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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 bioassay designed to test the bioavailability of the hapten-protein conjugate molecules attached to the SU-8 surface and scanned image obtained for the microarray.
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biosensors-02-00291-f004: Scheme of the bioassay designed to test the bioavailability of the hapten-protein conjugate molecules attached to the SU-8 surface and scanned image obtained for the microarray.

Mentions: To evaluate the bioavailability of the rabbit serum, two formats were studied in planar. For the direct format immunoassay, anti-gestrinone antibody in PBS 1× at different concentrations (1, 2 and 10 µg/mL) was immobilized on the SU-8 surface. After a blocking step with OVA or ethanolamine, HRP-h-G in PBS-T was spread out over the microarray using a coverslip and incubated for 10 min, then washed with PBS-T and water, and developed with TMB. Using this format, a positive signal was only observed for a protein-hapten conjugate concentration of 50 µg/mL. It was expected that the directly adsorbed antibody would be less efficient in reacting with the antigen that the liquid phase antibody due to denaturation and loss of its binding capacity. To test this, the indirect format was assayed (Figure 4); HRP-h-G in PBS 1× at 1, 5, 10 and 20 µg/mL was immobilized on the SU-8 planar surface accordingly to the procedure described above. Then, the surface was blocked with OVA and different dilutions of anti-gestrinone rabbit serum in PBS-T (from 1/100,000 to 1/200) were spread out, incubated for 10 min and washed. Finally, GAR-Au antibody diluted 1/200 in PBS-T was added, incubated for 10 min and washed again. Development with silver enhancer solution produced positive spots for anti-gestrinone serum dilutions up to 1/10,000, which corresponds to an anti-gestrinone antibody concentration of 100 ng/mL. The amount of specific antibody contained in the rabbit antiserum was determined from an aliquot of the serum that was purified by an affinity column and quantified by UV-vis spectrometry; the amount of specific antibody resulted in 1 mg/mL in the serum before any dilution.


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 bioassay designed to test the bioavailability of the hapten-protein conjugate molecules attached to the SU-8 surface and scanned image obtained for the microarray.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00291-f004: Scheme of the bioassay designed to test the bioavailability of the hapten-protein conjugate molecules attached to the SU-8 surface and scanned image obtained for the microarray.
Mentions: To evaluate the bioavailability of the rabbit serum, two formats were studied in planar. For the direct format immunoassay, anti-gestrinone antibody in PBS 1× at different concentrations (1, 2 and 10 µg/mL) was immobilized on the SU-8 surface. After a blocking step with OVA or ethanolamine, HRP-h-G in PBS-T was spread out over the microarray using a coverslip and incubated for 10 min, then washed with PBS-T and water, and developed with TMB. Using this format, a positive signal was only observed for a protein-hapten conjugate concentration of 50 µg/mL. It was expected that the directly adsorbed antibody would be less efficient in reacting with the antigen that the liquid phase antibody due to denaturation and loss of its binding capacity. To test this, the indirect format was assayed (Figure 4); HRP-h-G in PBS 1× at 1, 5, 10 and 20 µg/mL was immobilized on the SU-8 planar surface accordingly to the procedure described above. Then, the surface was blocked with OVA and different dilutions of anti-gestrinone rabbit serum in PBS-T (from 1/100,000 to 1/200) were spread out, incubated for 10 min and washed. Finally, GAR-Au antibody diluted 1/200 in PBS-T was added, incubated for 10 min and washed again. Development with silver enhancer solution produced positive spots for anti-gestrinone serum dilutions up to 1/10,000, which corresponds to an anti-gestrinone antibody concentration of 100 ng/mL. The amount of specific antibody contained in the rabbit antiserum was determined from an aliquot of the serum that was purified by an affinity column and quantified by UV-vis spectrometry; the amount of specific antibody resulted in 1 mg/mL in the serum before any dilution.

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