title: Monitoring the Switching of Single BSA-ATTO 488 Molecules Covalently End-Attached to a pH-Responsive PAA Brush.
abstract: We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the biomolecule can be switched from inside to outside the brush and vice versa by a change in pH. For this, we grafted ultrathin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a bis-N-succinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble, and it is swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface under basic pH conditions, and fluorescence recurred (in-state) when the chains collapsed under acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single-molecule level, and we demonstrate that this switching is fast but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush.
authors.affiliation: Membrane Science and Technology, and ‡Nanobiophysics, Mesa+ Institute for Nanotechnology, University of Twente , P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Namik
authors.initials: N
authors.lastname: Akkilic
authors.affiliation: Membrane Science and Technology, and ‡Nanobiophysics, Mesa+ Institute for Nanotechnology, University of Twente , P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Molenaar
authors.affiliation: Membrane Science and Technology, and ‡Nanobiophysics, Mesa+ Institute for Nanotechnology, University of Twente , P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Mireille M A E
authors.initials: MM
authors.lastname: Claessens
authors.affiliation: Membrane Science and Technology, and ‡Nanobiophysics, Mesa+ Institute for Nanotechnology, University of Twente , P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Christian
authors.initials: C
authors.lastname: Blum
authors.affiliation: Membrane Science and Technology, and ‡Nanobiophysics, Mesa+ Institute for Nanotechnology, University of Twente , P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Wiebe M
authors.initials: WM
authors.lastname: de Vos