Method of determining affinity or kinetic properties in solution

A method for determining affinity and kinetic parameters associated with a first binding interaction between an analyte and a binding partner, wherein the first binding interaction takes place under nonequilibrium conditions in a nonequilibrium solution, comprising the steps of:

• forming the nonequilibrium solution in which the first interaction occurs by mixing the analyte and the binding partner to form the nonequilibrium solution, or by causing dissociation of a complex of the analyte and the binding partner to form the nonequilibrium solution;
• sequentially contacting the nonequilibrium solution with a plurality of sensor surfaces, each sensor surface having (i) an additional amount of the binding partner or analogue thereof, immobilized thereon, or (ii) an additional amount of the analyte or analogue thereof, immobilized thereon;
• monitoring a second binding interaction between (i) the additional amount of the binding partner or analogue thereof immobilized on the plurality of sensor surfaces with the analyte of the nonequilibrium solution, or (ii) the additional amount of the analyte or analogue thereof immobilized on the plurality of sensor surfaces with the binding partner of the nonequilibrium solution, over a period of time;
• determining a free analyte or a free binding partner concentration in the nonequilibrium solution at two or more points in time;
• and therefrom
• determining the affinity or kinetic parameters associated with the first binding interaction between the analyte and the binding partner.