7.5 Experimental Section
145
thoroughly rinsed with Milli-Q water. For PEG 500 NH 2 patterning on functionalized
polymer film, the oxidized stamps were inked by soaking them into 200 μM PEG
solution for 60 min (PB, pH = 7.4). Before printing, the stamps were blown dry in
a stream of nitrogen. The stamps were applied on functionalized polymer film, then
carefully removed, and rinsed with Milli-Q water. The PEG patterned films were
immersed in fluoresceinamine solution (100 μM, PB: pH = 7.4) or BSA solution
(200 μM, PB: pH = 7.4). Then the samples were taken out, rinsed with PB and
Milli-Q water, and dried in a stream of nitrogen. Finally, the samples were dried
inside a vacuum oven for 1 day prior to the fluorescence microscopy experiments.
DNA immobilization (100 nM) in phosphate buffer on PEG 500 NH 2 patterned films
was carried out as described for coupling fluoresceinamine or BSA. Afterwards, the
hybridization of target DNA with dye (100 nM) was carried out in phosphate buffer.
Then the films were rinsed with PB (pH = 7.4) and Milli-Q water, and dried in a
stream of nitrogen. All experiments were carried out at T = 25 ± 2°.
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.
The fluorescence emission of these dyes was recorded with photomultiplier tubes
(Hamamatsu R6357) after spectral filtering with a 500–550 nm bandpass filter and a
650 nm long-pass filter for fluoresceinamine and Cy5-labeled DNA and BSA-Alexa
Fluor
® 594, respectively. Images were acquired with maximum pinhole diameters.
Atomic Force Microscopy (AFM). The contact mode AFM measurements were
carried out with a NanoScope X multimode AFM (Digital Instruments/Veeco, Santa
Barbara, CA) using a 100 μm scanner and microfabricated silicon tips/cantilevers
(Nanosensors, Wetzlar, Germany) in ambient atmosphere (ca. 30% relative humidity,
24 °C temperature) as described in Chap. 4.
References
1. Veiseh M, Zareie MH, Zhang MQ (2002) Highly selective protein patterning on gold-silicon
for biosensor applications. Langmuir 18:6671–6678
2. Prime KL, Whitesides GM (1991) Self-assembled organic monolayers-model systems for
studying adsorption of proteins at surfaces. Science 252:1164–1167
3. O’Neill C, Jordan P, Riddle P, Ireland G (1990) Narrow linear strips of adhesive substratum
are powerful induces of both growth and total focal contact area. Cell Sci 95:577–586
4. Hyun J, Zhu YJ, Liebmann-Vinson A, Beebe TP, Chilkoti A (2001) Microstamping on an activated polymer surface: Patterning biotin and streptavidin onto common polymeric biomaterials.
Langmuir 17:6358–6367
5. Bhatia SN, Yarmush ML, Toner MJ (1997) Controlling cell interactions by micropatterning in
co-cultures: Hepatocytes and 3T3 fibroblasts. Biomed Mater Res 34:189–199
145
thoroughly rinsed with Milli-Q water. For PEG 500 NH 2 patterning on functionalized
polymer film, the oxidized stamps were inked by soaking them into 200 μM PEG
solution for 60 min (PB, pH = 7.4). Before printing, the stamps were blown dry in
a stream of nitrogen. The stamps were applied on functionalized polymer film, then
carefully removed, and rinsed with Milli-Q water. The PEG patterned films were
immersed in fluoresceinamine solution (100 μM, PB: pH = 7.4) or BSA solution
(200 μM, PB: pH = 7.4). Then the samples were taken out, rinsed with PB and
Milli-Q water, and dried in a stream of nitrogen. Finally, the samples were dried
inside a vacuum oven for 1 day prior to the fluorescence microscopy experiments.
DNA immobilization (100 nM) in phosphate buffer on PEG 500 NH 2 patterned films
was carried out as described for coupling fluoresceinamine or BSA. Afterwards, the
hybridization of target DNA with dye (100 nM) was carried out in phosphate buffer.
Then the films were rinsed with PB (pH = 7.4) and Milli-Q water, and dried in a
stream of nitrogen. All experiments were carried out at T = 25 ± 2°.
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.
The fluorescence emission of these dyes was recorded with photomultiplier tubes
(Hamamatsu R6357) after spectral filtering with a 500–550 nm bandpass filter and a
650 nm long-pass filter for fluoresceinamine and Cy5-labeled DNA and BSA-Alexa
Fluor
® 594, respectively. Images were acquired with maximum pinhole diameters.
Atomic Force Microscopy (AFM). The contact mode AFM measurements were
carried out with a NanoScope X multimode AFM (Digital Instruments/Veeco, Santa
Barbara, CA) using a 100 μm scanner and microfabricated silicon tips/cantilevers
(Nanosensors, Wetzlar, Germany) in ambient atmosphere (ca. 30% relative humidity,
24 °C temperature) as described in Chap. 4.
References
1. Veiseh M, Zareie MH, Zhang MQ (2002) Highly selective protein patterning on gold-silicon
for biosensor applications. Langmuir 18:6671–6678
2. Prime KL, Whitesides GM (1991) Self-assembled organic monolayers-model systems for
studying adsorption of proteins at surfaces. Science 252:1164–1167
3. O’Neill C, Jordan P, Riddle P, Ireland G (1990) Narrow linear strips of adhesive substratum
are powerful induces of both growth and total focal contact area. Cell Sci 95:577–586
4. Hyun J, Zhu YJ, Liebmann-Vinson A, Beebe TP, Chilkoti A (2001) Microstamping on an activated polymer surface: Patterning biotin and streptavidin onto common polymeric biomaterials.
Langmuir 17:6358–6367
5. Bhatia SN, Yarmush ML, Toner MJ (1997) Controlling cell interactions by micropatterning in
co-cultures: Hepatocytes and 3T3 fibroblasts. Biomed Mater Res 34:189–199