A method of binding kinetics of a ligand to micropatterned proteins on a microfluidic chip

A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein–ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 μm diameter, was used to capture FITC–BSA–biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43 × 10−8 M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 μm, showed a consistent dissociation constant of FITC–BSA–biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.