Kinetics of antigen binding to arrays of antibodies in different sized spots

A fluorescence-based array biosensor has been developed which can measure t he binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar wave guide 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 wa s monitored continuously for 25 min. The resulting exponential rise in fluo rescence signal was determined by image analysis software and fitted to a r eaction-limited kinetics model, giving a k(f) of 3.6 x 10(5) M-1 s(-1). Dif ferent spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging f rom 80 to 1145 mum. It was demonstrated that under flow conditions, the red uction 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.