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Protein immobilization and detection on laser processed polystyrene surfaces
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10.1063/1.3627160
/content/aip/journal/jap/110/6/10.1063/1.3627160
http://aip.metastore.ingenta.com/content/aip/journal/jap/110/6/10.1063/1.3627160

Figures

Image of FIG. 1.
FIG. 1.

Simplified schematic diagram indicating the photodissociation path from an excited bound electronic state (W2(AB)*), correlated with a repulsive state (W3(A**+B**)), following VUV irradiation of an organic molecule (AB).

Image of FIG. 2.
FIG. 2.

(Color online) (a) AFM image of PS surface. (b) AFM image of irradiated PS surface with 20 laser pulses ( per pulse). The surface indicates major morphological changes following VUV irradiation.

Image of FIG. 3.
FIG. 3.

(Color online) (a) AFM image of BSA nano composites layered on PS. (b) AFM image of BSA irradiated with 2 laser pulses ( per pulse). (c) AFM image of BSA layer irradiated with 20 laser pulses. The BSA layer is removed by photodissociation. The surface morphology of the irradiated BSA-PS interface layer indicates similar features with the irradiated bare PS surface.

Image of FIG. 4.
FIG. 4.

(Color online) (a) AFM image of 3-5 nm thick, 30 nm wide nonirradiated BSA agglomerations. (b) Height distribution histogram of the BSA agglomerations consisting of two broad bands at 3.14 and 5.5 nm, respectively, for a 600 nm × 600 nm scan area. (c) Line profile analysis of BSA agglomerations for a 600 nm scan where a 30 nm wide protein nano composite is shown.

Image of FIG. 5.
FIG. 5.

(Color online) Surface parameters of bare PS and BSA-PS with the number of laser pulses. (▪) Area RMS (root mean square roughness) of PS. (•) Area RMS of BSA. (▴) Area average roughness of PS. (▾) Area average roughness of BSA. (♦) Average height of PS. (◂) Average height of BSA. The increment of the differences between the surface parameters of PS and BSA-PS with the laser energy confirms the formation of an interface layer, 5-15 nm thick, on PS following BSA photodissociation.

Image of FIG. 6.
FIG. 6.

(Color online) (a) ATR-FTIR spectrum of the 1635-1700 cm−1 band of BSA-PS following irradiation with 0 p, 1(1 p), 2 (2 p), 10 (10 p), and 20 (20 p) laser pulses. The 4 cm−1 shift, following irradiation with two laser pulses and the fixed position of the peak after prolonged irradiation, suggests photodissociation of the 3-5 nm BSA layer. The arrows indicate the position of the C=O bonds. (b) ATR-FTIR spectrum of the 3200-3370 cm−1 band of BSA-PS with a similar response of the band’s peak at 3300 cm−1.

Image of FIG. 7.
FIG. 7.

(Color online) ATR-FTIR spectrum of the non-irradiated and irradiated with 20 laser pulses bare PS substrate from 539 to 5000 cm−1. The two spectra coincide.

Image of FIG. 8.
FIG. 8.

(Color online) ATR-FTIR spectrum of the aromatic C-H stretching vibration mode at 3082.1 cm−1 of the PS substrate irradiated with 1 (1 p), 10 (10 p), and 20 (20 p) laser pulses, respectively. The fixed position of the peak confirms the instrumental stability. The spectra are normalized using the spectral range from 3070 to 3230 cm−1.

Image of FIG. 9.
FIG. 9.

(Color online) ATR-FTIR spectrum of the 1701.6 cm−1 band of bare PS with 0(0p), 1(1p), 10(10p), and 20(20p) laser pulses, indicating activation of the C=O group by atmospheric oxygen after irradiation. The spectra are normalized using the spectral range from 1490 to 1630 cm−1.

Image of FIG. 10.
FIG. 10.

(Color online) ATR-FTIR spectrum of the bare PS substrate from 1500-2300 cm−1 irradiated with 1(1 p), 10 (10 p), and 20 (20 p) laser pulses, respectively. The peaks at 1543, 2101.1, 2336.2, and 2361 cm−1 correspond to the presence of NH2 from the scission of [-N=C=N-] and C-N stressing modes, respectively. The formation of the NH II amide group is difficult to be identified since the vibrational spectra from 1500 to 1560 and from 1590 to 1650 cm−1 are overlapping with the phenyl ring band. The spectra are normalized using the spectral range from 1490 to 1630 cm−1.

Image of FIG. 11.
FIG. 11.

(Color) (a)-(d) Near field (Fresnel) edge diffraction pattern following laser irradiation with a non-focused laser beam with (a) 1, (b) 5, (c) 10, and (d) 20 laser pulses, respectively. Ten diffracted modes are developed along each one of the perpendicular directions of the two axes in the image plane. (e) Simulated 2-D Fresnel edge diffraction pattern along the axes (exact solution, Eq. (A11)) from the 100 μm × 100 μm metallic rectangular aperture in agreement with the experimental images (a)-(c). (f) First order simulated pattern, (Eq. (A16)).

Image of FIG. 12.
FIG. 12.

(Color online) Schematic layout of the diffraction geometry used in this experiment. AP: aperture plane, IP: image plane, P: virtual (apparent) position of the light source, O: origin of the coordinate system for aperture diffraction, : image recording position.

Image of FIG. 13.
FIG. 13.

(Color online) Intensity distribution of the diffracted mode as a function of the dimensionless parameter, . Calculations are for ten diffracted modes along the (or) axis. The results are in agreement with the diffraction pattern of Fig. 11. Upper, lower, and middle traces correspond to the second, exact numerical solution, and first order approximation (Eqs. (A15), (A11), and (A16), respectively).

Image of FIG. 14.
FIG. 14.

(Color) (a) Schematic layout of the near field (Fresnel) edge diffraction pattern formation following laser irradiation with a non-focused laser beam. (b) Near field (Fresnel) edge diffraction pattern [biotinylated-BSA (target), streptavidin (probe) labeled with AlexaFluor 546 (red)], following laser irradiation with a non-focused laser beam with 2 laser pulses. Five diffracted modes along each direction are developed. The size of the array is 100 μm × 100 μm. (c) Simulated 2-D Fresnel diffraction pattern (Eq. (A15)) from the metallic rectangular aperture. The simulated pattern is using the analytical approximation of the Fresnel integrals to second order harmonic terms, in agreement with the experimental results, shown in image (a).

Image of FIG. 15.
FIG. 15.

(Color online) Correlation between the water contact angle of bare PS (•) and BSA–PS substrates (▪) with the number of laser pulses ( per pulse).

Image of FIG. 16.
FIG. 16.

(Color online) (a) Force distance response of PS. (b) Force distance response of PS irradiated with 20 laser pulses ( per pulse). The Young’s modulus of the non-irradiated/irradiated PS areas is 2.6 ± 0.2 GPa and 11 ± 3 GPa (20 pulses), respectively.

Image of FIG. 17.
FIG. 17.

(Color online) Young’s modulus of the bare PS substrate and BSA-PS as a function of the number of laser pulses ( per pulse). The large increment of the Young’s modulus following irradiation with ten laser pulses indicates excessive carbonization of the surface layer, as specified from the penetration depth of the 157 nm photons.

Image of FIG. 18.
FIG. 18.

(Color online) (a) Force distance response of BSA layered on PS. (b) Force distance response of BSA on PS irradiated with 20 laser pulses. The Young’s modulus of the non-irradiated/ irradiated BSA-PS system is 1.2 ± 0.3 and 14 ± 5.0 GPa (20 pulses), respectively, indicating the formation of the interface layer with different functionality, in agreement with Figs. 3 and 5.

Image of FIG. 19.
FIG. 19.

(Color) Schematic lay-out of the repetitive steps of the bio-array fabrication. (a) Formation of the blocking BSA layer, followed by laser fabrication of the first spot array and immobilization of the IgG rabbit target. (b) Laser fabrication of the second spot array and immobilization of the biotinylated BSA target. (c) Laser fabrication of the third spot array and immobilization of the third IgG mouse target followed by the selective bio-targeting.

Image of FIG. 20.
FIG. 20.

(Color) Image of bioarray with three different proteins (red, green, blue) fabricated with the automated laser microstepper with one, two, and five laser pulses.

Image of FIG. 21.
FIG. 21.

(Color online) Intensity distribution of the fluorescence image taken with a CCD camera across one micro-spot, from one to thirty laser pulses.

Image of FIG. 22.
FIG. 22.

(Color online) Intensity distribution of the fluorescence image taken with a CCD camera across one micro-spot, fabricated with 40 to 1080 different laser pulses.

Tables

Generic image for table
Table I.

Virtual position, , of wavefronts for ten diffracted modes, , and the corresponding position at the aperture and image planes in the real and dimensionless spaces.

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/content/aip/journal/jap/110/6/10.1063/1.3627160
2011-09-19
2014-04-24
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Protein immobilization and detection on laser processed polystyrene surfaces
http://aip.metastore.ingenta.com/content/aip/journal/jap/110/6/10.1063/1.3627160
10.1063/1.3627160
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