Ultra-thin, densely packed polymer brushes (PBs) with ultra-low fouling properties play a crucial role in recent developments of surface modification strategies for biomedical and biosensing technologies. However, micro-defects within PB structures can significantly impair their ultra-low fouling performance, thereby reducing their effectiveness and the application potential. Reliable detection of these micro-defects is essential for further evaluation and optimization of brush-based surfaces. In this study, we present a straightforward spectroscopic method for detection of micro-defects in PBs using Raman mapping. We employed random copolymer brushes of carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) as examples of state-of-the-art ultra-low fouling PB systems prepared on a surface-enhanced Raman scattering (SERS)-active Ag nano-island layer fabricated on Au-coated glass substrates. Methylene blue, drop-deposited on the PB surface, is demonstrated as a suitable reporter molecule, providing spectroscopic information via both Raman and fluorescence signals. Analysis of the spectroscopic maps enabled not only the detection of micro-defects but also the differentiation between "shallow" and "deep" defects, providing detailed insights into the structural integrity of ultra-low fouling PBs.

Charles University Faculty of Mathematics and Physics Department of Macromolecular Physics 5 Holešovičkách 747 2 180 00 Prague Czech Republic

Charles University Faculty of Mathematics and Physics Institute of Physics Ke Karlovu 5 121 16 Prague Czech Republic

Institute of Physics of the Czech Academy of Sciences Na Slovance 1999 2 182 00 Prague Czech Republic

Institute of Physics of the Czech Academy of Sciences Na Slovance 1999 2 182 00 Prague Czech Republic; Charles University Faculty of Mathematics and Physics Institute of Physics Ke Karlovu 5 121 16 Prague Czech Republic

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