A total internal-reflection technique for the examination of protein adsorption

A total internal-reflection fluorescence (TIRF) technique for examining protein adsorption at solid-liquid interfaces is described. For the representative case of adsorption of bovine gamma-globulin onto silicone rubber, the applicability of the technique to serveral important aspects of protein adsorption phenomena is demonstrated. Specifically, in addition to the tightly adsorbed protein layer observed herein and previously by maany others, the presence of a loosely adsorbed protein layer was also noted under conditions of either static or flowing protein solutions. Taking advantage of the continuous real-time measurements possible with TIRF, a rate constant for the protein adsorption step during laminar flow was estimated at both 25 and 37 degrees C. These results serve to demonstrate that the TIRF technique represents a significant and versatile new tool for the study of protein adsorption phenomena.