Porous SiO₂Interferometric Biosensor for Quantitative Determination of Protein Interactions:  Binding of Protein A to Immunoglobulins Derived from Different Species

Abstract

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 × 10 7 M -1 using an equilibrium Langmuir model. Kinetic rate constants are calculated to be k d = 2.1 ± 0.2 × 10 -4 s -1 and k a = 1.2 ± 0.4 × 10 4 M -1 s -1 using non-linear least squares analysis, yielding an equilibrium binding constant of K a = 5.5 ± 1.5 × 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 non-linear 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.

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