Patent ID: 8906672
Filing Date: 2014-12-09
Classification: B01L,G01N,Y10S,Y10T

Abstract:
1. A method for determining an affinity or kinetic constant for ligand binding comprising the steps of: creating a gradient sample by mixing a first gradient component solution comprising an analyte having a starting concentration with a second gradient component solution in a channel connected to an inlet of a flow cell such that an analyte concentration gradient is generated thereby providing a range of analyte concentrations within the gradient sample; injecting, in a single, continuous injection, the gradient sample into the flow cell and onto a first sensing surface and a second sensing surface, wherein the first sensing surface comprises a ligand immobilized thereon and the second sensing surface is free of immobilized ligand; adding to one of the gradient component solutions a material that changes the bulk refractive index thereof; recording a first response at the first sensing surface and a second response at the second sensing surface, wherein the first response includes a binding response component elicited by an interaction between the analyte and the ligand thereby providing a binding response curve, and wherein the second response provides an actual bulk refractive index response of the gradient sample; determining a gradient injection profile based on the second response; extrapolating the range of analyte concentrations present at the first sensing surface by performing the following steps: generating an adjusted bulk refractive index response by dividing the actual bulk refractive index response by a maximum expected bulk refractive index response for a 100% pure solution of the material that changes the bulk refractive index; fitting an equation to the adjusted bulk refractive index response using a regression curve fitting procedure that suitably models the gradient injection profile of the gradient sample as a function of injection time thereby defining a gradient injection profile function; multiplying the starting concentration of the analyte by the gradient injection profile function in order to estimate the range of analyte concentrations at the first sensing surface as a function of time during the gradient injection; using the extrapolated range of analyte concentrations for determining at least one of a plurality of kinetic or affinity constants for the binding response component.