6.7 Experimental Section
121
room temperature for 24 h in vacuum before analysis. Mean film thicknesses were
determined using a custom-built spectroscopic ellipsometer using a He-Ne laser (λ
= 632.8 nm). The refractive index of PEG was approximated as 1.4638, while for
PNHSMA 1.50 was used [30]. The film thickness was 20–40 nm.
Patterning of Films. Microcontact printing stamps were prepared according to literature procedures [31]. The stamps were mildly oxidized in an ozone plasma reactor
for 50 min and were inked by soaking them in a 200 μM solution of PEG 500 -NH 2 in
PB buffer (pH = 7.4) for 60 min. Before reactive microcontact printing, the stamps
were blown dry in a stream of nitrogen. Then the PEG 500 -NH 2 loaded stamps were
applied for different time on the PNHSMA films (the stamp, surface area 1 cm
2 , was
loaded with a mass of 30 g). After careful removal of the stamp, the film surface was
rinsed with Milli-Q water.
Coupling of Fluoresceinamine. The preformed PEG pattern was backfilled with
fluoresceinamine by immersing the patterned film inside a fluoresceinamine solution (100 μM, PB buffer: pH = 7.4) for 30 min. Then the samples were taken
out, rinsed with PB buffer (pH = 7.4) and Milli-Q water, and dried in a stream of
nitrogen. Finally, the samples were dried in vacuum for 1 day before the fluorescence
experiment.
Coupling of BSA. BSA was coupled from solution (100 μM, PB buffer: pH = 7.4;
30 min) using the same procedure as described above for fluoresceinamine.
Coupling of DNA and hybridization. DNA immobilization was carried out in
buffer (100 nM in PB buffer: pH = 7.4) on patterned PNHSMA film following the
same procedure as for fluoresceinamine. Afterwards, the hybridization of dye-labeled
target DNA (100 nM) was carried out in phosphate buffer as described previously
[25].
Fourier Transform Infrared (FTIR) Spectroscopy. Transmission mode FTIR
spectra (spectral resolution of 4 cm
−1 , 1024 scans) were obtained using a BIORAD 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.
X-ray Photoelectron Spectroscopy (XPS). XPS spectra were recorded on a PHI
Quantum 2000 Scanning ESCA microprobe using a monochromated X-ray beam
(Al-anode; 100 μm diameter/ 25 watts X-ray beam) scanned over 700 μm × 300
μm area at a fixed take-off angle of 45°. Atomic concentrations were determined
by numerical integration of the relative peak areas using the Multipak software with
supplied sensitivity factors (C 1s : 0.314; O 1s : 0.733; N 1s : 0.499) [32].
Fluorescence Microscopy. Fluorescence microscopy images of dry samples on glass
cover slips were recorded at room temperature on a Zeiss LSM 510 confocal laser
scanning microscope using a Plan-Apochromat
® 63×/1.4 NA oil-immersion objective. Fluoresceinamine was excited with the 488 nm line of an Ar
+ laser, and Cy5labeled DNA and BSA conjugated with Alexa Fluor
® 594 with a 633 nm He-Ne laser.
121
room temperature for 24 h in vacuum before analysis. Mean film thicknesses were
determined using a custom-built spectroscopic ellipsometer using a He-Ne laser (λ
= 632.8 nm). The refractive index of PEG was approximated as 1.4638, while for
PNHSMA 1.50 was used [30]. The film thickness was 20–40 nm.
Patterning of Films. Microcontact printing stamps were prepared according to literature procedures [31]. The stamps were mildly oxidized in an ozone plasma reactor
for 50 min and were inked by soaking them in a 200 μM solution of PEG 500 -NH 2 in
PB buffer (pH = 7.4) for 60 min. Before reactive microcontact printing, the stamps
were blown dry in a stream of nitrogen. Then the PEG 500 -NH 2 loaded stamps were
applied for different time on the PNHSMA films (the stamp, surface area 1 cm
2 , was
loaded with a mass of 30 g). After careful removal of the stamp, the film surface was
rinsed with Milli-Q water.
Coupling of Fluoresceinamine. The preformed PEG pattern was backfilled with
fluoresceinamine by immersing the patterned film inside a fluoresceinamine solution (100 μM, PB buffer: pH = 7.4) for 30 min. Then the samples were taken
out, rinsed with PB buffer (pH = 7.4) and Milli-Q water, and dried in a stream of
nitrogen. Finally, the samples were dried in vacuum for 1 day before the fluorescence
experiment.
Coupling of BSA. BSA was coupled from solution (100 μM, PB buffer: pH = 7.4;
30 min) using the same procedure as described above for fluoresceinamine.
Coupling of DNA and hybridization. DNA immobilization was carried out in
buffer (100 nM in PB buffer: pH = 7.4) on patterned PNHSMA film following the
same procedure as for fluoresceinamine. Afterwards, the hybridization of dye-labeled
target DNA (100 nM) was carried out in phosphate buffer as described previously
[25].
Fourier Transform Infrared (FTIR) Spectroscopy. Transmission mode FTIR
spectra (spectral resolution of 4 cm
−1 , 1024 scans) were obtained using a BIORAD 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.
X-ray Photoelectron Spectroscopy (XPS). XPS spectra were recorded on a PHI
Quantum 2000 Scanning ESCA microprobe using a monochromated X-ray beam
(Al-anode; 100 μm diameter/ 25 watts X-ray beam) scanned over 700 μm × 300
μm area at a fixed take-off angle of 45°. Atomic concentrations were determined
by numerical integration of the relative peak areas using the Multipak software with
supplied sensitivity factors (C 1s : 0.314; O 1s : 0.733; N 1s : 0.499) [32].
Fluorescence Microscopy. Fluorescence microscopy images of dry samples on glass
cover slips were recorded at room temperature on a Zeiss LSM 510 confocal laser
scanning microscope using a Plan-Apochromat
® 63×/1.4 NA oil-immersion objective. Fluoresceinamine was excited with the 488 nm line of an Ar
+ laser, and Cy5labeled DNA and BSA conjugated with Alexa Fluor
® 594 with a 633 nm He-Ne laser.