Quantification of the kinetics and thermodynamics of protein adsorption using atomic force microscopy

Abstract Both in situ and ex situ methods for quantifying area fraction coverage of protein on a surface using atomic force microscopy were developed. The in situ method used a continuous fluid flow system to observe the kinetics of adsorption in real time. The ex situ method required immersing the sample in solution, drying the sample, and imaging in an ambient environment to obtain kinetic and isothermal data. These methods were developed using the plasma protein fibrinogen in a phosphate‐buffered saline solution on grade IV muscovite mica and highly ordered pyrolytic graphite (HOPG) substrates. Kinetic and quasiisothermal data were obtained and a Langmuir model was fit to the data. An adsorption rate constant of 2.2 × 10⁻⁴ mL · μg⁻¹s⁻¹ and a desorption rate constant of 8.3 × 10⁻⁵ s⁻¹ were found on an HOPG surface. Completely irreversible adsorption was found on the mica surface with an adsorption rate constant of 2.7 × 10⁻⁴ mL · μg⁻¹s⁻¹. Additionally, protein conformation and assembly orientation on these surfaces were documented where fibrinogen on HOPG formed a network‐like structure, whereas fibrinogen on mica was more random. Also, nano‐topographical factors (ledges) were seen as sites of preferential adsorption. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res 72A: 246–257, 2005