4.6 Experimental Section
87
using a P6700 spin-coater (Specialty Coating Systems Inc). All spin-coated samples
were dried at room temperature for 24 h in vacuum before analysis. The absence
of residual solvent was verified by IR spectroscopy. Mean film thicknesses were
determined using a custom-built spectroscopic ellipsometer using a He–Ne laser (λ
= 632.8 nm). The refractive index of the PEG films was approximated as 1.4638,
while for PNHSMA and NHS-C 10 1.50 and 1.45, respectively, were utilized [32].
PEG 500 -NH 2 Coupling and Studies of Kinetics. PEG 500 -NH 2 solutions in PB buffer
(pH = 7.4; ionic strength: 0.078 M) were prepared in freshly cleaned glassware with
concentrations between 1.0 × 10
−4 and 4.0 × 10
−4 M. The polymer films and freshly
rinsed SAMs were immersed into the corresponding PEG 500 -NH 2 solution. After a
reaction time between 5 min and 10 h the samples were taken out of the corresponding
solution and were thoroughly rinsed with Milli-Q water. All experiments were carried
out at T = 25 ± 2 °C.
Fluoresceinamine Coupling. The polymer films on glass or Si wafer were immersed
into a fluoresceinamine solution in PB buffer (pH = 7.4) (2.0 × 10
−4 M). After a
reaction time between 5 min and 1 h the samples were taken out of solution and were
thoroughly rinsed with PB buffer, and then Milli-Q water. All experiments were
carried out at T = 25 ± 2 °C.
DNA Immobilization and Hybridization. DNA solutions in phosphate buffer (PB)
were prepared with a concentration of 1.0 × 10
−7 M (pH = 7.4). For covalent
coupling to surface-bound reactive ester groups, the polymer films were immersed
into the probe DNA solution. After a reaction time of 60 min, the samples were taken
out and rinsed with PB buffer (pH = 7.4). For hybridization, the films were placed
in the target DNA solution in PB (T = 25 °C, pH = 7.4), and after 30 min the films
were taken out followed by a thorough rinse with PB buffer, and then Milli-Q water.
Fourier Transform Infrared (FTIR) Spectroscopy. Transmission mode FTIR
spectra (spectral resolution of 4 cm
−1 , 1024 scans) were obtained using a BIO-RAD
model FTS575C FTIR spectrometer equipped with a liquid nitrogen-cooled cryogenic mercury cadmium telluride (MCT) detector. Background spectra were obtained
using oxygen plasma-cleaned silicon wafers. The grazing incidence reflection FTIR
spectra of SAMs on gold were collected using a GIR accessory (BIO-RAD) at an
angle of incidence of 87° relative to the surface normal.
Atomic Force Microscopy (AFM). The AFM measurements were carried out in
intermittent contact (tapping) mode using a NanoScope X multimode AFM (Digital
Instruments/Veeco, Santa Barbara, CA) equipped with a 100 μm scanner. Microfabricated silicon tips/cantilevers (Nanosensors, Wetzlar, Germany) were used in
ambient atmosphere (ca. 30% relative humidity, 24 °C temperature) as described in
Chap. 4. For the determination of the film thicknesses by profilometry, the film was
removed by scratching the samples using sharp tweezers (width of scratch between
15 and 30 μm). Surfaces of films that were immersed in buffer for swelling (60 min)
were quickly dried in a stream of nitrogen before scratching and subsequent AFM
analysis. The captured AFM height images were subjected to a first-order plane fit
87
using a P6700 spin-coater (Specialty Coating Systems Inc). All spin-coated samples
were dried at room temperature for 24 h in vacuum before analysis. The absence
of residual solvent was verified by IR spectroscopy. Mean film thicknesses were
determined using a custom-built spectroscopic ellipsometer using a He–Ne laser (λ
= 632.8 nm). The refractive index of the PEG films was approximated as 1.4638,
while for PNHSMA and NHS-C 10 1.50 and 1.45, respectively, were utilized [32].
PEG 500 -NH 2 Coupling and Studies of Kinetics. PEG 500 -NH 2 solutions in PB buffer
(pH = 7.4; ionic strength: 0.078 M) were prepared in freshly cleaned glassware with
concentrations between 1.0 × 10
−4 and 4.0 × 10
−4 M. The polymer films and freshly
rinsed SAMs were immersed into the corresponding PEG 500 -NH 2 solution. After a
reaction time between 5 min and 10 h the samples were taken out of the corresponding
solution and were thoroughly rinsed with Milli-Q water. All experiments were carried
out at T = 25 ± 2 °C.
Fluoresceinamine Coupling. The polymer films on glass or Si wafer were immersed
into a fluoresceinamine solution in PB buffer (pH = 7.4) (2.0 × 10
−4 M). After a
reaction time between 5 min and 1 h the samples were taken out of solution and were
thoroughly rinsed with PB buffer, and then Milli-Q water. All experiments were
carried out at T = 25 ± 2 °C.
DNA Immobilization and Hybridization. DNA solutions in phosphate buffer (PB)
were prepared with a concentration of 1.0 × 10
−7 M (pH = 7.4). For covalent
coupling to surface-bound reactive ester groups, the polymer films were immersed
into the probe DNA solution. After a reaction time of 60 min, the samples were taken
out and rinsed with PB buffer (pH = 7.4). For hybridization, the films were placed
in the target DNA solution in PB (T = 25 °C, pH = 7.4), and after 30 min the films
were taken out followed by a thorough rinse with PB buffer, and then Milli-Q water.
Fourier Transform Infrared (FTIR) Spectroscopy. Transmission mode FTIR
spectra (spectral resolution of 4 cm
−1 , 1024 scans) were obtained using a BIO-RAD
model FTS575C FTIR spectrometer equipped with a liquid nitrogen-cooled cryogenic mercury cadmium telluride (MCT) detector. Background spectra were obtained
using oxygen plasma-cleaned silicon wafers. The grazing incidence reflection FTIR
spectra of SAMs on gold were collected using a GIR accessory (BIO-RAD) at an
angle of incidence of 87° relative to the surface normal.
Atomic Force Microscopy (AFM). The AFM measurements were carried out in
intermittent contact (tapping) mode using a NanoScope X multimode AFM (Digital
Instruments/Veeco, Santa Barbara, CA) equipped with a 100 μm scanner. Microfabricated silicon tips/cantilevers (Nanosensors, Wetzlar, Germany) were used in
ambient atmosphere (ca. 30% relative humidity, 24 °C temperature) as described in
Chap. 4. For the determination of the film thicknesses by profilometry, the film was
removed by scratching the samples using sharp tweezers (width of scratch between
15 and 30 μm). Surfaces of films that were immersed in buffer for swelling (60 min)
were quickly dried in a stream of nitrogen before scratching and subsequent AFM
analysis. The captured AFM height images were subjected to a first-order plane fit