Fabrication of bio-arrays with vacuum ultraviolet light
Laser fabrication bioarrays at 157 nm in three steps.
The bovine serum albumin (BSA)-polystyrene (PS) interface layer is laser photo activated at 157 nm for site selective multiple target-protein immobilization. The 5-15 nm photon induced interface layer has different chemical, wetting and stiffness properties than the PS photon processed surface. The irradiated areas exhibit target-protein binding, followed by localized probe-target protein detection.
The photon induced chemical modification of the BSA-PS interface layer is identified by:
(1) Morphological, imaging and analysis of surface parameters with Atomic Force Microscopy (AFM).
(2) Spectroscopic shift (4 cm-1), of the amide I group and formation of new C=N, NH2, C-O, C=O, O-C=O groups following irradiation, identified with Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy.
(3) The different hydrophilic/hydrophobic and force-distance response of the bare PS and BSA-PS surfaces.
Near field edge diffraction (Fresnel) fluorescence imaging specifies the threshold photon energy and the fluence required to detect optically the protein detection on the photon induced BSA-PS interface layer. By approximating the Fresnel integrals with analytical functions, the threshold photon energy and the fluence are expressed as the sum of zero, first and second order harmonic terms of two characteristic diffracted modes and they are specified to be 8.73x10-9 J and 623 Jm-2 respectively. Furthermore, a bioarray of three probe-target proteins is fabricated with 1.5 micron spatial resolution using an 157 nm laser microstepper. The methodology eliminates the use of intermediate polymer layers between the blocking BSA protein and the PS substrate in bioarray fabrication.
Simplified schematic diagram indicating the photo dissociation 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).
(a) AFM image of PS surface.
(b) AFM image of irradiated PS surface with 20 laser pulses
(a) AFM image of BSA nano composites layered on PS.
(b) AFM image of BSA irradiated with 2 laser pulses.
(c) AFM image of BSA layer irradiated with 20 laser pulses.
Surface parameters of bare PS and BSA-PS with the number of laser pulses.
Area RMS of (black) PS and (red) BSA.
(green) Area average roughness of PS & (blue) BSA.
(light blue) Average height of PS & (magenta) BSA.
(a) AFM image of 3-5 nm thick, 30 nm wide non irradiated 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 x 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.
Left: ATR-FTIR spectrum of non- irradiated and irradiated with 20 laser pulses bare PS substrate from 539 to 5000 cm-1.
RIght:ATR-FTIR spectrum of the aromatic C-H stretching vibration mode at 3082.1 cm-1 of the PS substrate irradiated with 1 (1p), 10(10p) and 20(20p) laser pulses respectively.
Left: 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.
Right: ATR-FTIR spectrum of the bare PS substrate from 1500-2300 cm-1irradiated with 1(1p),10(10p) and 20(20p) laser pulses respectively. The peaks at 1543, 2101.1, 2336.2, 2361 cm-1 correspond to the presence of NH2 from the scission of [-N=C=N-] and C-N stressing modes respectively.
Left:ATR-FTIR spectrum of the 1635-1700 cm-1 band of BSA -PS following irradiation with 0 (BSA-PS non exposed), 1(1p), 2(2p), 10(10p), 20(20p) laser pulses.
Right: ATR-FTIR spectrum of the 3200-3370 cm-1 band of BSA-PS with similar response of the band's peak at 3300 cm-1.
Schematic lay-out 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, P1 : Image recording position.
Near field (Fresnel) edge diffraction pattern following laser irradiation with a non-focused laser beam with 1 (a), 5 (b), 10 (c) and 20 (d) 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 from the metallic rectangular aperture in agreement with the experimental images 11(a-c).
(f) First order simulated pattern.
Intensity distribution of the diffracted mode (n,0) as a function of the dimensionless parameter u.
(a) Schematic lay-out 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, [bionytilated- BSA (target), streptavidin (probe) labelled with AlexaFluor 546 (red)], following laser irradiation with non-focused laser beam with 2 laser pulses.
(c) Simulated 2-D Fresnel diffraction pattern from the metallic rectangular aperture.
Correlation between the water contact angle of bare PS (black) and BSA-PS substrates (red) with the number of laser pulses.
Young's modulus of the bare PS substrate as a function of the number of laser pulses.
(a) Force distance response of PS.
(b) Force distance response of PS irradiated with 20 laser pulses. The Young's modulus of the non-irradiated/irradiated PS areas is 2.6±0.2 GPa and 11±3 GPa (20 pulses) respectively.
(c) Force distance response of BSA layered on PS.
(d) 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.
Image of bio-array with three different proteins (red, green, blue) fabricated with the automated laser micro-stepper with one, two and five laser pulses.
Left:Intensity distribution of the fluorescence image taken with a CCD camera across one micro-spot, from one to thirty laser pulses.
Right:Intensity distribution of the fluorescence image taken with a CCD camera across one micro-spot, fabricated with 40 to 1080 different laser pulses.
Protein immobilization and detection on laser processed polystyrene surfaces.
E. Sarantopoulou, P. S. Petrou, Z. Kollia, D. Palles, N. Spyropoulos-Antonakakis,
S. Kakabakos and A. C. Cefalas,
J. Appl. Phys. 110(6), 064309 (2011). DOI:10.1063/1.3627160
157nm laser ablation of polymeric layers for fabrication of biomolecule microarrays.
A.M. Douvas, P.S. Petrou, S.E. Kakabakos, K. Misiakos, P. Argitis, E. Sarantopoulou,
Z. Kollia and A.C. Cefalas
Anal. Bioanal. Chem., 381 (5), 1027 (2005). DOI:10.1007/s00216-004-2985-3
Nano -engineering of bio-arrays at 157 nm.
A.C. Cefalas*, E. Sarantopoulou, Z. Kollia, P. Petrou and S. Kakabakos,
11th Trends in Nanotechnology International Conference, TNT 2010, Braga,
Portugal, 06-10 September, 2010.