SURFACE AND INTERFACE ANALYSIS Surf. Interface Anal. 2001; 31: 724–733 Static time-of-flight secondary ion mass spectrometry and x-ray photoelectron spectroscopy characterization of adsorbed albumin and fibronectin films Caren D. Tidwell, 1 David G. Castner, 1* Stephen L. Golledge, 1 Buddy D. Ratner, 1 Klaus Meyer, 2 Brigit Hagenhoff 2 and Alfred Benninghoven 2 1 Departments of Bioengineering and Chemical Engineering, Box 351750, University of Washington, Seattle, Washington 98195-1750, USA 2 Physikalisches Institut der Universit ¨ at M ¨ unster, Wilhelm-Klemm-Strasse 10, D-48149, M ¨ unster, Germany Received 16 August 2000; Revised 30 March 2001; Accepted 4 April 2001 Static time-of-flight secondary ion mass spectrometry (ToF-SIMS), monochromatized x-ray photoelectron spectroscopy (XPS) and 125 I radiolabeling have been used to characterize albumin films adsorbed onto titanium, gold, polytetrafluoroethylene and r.f. glow discharge-deposited tetrafluoroethylene (TFE) substrates. A comparison between albumin and fibronectin films also was made. The intensities of characteristic amino acid mass fragments (immonium ions) detected in the static ToF-SIMS experiments depended on the protein type, the substrate type and the adsorption conditions, demonstrating the sensitivity of static ToF-SIMS for probing the structure of adsorbed protein films. Based on the results from albumin and fibronectin, static ToF-SIMS can provide information about the identity of adsorbed proteins and their conformation, orientation, denaturation, etc. X-ray photoelectron spectroscopy can distinguish pure protein films, but the higher molecular specificity of static ToF-SIMS is more useful than XPS for examining complex protein films. The 125 I radiolabeling experiments and the XPS atomic percentage of nitrogen were used to quantify the amount of adsorbed protein. Copyright  2001 John Wiley & Sons, Ltd. KEYWORDS: ToF-SIMS; XPS; albumin; fibronectin INTRODUCTION Surface phenomena direct biological responses, as is evi- dent from biochemical, cellular and some clinical responses to biomedical implants. This surface control is mediated through an adsorbed protein layer. Convincing evidence often is presented showing that surface structures correlate with bioresponses. 1 Although clinically used implant materi- als are engineered for obvious mechanical and permeability requirements, little consideration is given to the interfacial reactions between these materials and the physiological envi- ronment. For example, it has not been determined yet why, at a molecular level, some biomaterials are blood-compatible and others fail due to thrombosis or thromboembolism. These problems of surface-induced bioreaction are difficult to solve due to the limited understanding of the fundamental phenomena controlling interfacial biology. The initial event that occurs upon exposure of a biomaterial to a physiological environment is the rapid adsorption of proteins to the material from the surrounding fluid phase. 1 Material surface properties affect biological Ł Correspondence to: D. G. Castner, Department of Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195-1750, USA. E-mail: castner@nb.engr.washington.edu Contract/grant sponsor: National Center for Research Resources; Contract/grant number: RR-01296. interactions via the composition, structure and conformation of the adsorbed protein layer. 2–6 The composition of the adsorbed protein layer differs from the fluid phase composition, varies significantly with the type of substrate and has been demonstrated to undergo conformational and orientational changes with time. Polymer surface properties can affect the amounts and types of proteins bound, as well as the conformation, orientation or binding strength of the adsorbed protein. 7–9 Adsorbed proteins can retain a structure close to that in solution or may conformationally adjust in response to local environments. This time-dependent conformational readjustment has been hypothesized to be an element in the communication link between surface and cell that mediates biological response. 1,9 The small amount of protein present, its localization at a solid substrate and the biological complexity of the layer challenge traditional biochemical methods to provide insightful information on the conformation and orientation of the protein on the substrate and the interaction of the protein with the substrate. Changes in protein conformation after adsorption can be inferred from bulk adsorption experiments. For example, changes in the strength of interaction between a number of proteins and surfaces as measured by detergent elutability are most likely due to conformational alterations. 10 Sodium dodecyl sulfate (SDS) DOI: 10.1002/sia.1101 Copyright  2001 John Wiley & Sons, Ltd.