Microdialysis sampling method for evaluation of binding kinetics of small molecules to macromolecules

Here we present an application of microdialysis sampling for evaluation of the binding kinetics of small molecules to macromolecules. It is label-free, and no immobilization of any interaction partner is required. The method was established by the coupling of a binding reaction with a membrane transport in a miniature and dynamic microdialysis sampling system. A theoretical model was established to describe the quantitative relationship between the binding kinetics of small ligands to macromolecules and the enhanced mass transport of small ligands and was applied to estimate the binding kinetics. To demonstrate the proof-of-principle, we examined the binding kinetics of an abundant plasma protein human serum albumin (HSA) and a representative drug ketoprofen as an example. The primary binding constant of ketoprofen to HSA was estimated as 1.63 (+/-0.12) x 10(6) M(-1). The estimated association and dissociation rate constants (k1 and k(-1)) were about 3.71 x 10(5) M(-1) s(-1) and 0.227 s(-1), respectively. The results suggest a fast binding of ketoprofen to HSA and a fast dissociation of the formed complex, which are consistent with the reversible binding property of drug and HSA (k(-1) in the order of s(-1)). This is the first report on binding-kinetics measurement using microdialysis sampling.