title: Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization.
abstract: A successive multiple ionic polymer layer (SMIL) coating composed of four layers improved the capillary electrophoretic separation of a recombinant major birch pollen allergen and closely related variants when poly(acrylamide-co-2-acrylamido-2-methyl-1-propansulfonate) (55% PAMAMPS) replaced dextran sulfate as terminal SMIL layer. 55% PAMAMPS decelerated the electroosmotic flow (EOF) due to its lower charge density. Atomic force microscopy (AFM) was used to investigate SMIL properties directly on the inner capillary surface and to relate them to EOF measurements and results of associated CZE separations of a mixture of model proteins and peptides that were performed in the same capillary. For the first time, AFM-based biosensing topography and recognition imaging mode (TREC) under liquid conditions was applied for a sequential characterization of the inner surface of a SMIL coated capillary after selected treatments including pristine SMIL, SMIL after contact with the model mixture, after alkaline rinsing, and the replenishment of the terminal polyelectrolyte layer. A cantilever with tip-tethered avidin was used to determine the charge homogeneity of the SMIL surface in the TREC mode. SMIL coated rectangular capillaries with 100 μm internal diameter assured accessibility of the inner surface for this cantilever type. Observed changes in CZE performance and EOF mobility during capillary treatment were also reflected by alterations in surface roughness and charge distribution of the SMIL coating. A renewal of the terminal SMIL layer restored the original surface properties of SMIL and the separation performance. The alliance of the novel TREC approach and CZE results allows for an improved understanding and a comprehensive insight in effects occurring on capillary coatings.
authors.affiliation: Division of Chemistry and Bioanalytics, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria; Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
authors.forename: Lorenz G
authors.initials: LG
authors.lastname: Stock
authors.affiliation: Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Leitner
authors.affiliation: Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Traxler
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Bonazza
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier, Place Eugène Bataillon, CC 1706, 34095 Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Leclercq
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier, Place Eugène Bataillon, CC 1706, 34095 Montpellier, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Cottet
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
authors.forename: Gernot
authors.initials: G
authors.lastname: Friedbacher
authors.affiliation: Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
authors.affiliation: Division of Chemistry and Bioanalytics, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria; Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, Hellbrunnerstrasse 34, 5020 Salzburg, Austria. Electronic address: hanno.stutz@sbg.ac.at.
authors.forename: Hanno
authors.initials: H
authors.lastname: Stutz
mesh_terms: D000485:Allergens / Q000032:analysis*; D029662:Betula; D053841:Electroosmosis; D019075:Electrophoresis, Capillary; D007477:Ions; D011058:Pollen; D011108:Polymers / Q000737:chemistry*; D011506:Proteins / Q000032:analysis
chemical_list: D000485:Allergens; D007477:Ions; D011108:Polymers; D011506:Proteins
keywords: Atomic force microscopy; Capillary zone electrophoresis; Protein separation; SMIL coating; Square capillary; Topography and recognition imaging