88
4 Reactive Thin Polymer Films as Platforms for the Immobilization …
followed by a cross-section analysis. An accurate calibration of the AFM scanner
in the z-direction was ensured by using a set of three vertical calibration standards
(TGZ 01-03, Silicon-MDT, Moscow, Russia).
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).
Fluorescence Microscopy. Fluorescence micrographs were taken using a Zeiss
LSM 510 confocal fluorescence microscope with a BP 500-550 IR filter for fluoresceinamine, a LP 650 filter for Cy5-labeled target DNA and BSA conjugated
with Alexa Fluor
® 594 with a 633 nm He–Ne laser. For excitation an Ar laser (30
mW) was used. A 500–550 nm bandpass filter was selected for experiments with
fluoresceinamine and a 650 nm long-pass filter was selected for the labeled target
Cy5-labeled DNA and BSA-Alexa Fluor
® 594, respectively.
SPR and SPFS Setups. The SPR experiments were carried out in the group of Prof.
Dr. W. Knoll at the Max-Planck-Institute for Polymer Research (Mainz, Germany)
on a custom-built SPR setup [33], which is based on the configuration introduced by
Kretschmann and Raether. A refractive index of n = 1.464 was used for the polymer
film and n = 1.375 for the DNA adlayers. The SPFS setup used has been recently
described in detail by Knoll et al. [34].
Experimental Procedure for Non-Specific Adsorption of Listeria. The measurements were carried out by C. Ruengruglikit and Prof. Dr. Q. Huang (Department
of Food Science, Rutgers University, New Brunswick, NJ). At room temperature,
0.4 μl of 0.025 mg/ml protein G in 1:1 glycerol:PBS solution were pipetted on a
PNHSMA film or PEG 500 -NH 2 -coated PNHSMA film, followed by rinsing with PBS
buffer. 0.2 μl of 500 μg/ml anti-listeria antibody was spotted on top of protein G in
a dilution buffer (1:1 glycerol/PBS buffer) and was incubated for at least 24 h at 4
°C. Then the surfaces were blocked with 1% w/v BSA and were incubated at room
temperature for 2 h. Fluorescently-labeled heat-killed listeria monocytogenes cells
were prepared with 1 mg/ml EtBr in saline by mixing serially diluted cell solution
in saline (10, 100, 1000, 10000 dilution factors from stock solution). The solution
was mixed and set aside for 5 min. The cells were incubated for 40 min and unbound
cells were rinsed off with washing solution, then PBS buffer and deionized water,
respectively. Cells were detected by inverted fluorescent microscopy using a 40 ×
objective lens.
4 Reactive Thin Polymer Films as Platforms for the Immobilization …
followed by a cross-section analysis. An accurate calibration of the AFM scanner
in the z-direction was ensured by using a set of three vertical calibration standards
(TGZ 01-03, Silicon-MDT, Moscow, Russia).
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).
Fluorescence Microscopy. Fluorescence micrographs were taken using a Zeiss
LSM 510 confocal fluorescence microscope with a BP 500-550 IR filter for fluoresceinamine, a LP 650 filter for Cy5-labeled target DNA and BSA conjugated
with Alexa Fluor
® 594 with a 633 nm He–Ne laser. For excitation an Ar laser (30
mW) was used. A 500–550 nm bandpass filter was selected for experiments with
fluoresceinamine and a 650 nm long-pass filter was selected for the labeled target
Cy5-labeled DNA and BSA-Alexa Fluor
® 594, respectively.
SPR and SPFS Setups. The SPR experiments were carried out in the group of Prof.
Dr. W. Knoll at the Max-Planck-Institute for Polymer Research (Mainz, Germany)
on a custom-built SPR setup [33], which is based on the configuration introduced by
Kretschmann and Raether. A refractive index of n = 1.464 was used for the polymer
film and n = 1.375 for the DNA adlayers. The SPFS setup used has been recently
described in detail by Knoll et al. [34].
Experimental Procedure for Non-Specific Adsorption of Listeria. The measurements were carried out by C. Ruengruglikit and Prof. Dr. Q. Huang (Department
of Food Science, Rutgers University, New Brunswick, NJ). At room temperature,
0.4 μl of 0.025 mg/ml protein G in 1:1 glycerol:PBS solution were pipetted on a
PNHSMA film or PEG 500 -NH 2 -coated PNHSMA film, followed by rinsing with PBS
buffer. 0.2 μl of 500 μg/ml anti-listeria antibody was spotted on top of protein G in
a dilution buffer (1:1 glycerol/PBS buffer) and was incubated for at least 24 h at 4
°C. Then the surfaces were blocked with 1% w/v BSA and were incubated at room
temperature for 2 h. Fluorescently-labeled heat-killed listeria monocytogenes cells
were prepared with 1 mg/ml EtBr in saline by mixing serially diluted cell solution
in saline (10, 100, 1000, 10000 dilution factors from stock solution). The solution
was mixed and set aside for 5 min. The cells were incubated for 40 min and unbound
cells were rinsed off with washing solution, then PBS buffer and deionized water,
respectively. Cells were detected by inverted fluorescent microscopy using a 40 ×
objective lens.