Porous SiO_(2) Interferometric Biosensor for Quantitative Determination of Protein Interactions: Binding of Protein A to Immunoglobulins Derived from Different Species

摘要

Determination of kinetic and thermodynamic protein binding constants using interferometry from a porous Si Fabry-Perot layer is presented. A protein A capture probe is adsorbed within the pores of an oxidized porous Si matrix, and binding of immunoglobulin G (IgG) antibodies derived from different species is investigated. The relative protein A/IgG binding affinity is human > rabbit > goat, in agreement with literature values. The equilibrium binding constant (K_(a)) for human IgG binding to surface-immobilized protein A is determined to be (3.0 +- 0.5) X 10~(7) M~(-1) using an equilibrium Langmuir model. Kinetic rate constants are calculated to be k_(d) velence (2.1 +- 0.2) X 10~(-4) s~(-1) and k_(a) velence (1.2 +- 0.4) X 10~(4) M~(-1) s~(-1) using nonlinear least-squares analysis, yielding an equilibrium binding constant of K_(a) velence (5.5 +- 1.5) X 10~(7) M~(-1). Both steady-state and time-dependent measurements yield equilibrium binding constants that are consistent with literature values. Kinetic rate constants determined through nonlinear least-squares analysis are also in agreement with protein A/IgG binding on a surface. Dosing with a high concentration of analyte leads to deviations from ideal binding behavior, interpreted in terms of restricted analyte diffusion within the porous SiO_(2) matrix. It is shown that the diffusion limitations can be minimized if the kinetic measurements are performed at low analyte concentrations or under conditions in which the protein A capture probe is not saturated with analyte. Potential limitations of the use of porous SiO_(2) interferometers for quantitative determination of protein binding constants are discussed.