Polydopamine (PDA) films have attracted a rapidly increasing research attention during the last years due to its simple and rapid deposition under alkaline conditions in substrate independent manner providing a universal coating for materials with different chemical and physical properties . Furthermore, this polymerized layer is enriched with functional groups that enable immobilization of primary amine or thiol-based biomolecules via a simple dipping process . Although these fascinating aspects justify PDA wide and successful application as a versatile coating for biomolecule immobilization, several aspects have not been deeply investigated leaving some key details unclear and thus limiting PDA practical applications. A number of approaches are commonly used for the growth of PDA , but the effect of deposition conditions on film properties which in turn influence biomolecule immobilization has not been systematically investigated yet. In the present work, a detailed investigation by X-Ray Photoelectron Spectroscopy (XPS) of PDA coatings deposited by different synthetic schemes (namely by autoxidation in air, under a pure oxygen environment, in the presence of a strong oxidizing agent for different time intervals (1, 3, 5, 8, 18, and 24 hours), and by electrochemical oxidation) is performed aimed at investigating film thickness and chemical composition as a function of polymerization conditions. Comparative spectroscopic analysis of PDA films revealed significant differences in terms of deposition kinetics and abundance of chemical components and allowed selection of synthesis conditions making PDA chemical structure richer in functionalities mainly involved in conjugation of biomolecules. The high suitability of the selected PDA film for bioconjugation was verified using a biomolecule conjugated to Horseradish Peroxidase or to fluorophore, obtaining also an estimation of immobilization time-stability within 4 weeks and a quantitative evaluation of immobilization extent. Moreover, further insight on biomolecule anchoring was provided by the comparison of XPS data on PDA samples before and after interaction with biomolecule .  H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426–430.  Y. Liu, K. Ai, L. Lu, Chem. Rev. 114 (2014) 5057–5115.  H.W. Kim, B.D. McCloskey, T.H. Choi, C. Lee, M.J. Kim, B.D. Freeman, H.B. Park, ACS Appl. Mater. Interfaces. 5 (2013) 233–238  S. Rella, E. Mazzotta, A. Caroli, M. De Luca, C. Bucci, C. Malitesta, Appl. Surf. Sci. 447 (2018) 31–39.
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