The efficiency of a pre-absorbed bovine serum albumin (BSA) layer in blocking the non-specific adsorption of different proteins on hydrophobic and hydrophilic Surfaces was evaluated qualitatively and quantitatively using infrared reflection spectroscopy supported by spectral simulations. A BSA layer with a surface coverage of 35% of a close-packed monolayer exhibited a blocking efficiency of 90-100% on a hydrophobic and 68-100% on a hydrophilic Surface, with respect to the non-specific adsorption of concanavalin A (Con A), immunoglobulin G (IgG), and staphylococcal protein A (SpA). This BSA layer was produced Using a solution concentration of 1 mg/mL and 30 min incubation time. BSA layers that were adsorbed at conditions commonly employed for blocking (a 12 h incubation time and a Solution concentration of 10 mg/mL) exhibited a blocking activity that involved competitive adsorption-desorption. This activity resulted from the formation of BSA-phosphate Surface complexes, which correlated with the conformation of adsorbed BSA molecules that was favourable for blocking. The importance of optimisation 017 the adsorbed BSA layer for different Surfaces and proteins to achieve efficient blocking was addressed in this study. (C) 2009 Elsevier Inc. All rights reserved.