Adsorption and Stability of Streptavidin on Cluster-Assembled Nanostructured TiO x Films Luca Giorgetti,* ,†,‡ Gero Bongiorno, † Alesssandro Podestà, † Giuseppe Berlanda, † Pasquale Emanuele Scopelliti, † Roberta Carbone, § and Paolo Milani* ,† CIMAINA and Dipartimento di Fisica, UniVersity of Milano, Via Celoria 16, 20133 Milano, Italy, SEMM, Scuola Europea di Medicina Molecolare, and Department of Experimental Oncology, European Institute of Oncology, Campus IFOM-IEO, Via Adamello 16, 20139 Milan, Italy, and Tethis srl, Via Russoli 3, 20143 Milano, Italy ReceiVed June 18, 2008. ReVised Manuscript ReceiVed August 4, 2008 The study of the adsorption of proteins on nanostructured surfaces is of fundamental importance to understand and control cell-surface interactions and, notably, cell adhesion and proliferation; it can also play a strategic role in the design and fabrication of nanostructured devices for postgenomic and proteomic applications. We have recently demonstrated that cluster-assembled nanostructured TiO x films produced by supersonic cluster beam deposition possess excellent biocompatibility and that these films can be functionalized with streptavidin, allowing the immobilization of biotinylated retroviral particles and the realization of living-cell microarrays for phenotype screening. Here we present a multitechnique investigation of the adsorption mechanisms of streptavidin on cluster-assembled TiO x films. We show that this nanostructured surface provides an optimal balance between adsorption efficacy and protein functionality. By using low-resolution protein arrays, we demonstrate that a layer of adsorbed streptavidin can be stably maintained on a cluster-assembled TiO x surface under cell culture conditions and that streptavidin retains its biological activity in the adsorbed layer. The adsorption mechanisms are investigated by atomic force microscopy in force spectroscopy mode and by valence-band photoemission spectroscopy, highlighting the potential role of the interaction of the exposed carboxyl groups on streptavidin with the titanium atoms of the nanostructured surface. Introduction The immobilization of proteins on solid surfaces has a fundamental influence on cell adhesion and proliferation: the protein-material interface can be thus considered to be the playground where the mechanisms governing biocompatibility can be explored and where the strategies for the fabrication of effective protein and cell microarrays can be identified and tested. 1,2 Protein-surface interaction is determined by the chemistry and morphology of the substrate in a complex way that is far from being understood: in particular, it is not clear how the interplay between parameters such as surface chemistry and surface topography influence the amount and conformation of adsorbed proteins. 2-5 To elucidate the role of substrate topography and to fabricate biocompatible interfaces capable of mimicking the physiological conditions of the extracellular environment, a large number of studies have been devoted to the investigation of cell interactions with artificially produced nanostructures such as pits, pillars, grooves, dots, and random structures obtained by chemically or physically etching metallic, semiconducting, and polymeric surfaces. 6,7 Particular efforts have been devoted to the topo- graphical modification of titanium and titanium dioxide surfaces because these materials are among the most studied and most well characterized biomaterials. 8 In parallel, different strategies for the functionalization of surfaces with molecular groups favoring protein adhesion have been proposed. 9 Recently, we demonstrated that nanostructured TiO x (ns-TiO x ) films obtained by supersonic cluster beam deposition have excellent biocompatibility by performing long-term experiments with a range of cancer and primary cells. 10 Ns-TiO x films resulting from a random stacking of nanoparticles are characterized, on the nanoscale, by granularity and porosity mimicking those of extra-cellular matrix (ECM) structures. 10,11 Their large nanoscale porosity, along with the abundance of adsorption sites and defects, 12 makes cluster-assembled TiO x a promising candidate as a substrate for the adsorption and stable docking of proteins. By exploiting these properties, we employed ns-TiO x films as multifunctional substrates for macromolecule functionalization and cell culture in the context of a surface-mediated gene transduction protocol. 11 In particular, we demonstrated the feasibility of a retroviral microarray technology in which biotinylated retroviruses were docked and localized onto the * Corresponding authors: luca.giorgetti@ifom-ieo-campus.it, pmilani@ mi.infn.it. † University of Milano. ‡ SEMM and European Institute of Oncology. § Tethis srl. (1) Gallagher, W. M.; Lynch, I.; Allen, L. T.; Miller, I.; Penney, S. C.; O’Connor, D. P.; Pennington, S.; Keenan, A. K.; Dawson, K. A. Biomaterials 2006, 27, 5871–5882. (2) Lynch, I. Physica A 2007, 373. (3) Xu, L. C.; Siedlecki, C. A. Biomaterials 2007, 28, 3273–3283. (4) Rechendorff, K.; Hovgaard, M. B.; Foss, M.; Zhdanov, V. P.; Besenbacher, F. Langmuir 2006, 22, 10885–10888. (5) Han, M.; Sethuraman, A.; Kane, R. S.; Belfort, G. Langmuir 2003, 19, 9868–9872. 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G.; Amati, M.; Bongiorno, G.; Mazza, T.; Policicchio, A.; Formoso, V.; Maccallini, E.; Colavita, E.; Chiarello, G.; Finetti, P.; Sutara, F.; Skala, T.; Piseri, P.; Prince, K. C.; Milani, P. J. Chem. Phys. 2008, 128, 094704. 11637 Langmuir 2008, 24, 11637-11644 10.1021/la801910p CCC: $40.75  2008 American Chemical Society Published on Web 09/27/2008