Polarized gonio fluorimetric measurements of DNA-chip: a step forward towards fluorescence quantification

Quantifying hybridization and therefore fluorescence signals has become a key-issue in DNA-chip technology. Thus a better understanding of fluorescence near a surface has become a necessity. To study this issue, we modeled the fluorophore after an electromagnetic dipole radiating over the substrate; we then developed a simulation code which enabled us to calculate the observation-angle-dependent-intensity radiated by a population and altitude. In the mean time we developed a polarized-gonio-fluoimeter which permits angular fluorescence patterns and fluorescence polarization measurements. We studied DNA-chips obtained by covalent grafting of labeled oligonucleotides. Simulation curves perfectly matched experimental ones, enabling an accurate determination of fluorophore localization on the substrate. Once achieved a better understanding of the fluorophore emission, we designed and realized a thin-film-coated microscope slide dedicated to the enhancement of DNA-chip fluorescence. This substrate was used in a c-DNA gene analysis. Fluorescence enhancement was clearly observed enabling the detection of Cart1 and P2A which are undetectable when using non-coated microscope slides.