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Biomolecular Interaction Analysis of Gestrinone-anti-Gestrinone Using Arrays of High Aspect Ratio SU-8 Nanopillars.

Ortega FJ, Bañuls MJ, Sanza FJ, Casquel R, Laguna MF, Holgado M, López-Romero D, Barrios CA, Maquieira Á, Puchades R - Biosensors (Basel) (2012)

Bottom Line: After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed.The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored.The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).

View Article: PubMed Central - PubMed

Affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico, Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, Valencia 46022, Spain. fraorhi1@upvnet.upv.es.

ABSTRACT
In this paper, label-free biosensing for antibody screening by periodic lattices of high-aspect ratio SU-8 nano-pillars (BICELLs) is presented. As a demonstration, the determination of anti-gestrinone antibodies from whole rabbit serum is carried out, and for the first time, the dissociation constant (KD = 6 nM) of antigen-antibody recognition process is calculated using this sensing system. After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed. The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored. The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).

No MeSH data available.


Related in: MedlinePlus

Scheme of the procedure employed to test the selective antibody immobilization on SU-8 planar chips using microarrays and Cy5 labeling.
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Related In: Results  -  Collection

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biosensors-02-00291-f002: Scheme of the procedure employed to test the selective antibody immobilization on SU-8 planar chips using microarrays and Cy5 labeling.

Mentions: With regard to the bioreceptor surface attachment, the epoxy groups in the SU-8 polymer allow the direct protein immobilization by the nucleophilic attack of the protein amine moieties. Thus, the first stage was to study the immobilization process. To evaluate the immobilization and biorecognition processes, planar chips consisting of glass slides coated with a layer of SU-8 photoresist were employed because our previous experience with BICELLs showed that the protocols developed in planar substrates constituted a good starting point to be applied later in the nanostructures functionalization. Microarrays (3 × 3 spots) of Atto-labeled streptavidin and Cy5-labeled goat anti-rabbit antibody were created—ranging from 0 to 50 µg/mL—on these chips with a non-contact printing automatic arrayer. Different variables such as printing buffer (carbonate buffer, phosphate buffered saline and sodium citrate saline), incubation time (from 15 min to 16 h) and washing steps were analyzed to improve the protein immobilization. The best immobilization result was obtained for 1 µg/mL of labeled probe in PBS 1× as the immobilization buffer, incubating in a humid, dark chamber for 1 h and washing by immersion in DI-H2O. The immobilization yield was calculated from a calibration curve, as explained in the Experimental Section, resulting a 50% of a closely packed monolayer for both the protein [26] and the antibody [27], which corresponds to a coating density of 3.3 and 1.0 pmol/cm2, respectively. These values were in accordance with others reported in the literature for protein and antibody immobilization on SU-8 [19,28]. Also, the selectivity of the immobilization against SiO2, the material creating the nanopillar platform, was evaluated. To this end, only half of the surface of the SiO2 planar chips was partially coated with SU-8, and an immobilization protocol was applied to both parts of the chip. After incubating and washing, the fluorescence was read (ESI, Table S1), observing protein immobilization only on the coated surface (Figure 2). Finally, the chip was washed under acidic conditions (glycine buffer, pH 2.4, 2 h), observing that the fluorescence in the microarrays decreased in less than 10% of the cases, indicating the robustness of the attachment (ESI, Table S1).


Biomolecular Interaction Analysis of Gestrinone-anti-Gestrinone Using Arrays of High Aspect Ratio SU-8 Nanopillars.

Ortega FJ, Bañuls MJ, Sanza FJ, Casquel R, Laguna MF, Holgado M, López-Romero D, Barrios CA, Maquieira Á, Puchades R - Biosensors (Basel) (2012)

Scheme of the procedure employed to test the selective antibody immobilization on SU-8 planar chips using microarrays and Cy5 labeling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263551&req=5

biosensors-02-00291-f002: Scheme of the procedure employed to test the selective antibody immobilization on SU-8 planar chips using microarrays and Cy5 labeling.
Mentions: With regard to the bioreceptor surface attachment, the epoxy groups in the SU-8 polymer allow the direct protein immobilization by the nucleophilic attack of the protein amine moieties. Thus, the first stage was to study the immobilization process. To evaluate the immobilization and biorecognition processes, planar chips consisting of glass slides coated with a layer of SU-8 photoresist were employed because our previous experience with BICELLs showed that the protocols developed in planar substrates constituted a good starting point to be applied later in the nanostructures functionalization. Microarrays (3 × 3 spots) of Atto-labeled streptavidin and Cy5-labeled goat anti-rabbit antibody were created—ranging from 0 to 50 µg/mL—on these chips with a non-contact printing automatic arrayer. Different variables such as printing buffer (carbonate buffer, phosphate buffered saline and sodium citrate saline), incubation time (from 15 min to 16 h) and washing steps were analyzed to improve the protein immobilization. The best immobilization result was obtained for 1 µg/mL of labeled probe in PBS 1× as the immobilization buffer, incubating in a humid, dark chamber for 1 h and washing by immersion in DI-H2O. The immobilization yield was calculated from a calibration curve, as explained in the Experimental Section, resulting a 50% of a closely packed monolayer for both the protein [26] and the antibody [27], which corresponds to a coating density of 3.3 and 1.0 pmol/cm2, respectively. These values were in accordance with others reported in the literature for protein and antibody immobilization on SU-8 [19,28]. Also, the selectivity of the immobilization against SiO2, the material creating the nanopillar platform, was evaluated. To this end, only half of the surface of the SiO2 planar chips was partially coated with SU-8, and an immobilization protocol was applied to both parts of the chip. After incubating and washing, the fluorescence was read (ESI, Table S1), observing protein immobilization only on the coated surface (Figure 2). Finally, the chip was washed under acidic conditions (glycine buffer, pH 2.4, 2 h), observing that the fluorescence in the microarrays decreased in less than 10% of the cases, indicating the robustness of the attachment (ESI, Table S1).

Bottom Line: After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed.The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored.The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).

View Article: PubMed Central - PubMed

Affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico, Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, Valencia 46022, Spain. fraorhi1@upvnet.upv.es.

ABSTRACT
In this paper, label-free biosensing for antibody screening by periodic lattices of high-aspect ratio SU-8 nano-pillars (BICELLs) is presented. As a demonstration, the determination of anti-gestrinone antibodies from whole rabbit serum is carried out, and for the first time, the dissociation constant (KD = 6 nM) of antigen-antibody recognition process is calculated using this sensing system. After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed. The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored. The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).

No MeSH data available.


Related in: MedlinePlus