The adsorption kinetics and surface coverage of free biotin with a thin gold film have been exploited by means of surface plasmon resonance sensing. The material was dissolved in phosphate buffered saline (PBS) aqueous solution and ethanol-PBS mixtures. A micro-fluidic flow cell was used for solute admission. Experimental conditions eliminate contact to ambient air and surface contaminations and maintain a stable, initially hydrophilic metal surface and reproducible adsorption conditions. In the initial binding phase, and at higher solute concentration, biotin films are composed from up to three mono-layers. Rinsing with PBS buffer removes the weakly bound fraction and leaves a single molecular layer at 1.1 nm thickness, along with concentration dependent surface coverage. The binding kinetics is well described by a Langmuir adsorption isotherm. The equilibrium constant K-eq determines to 2.94 x 10(3) mol(-1), and the free adsorption energy Delta G(ads) to -4.7 kcal/mol. Both values are well within the parameter range for alkene-thiolate adsorption to microcrystalline gold films. This supports a chemisorptive binding state, along with the possibility of molecular self-assembly. Both, Neutravidin (NA) and human serum (HS) effectively immobilize onto a biotin monolayer beneath. In the reversed binding sequence, biotin firmly attaches also to NA, but not to HS. Hence, the direct binding route of biotin onto thin gold films can be favorably used in the design of simplified immuno-assays, solely relying on biotinylated antibodies, while avoiding the need for immobilization of an intermediate Neutravidin linker layer. (C) 2013 Elsevier B.V. All rights reserved.