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1. WO2007105081 - PROCÉDÉ DE MESURE D'INTERACTIONS MOLÉCULAIRES EN MESURANT LA LUMIÈRE RÉFLÉCHIE PAR DES SURFACES PLANES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

CLAIMS

1. Method for the determination of the binding constant of two interacting molecular species and/or of the concentration of ligand in solution by using the measure of the intensity of reflected light, comprising the following steps: a) a planar surface, flat or rough, of a transparent solid material constituted by a hydrophobic amorphous polymer, having a refractive index between 1.3200 and 1.3500, preferably between 1.3300 and 1.3350, is brought into contact with an aqueous or non-aqueous solution of a mixture containing from 1 nanogram/ml to 10 milligrams/ml in concentration of molecules with a function of receptor or reagent, such as antibodies or other proteic or peptidic complexes, or nucleic acids, or lipids, or amphiphilic surfactants or block polymers ended with a receptor or reagent, optionally mixed to other molecules (spacers) having no receptor function, optionally measuring at each addition the light intensity reflected from the interface between the aqueous solution and the said solid material and reporting the measured value on a diagram as a function of time or as a function of the receptor concentration progressively added, optionally repeating this procedure by bringing the surface into contact with other aqueous or non-aqueous solutions of the said molecules;
b) addition to the solution obtained in step (a) of a sequence of known volumes of an aqueous solution of a ligand, measuring at each addition the light intensity reflected from the interface between the aqueous solution and the polymeric material and reporting the measured value on a diagram as a function of the time or as a function of the ligand concentration JT0] progressively added, and fitting the reflected light intensity data / as a function of the ligand additions with the Fresnel formulas for thin layer reflection to obtain the adhered mass, and with the Langmuir formula for adsorption to obtain the ligand-receptor affinity constant at equilibrium.
2. Method according to claim 1 wherein the amphiphilic non ionic, or ionic, surfactants are surfactants which produce a monolayer (self assembled monolayer) on the polymeric surface.
3. Method according to either one of claims 1-2 wherein the ligand-receptor couple is chosen among proteins, nucleic acids, glycoproteins, carbohydrates, hormones.
4. Method according to claim 3 wherein the ligand-receptor couple of molecules is chosen among antibody/antigen, enzyme/inhibitor, carbohydrate/carbohydrate, protein/DNA, DNA/DNA, peptide/peptide.
5. Method according to either one of claims 1-4 wherein the receptors are immobilized and/or chemically modified through methods of the known art, such as chemical methods or electromagnetic irradiation or plasma treatment methods.
6. Method according to either one of claims 1-5 wherein the solution in contact with the surface of the solid material and containing the molecules or the molecular complexes having the function of receptors and/or ligands, is optically transparent, or turbid and/or absorbing.
7. Method according to either one of claims 1-6 wherein the surface of the solid material presents a regular or irregular roughness, having characteristic size of the depth and of the width of the surface roughness between 10 nanometers and 3 millimeters, and the intensity of the light emitted from the surface in presence of the ligand-receptor interaction is measured not only in the direction of the geometrical reflection but also in a direction different from that of the geometrical reflection.

8. Method according to either one of claims 1-7 wherein the incidence angle and the polarization of the incident light are selected in order to optimize the ratio between the signal of reflected light in presence of the ligand-receptor interaction and the background noise due to reflection and scattering of light.
9. Method according to claim 8 wherein the reflected light is filtered by means of a polarizer whose orientation is chosen in order to optimize the ratio between the signal of reflected light in presence of the ligand-receptor interaction and the background noise due to reflection and scattering of light. 10. Method according to either one of claims 1-9 wherein the surface of the polymeric amorphous material is embodied in a cell for measures in the absence of flow, or is embodied in a flow cell, or is embodied in an immersion probe.
11. Method according to either one of claims 1-10 wherein the surface of the polymeric material is divided in smaller areas, on which are adsorbed or immobilized different receptors.