The mass-transfer processes that affect kinetic measurements of biospecific interactions between one species in a flowing solution and another species immobilized in a thin hydrogel instrument were analysed by means of convection-diffusion-reaction models. The specific purpose was to identify experimental design considerations for kinetic measurements using the BIAcore(TM) instrument. Numerical solutions identified three different regimes of operation. A kinetic regime exists at low values of Damkohler number (Da), when the intrinsic kinetics are slow and the diffusion is relatively fast. This allows for the accurate determination of kinetic constants. A limiting value of Da, above which mass-transfer limitations appear, is presented as a function of Peclet number, Pe. At higher Da values, the reaction occurs in the mass-transfer-controlled regime where the reaction-rate is independent of the intrinsic kinetics. It was observed that, frequently, the reaction occurred in an intermediate regime where, although the mass-transfer rate was not strictly limiting, substantial concentration gradients were present. Analysing the data in this regime by direct application of kinetic equations underestimates the association rate constant. Even when the reaction was not limited by mass-transfer in the flow channel, it may have been affected by steric hindrance to transport in the hydrogel, if a large concentration of capturing antibody or ligand was immobilized. The primary effect of the hindrance was to lower the soluble-species (analyte) concentration in the hydrogel when compared to the bulk solution. Non-uniformity of conditions within the hydrogel in the presence of steric hindrance had a significant effect on the observed reaction. The effect was most prominent at higher analyte concentration, when the rate constant showed an apparent reduction as the reaction progressed.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- operating regimes
- steric hindrance