Comparative Methods for the Evaluation of Protein Adsorption Eva Servoli, * Devid Maniglio, Maria Rosa Aguilar, Antonella Motta, Blanca Vazquez, Julio San Roman, Claudio Migliaresi Introduction Soon upon contacting biological fluids, implanted materi- als adsorb proteins according to the Vroman effect, [1] where proteins compete for binding to the surface of the biomaterial, with larger and surface active proteins displacing low-molecular-weight ones. Material surface properties can regulate the amount, composition, dis- tribution and conformation of the protein layer, which in turn highly affects the subsequent biological response, eliciting phenomena of defence or acceptance. Many physical and chemical techniques for surface modifications are currently used to control protein adsorption on materials for body implants or blood contact; homogeneous surfaces as well as patterned substrates with features down to nanometric scale can be produced. [2] A deep understanding of protein-material interaction can help to correlate material properties to human tissue responses, thus promoting the development of effective biomedical devices for tissue replacement and regenera- tion as well as efficient diagnostic and therapeutic tools. In particular, this work is intended as a critical study of protein adsorption through a comparative analysis of detection strategies, which took into account several experimental conditions (static vs. dynamic, individual vs. competitive adsorption, variation of substrate surface properties). Starting from simple adsorption models, the complexity of the process has been gradually increased to understand the contribution of individual parameters and their interplay. The study has been carried out on fibroin, Full Paper E. Servoli, D. Maniglio, A. Motta, C. Migliaresi Department of Materials Engineering and Industrial Technologies and INSTM Research Unit, University of Trento, via Mesiano 77, 38100 Trento, Italy Fax þ39 461 88 1977; E-mail: servolie@ing.unitn.it M. R. Aguilar, B. Vazquez, J. San Roman Institute of Polymer Science and Technology, CSIC, C/ Juan de la Cierva 6, 28006 Madrid, Spain AFM, Immunogold labeling and SPR have been successfully applied to the analysis of protein adsorption on fibroin substrates, chosen as a paradigmatic biomaterial. The process was evaluated both under static and dynamic conditions, also considering the effect of protein competition. AFM via antibody-modified tips provided maps of protein adsorption and information on protein-protein interactions. Double labeling Immunogold imaged protein distribution upon competitive adsorption. SPR described the dynamics of the process, offering also quantitative data both from individual and two-component protein solutions. The integ- ration of these analytical techniques and the critical evaluation of the results can provide a detailed description of protein adsorption. Macromol. Biosci. 2009, 9, 000–000 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/mabi.200800301 1 Early View Publication; these are NOT the final page numbers, use DOI for citation !! R