Biotin-avidin binding kinetics measured by single-molecule imaging.

The high affinity of avidin for biotin has made it useful for many bioanalytical applications involving the immobilization of proteins, vesicles, and other biomolecules to surfaces. To understand the formation and stability of the resulting biotin-avidin complex, it is useful to know the kinetics of the binding reaction, especially for situations where the complex is formed at a liquid-solid interface typically used in sensor or separation applications. In this work, a single-molecule fluorescence method is developed for measuring the kinetics and affinity constant for the binding of neutravidin, a deglycosylated variant of avidin, to surface-immobilized biotin. Biotin was immobilized using succinimidyl ester chemistry onto amine sites on glass surfaces. The surface density of biotin was controlled by the extreme dilution of 3-aminopropyltriethoxysilane into a monolayer of 2-cyanoethyltriethoxysilane. The resulting biotin binding sites are spaced apart by micrometer distances, and this avoids crowding effects and makes the resolution of single molecules possible. The binding and unbinding of individual tetramethylrhodamine-labeled neutravidin molecules is measured in situ by total-internal-reflection fluorescence (TIRF) microscopy imaging. Single-molecule detection and counting is readily achieved by this measurement, where quantitative control is established by determining the probabilities of false positive and negative events based on the intensity distributions of background and single-molecule spots and by comparing the bound molecule populations with the independently measured density of binding sites on the surface. The kinetics of binding and unbinding are evaluated by intermittent imaging and counting the number of bound neutravidin molecules versus time, following introduction of a neutravidin solution or its replacement by buffer over the low-density biotinylated surface. The neutravidin binding kinetics were found to be fast, essentially diffusion-controlled, while the stability of the complex and its dissociation rate appear to be influenced by the chemistry of biotin immobilization.