The authors recently demonstrated that fully functional Escherichia coli 0157:H7 antibody thin films can be prepared using a simple pneumatic nebulizer on glass surface [1]. This paper focuses on the investigation of the morphology and physical properties of these films with the aim to better understand their performance. A series of E. coli 0157:H7 antibody spray-on thin films were investigated by ellipsometry, X-ray photoelectron spectroscopy (XPS), immunoassays, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), fluorescence microscopy, atomic force microscope (AFM) and contact angle analysis. These data were compared to measurements on films prepared with the biotin-avidin covalent bonding scheme. The investigation showed that films created by a 2 min pneumatic spray deposition time can capture antigens similar as the avidin-biotin wet-chemical method. The results also suggests that an influential factor for the comparable capture cell ability between sprayed and covalent films is an increased antibody surface coverage for the sprayed films (non-equilibrium technique), which compensates for the lack of its antibody orientation. There was no significant antibody denaturation detected on any of the sprayed films. Both techniques led to the formation of cluster-aggregates, a factor that seems unavoidable due to the natural tendency of protein to cluster. The avidin-biotin bridge films generally had a higher roughness, which manifested itself in a higher wettability compared to the sprayed films. (C) 2014 Elsevier B.V. All rights reserved.