Analytical measuring and evaluation method for molecular interactions

1. A method for determining kinetic constants for a molecular interaction between a first binding partner L and a second binding partner A forming a complex LA, wherein the first binding partner L (ligand) is immobilized on a sensor surface of a biosensor, the sensor surface is contacted with a solution containing the second binding partner A (analyte), and binding of the second binding partner A to the immobilized binding partner L on the sensor surface is detected over time providing response data, the method comprising:

raising or lowering several times an analyte concentration c ti (A) of the second binding partner A in a stepwise fashion and before an equilibrium interaction state for the respective analyte concentration c ti (A) has been reached, and without any regeneration of the sensor surface between each raising or lowering of the analyte concentration; and

for each stepwise raising of the analyte concentration, comparing an initial rate of association between the ligand and the analyte to the associated response data, to ascertain association related kinetic constants of the interaction,

for each stepwise lowering of the analyte concentration, comparing an initial rate of disassociation between the ligand and the analyte to the associated response data, to ascertain dissociation related kinetic constants of the interaction.

2. The method of claim 1 , wherein the initial rate of association or the initial rate of disassociation is ascertained using actual measured values of the rate of complexation between the ligand and the analyte, the actual measured values being acquired prior to the rate of complexation reaching equilibrium.

3. The method of claim 2 , wherein initial association and dissociation rates are ascertained under an assumption of ideal conditions that no analyte depletion occurs upon association and that no analyte enrichment occurs upon dissociation.

4. The method of claim 1 , wherein the method is practiced in a cuvette system with a measurement chamber, and wherein the analyte concentration of a solution in the measurement chamber is kept constant during an association or dissociation process by continuously removing a portion of the solution from the measurement chamber through a first hollow needle and adding a solution having the analyte concentration through a second hollow needle.

5. The method of claim 1 , wherein the analyte concentration is constant at each i-th step, and determining k ass and k diss is performed from the relationship k′ on,i =k ass *c 0,i (A)+k diss , wherein k′ on,i is an exponent of R ass (t) at each i-th step and c 0,i (A) is a starting analyte concentration at each step.

6. The method of claim 1 , wherein said raising or lowering of the analyte concentration c ti (A) of the second binding partner A is started at any time point.

7. The method of claim 1 , wherein said analyte concentration c ti (A) of the second binding partner A is the same during more than one of said individual steps by successive impingements of solutions or sample loops that contain the same analyte concentration.

8. The method of claim 1 , wherein said analyte concentration c ti (A) of the second binding partner A is varying over time within one or more of said individual steps by controlled activation of sample loops that contain different analyte concentrations or by mixing of solutions via frequent switching of specific valves that are connected to different solutions.

9. The method of claim 1 , wherein the kinetic constants comprise the association rate constant k ass and the dissociation rate constant k diss .

10. The method of claim 1 , wherein the kinetic constants comprise a dissociation equilibrium constant K D =k diss /k ass and an association equilibrium constant K A =1/K D .

11. A method for detecting the formation of analyte-ligand complexes, wherein the ligand is immobilized on a sensor surface of a biosensor and the sensor surface is contacted with a solution containing the analyte, the method comprising:

raising or lowering several times a concentration of the analyte in a stepwise fashion before an equilibrium interaction state of the respective analyte concentration has been reached, and without any regeneration of the sensor surface between each raising or lowering of the analyte concentration;

for each stepwise raising of the analyte concentration, determining an association rate constant k ass ; and

for each stepwise lowering of the analyte concentration, determining an disassociation rate constant k diss .

12. A method in accordance with claim 11 further comprising:

for each stepwise raising of the analyte concentration, comparing an initial rate of association between the ligand and the analyte to the associated response data to ascertain the association related kinetic constants of the interaction; and

for each stepwise lowering of the analyte concentration, comparing an initial rate of disassociation between the ligand and the analyte to the associated response data to ascertain the dissociation related kinetic constants of the interaction.

13. A method in accordance with claim 11 further comprising determining an initial concentration of the analyte based on an initial rate of association between the ligand and the analyte.