Quantifying adsorbed protein on surfaces using confocal fluorescence microscopy

The quantification and analysis of protein adsorption on solid surfaces are of significant importance in many areas of biosensors, biomaterials, and biomedical devices research. The accurate, in situ, measurement of multiple physicochemical properties from the thin protein layers which adsorb on surfaces is critical to understanding biocompatibility, surface chemistry factors, and the performance of implanted medical devices. To implement such studies, new tools and simple protocols based on instrumentation available in typical bioscience laboratories are desirable. In this work, we have developed an approach using confocal fluorescence microscopy to quantify the amount of bovine serum albumin (BSA) adsorbed onto a flat hydrophilic glass surface, under different pH conditions. This approach which can be implemented using most confocal fluorescence microscopes is described in detail and its limitations are discussed. This quantitative method coupled with the Langmuir model allowed for the determination of adsorption parameters at pH 2.0, 4.0, 7.4, and 9.2. The adsorption parameters were validated by comparison with literature values obtained from different techniques for a similar protein–surface system. The Derjaguin–Landau–Verwey–Overbeek (DLVO) theory was then used for a detailed analysis of these parameters, to understand in general terms how pH affected the surface adsorption interactions.