Determination of binding constants by equilibrium titration with circulating sample in a surface plasmon resonance biosensor.

A commercial surface plasmon resonance biosensor, BIACORE X, is employed as a detector in a closed loop of a small sample volume. The sample is continuously circulated by an external syringe pump over two sensor spots, one functionalized with immobilized binding sites to a soluble binding partner in the mobile phase and one serving as a reference surface. A binding isotherm for the interacting macromolecules can be obtained by a stepwise titration of the soluble reactant into the circulating loop, each step followed by observation of the signal increase until equilibrium is attained. Binding constants can be measured under conditions free of mass transport artifacts and without the requirement for regeneration of the immobilized binding sites. This procedure is similar to the stepwise titration procedure described for the cuvette-based sensor design (D. R. Hall and D. J. Winzor, 1997, Anal. Biochem. 244, 152-160). In the presented configuration, the high baseline stability of the instrument combined with the availability of a reference surface for the detection of nonspecific binding permits refractive index changes upon addition of the aliquots to be measured, as well as accounting for temperature or instrumental drifts, and allows for a very long experimental time. This feature extends the applicability of equilibrium titration to systems with higher affinity or slower dissociation rate constants. Furthermore a solution competition titration is described that avoids artifacts from the immobilization procedure to provide a method for measurement of binding constants in solution. Kinetic information on the complex dissociation can also be obtained by combination of sample delivery via the external pump with the injection of competitor via the microfluidics of the biosensor. The rapid injection of high concentrations of competitor allows the observation of fast dissociation processes under conditions minimizing rebinding.