Kinetics of Antigen Binding to Arrays of Antibodies in Different Sized Spots

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Abstract
A fluorescence-based array biosensor has been developed which can measure the binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar waveguide was used to capture a Cy5-labeled antigen present in a solution that was continuously flowed over the surface. The CCD image of the waveguide was monitored continuously for 25 min. The resulting exponential rise in fluorescence signal was determined by image analysis software and fitted to a reaction-limited kinetics model, giving a kf of 3.6 × 105 M-1 s-1. Different spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging from 80 to 1145 μm. It was demonstrated that under flow conditions, the reduction of spot size did not alter the association rate of the antigen with immobilized antibody; however, as the spot width decreased to < 200 nm, the signal intensity also decreased.