferometry with a He–Ne laser
(λ = 633 nm; Thorlabs, USA) through a removable quartz glass
lid. Di-tert-butyl peroxide (TBPO, 98%; Aldrich,
Germany) is used as an initiator. TBPO is kept at room temperature
in a glass jar connected to the reaction chamber via a needle valve
(Swagelok, USA) to be able to set the desired flow rate of 1 sccm.
Twenty-five mm above the substrates, a Ni–Cr wire wound in
12 parallel lines (20 mm wire separation) functions as a heated filament
(200 °C) to cleave the initiator molecules entering the reaction
chamber. N-isopropylacrylamide (NIPAAm, 99%;
Aldrich, Germany) is used as monomer and di(ethylene glycol) divinyl
ether (DEGDVE, 99%; Aldrich, Germany) as cross-linker. NIPAAm and
DEGDVE are also kept in glass jars but heated to 85 and 70 °C,
respectively. The monomer and cross-linker vapors are flown into the
reaction chamber through a heated mixing line (90 °C). Needle
valves (Swagelok, USA) are used to set flow rates and achieve controlled
composition. Since the deposition rate depends on the individual flow
rates, substrate temperature, and working pressure, the film thickness
increase as monitored in situ by laser interferometry
was used to stop the deposition at different deposition times when
the desired thickness was achieved.
Spectroscopic ellipsometry
(SE) in a wavelength range of 370–1000
nm (M-2000S, J.A. Woollam, USA) was used to determine the film thickness
and optical properties of the thin films in a controlled environment
(nitrogen, relative humidity, and water at set temperature). A temperature
controlled liquid stage (J. A. Woollam, USA) was used for performing
swelling experiments in deionized water. The recorded data were evaluated
with an optical model consisting of a c-Si semi-infinite layer on
the bottom (temperature-dependent), a 1.6 nm thick native SiO₂ layer in the middle, and the polymer film on top. The polymer
layer was modeled with a Cauchy function, and an Urbach tail was adopted
accounting for adsorption in the low wavelength region. The surrounding
medium was set to H₂O with temperature-dependent optical
properties. For the temperature-dependent swelling experiments, the
liquid stage and the mounted sample (already exposed to deionized
water) were precooled to 10 °C. The respective signal was then
recorded while applying a temperature ramp from 10 to 50 °C at
a heating rate of 0.5 °C/min. Directly after deposition, the
thin film samples were rinsed for 30 s with deionized water for equilibration.
Despite rinsing, the first and second heating experiments showed differences
in shape and position of the transition. As equilibration has been
earlier reported to be needed for the study of temperature-dependent
behavior of iCVD thin films,^{16 (link)} the third
heating experiment was used for the determination of the LCST, as
all of the further heating ramps give similar results. This effect
was attributed to the removal of loosely attached material and the
rearrangement of polymer chains in the first couple of heating cycles
for which rinsing is not sufficient while heavier rearrangements during
cooling/heating are (especially in films exhibiting a low amount of
cross-linking). As described in detail later, the film thickness changes
after rinsing, but together with the optical properties as recorded
by SE, it has not been observed to change after the first two heating
cycles applied for equilibration purposes. This hints to structural
rearrangements occurring during equilibration that do not affect the
amount of material present on the substrate. The ellipsometry measurements
in relative humidity and N₂ atmosphere were performed in
a THMS600 temperature stage (Linkam, UK) at room temperature (∼25
°C), with the gases being supplied from a custom-built mixing
setup. An SHT15 humidity sensor (Sensirion, Switzerland) was used
to monitor the relative humidity (RH) in the sample stage in situ; the samples were measured after equilibration in
the respective environment, so that the film thickness would not change
more than 0.5 nm in 5 min. The recorded optical data have been evaluated
using the same model as that in the liquid case but with the ambient
material being set to air (n ≈ 1). Likewise,
measurements to obtain information about the available free volume
detectable with water have been carried out similar to Perrotta et
al.^{17 (link),18 (link)} Therefore, the thin film samples have been
kept under a nitrogen atmosphere at a constant temperature (25 °C),
determining their optical properties. Subsequently, water vapor has
been introduced into the system in 10% RH steps, to which the films
respond by filling free volume with H₂O. Hence, the refractive
index first increases due to water permeation, which can be understood
as a measure for free volume of the respective thin film.
X-ray
reflectivity (XRR) measurements were performed on a PANalytical
Empyrean diffractometer. The diffractometer uses a copper sealed tube,
a multilayer mirror for monochromatizing the beam (λ = 0.154
nm), a beam mask of 10 mm, and a 1/32° divergence slit on the
incident beam side. On the diffracted beam side, a receiving slit
of 0.1 mm and a 0.02 rad Soller slit were used in front of a PANalytical
PIXcel 3D detector in point detector mode. The critical angle of total
reflection was read out of the XRR patterns as the angle 2θ
slightly above the maximum intensity where the intensity drops to
half its maximum value.^{19 (link)} All of the XRR
measurements have been performed at room temperature (∼25 °C)
and at a relative humidity of ∼40%.
Absorbance spectra
of several samples were collected in transmission
mode on a Bruker IFS 66 v/s Fourier transform infrared (FTIR) spectrometer.
The measurements were run in the wavenumber range 1000–4000
cm^–1 at a resolution of 4 cm^–1 and a zero filling factor of 8.