Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging.
Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated.
Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria.
Michael
M
Leitner
Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria; Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
Lorenz G
LG
Stock
Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria.
Lukas
L
Traxler
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.
Laurent
L
Leclercq
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
Klaus
K
Bonazza
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
Gernot
G
Friedbacher
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.
Hervé
H
Cottet
Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria; Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
Hanno
H
Stutz
Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: andreas.ebner@jku.at.
Andreas
A
Ebner
D000268:Adhesiveness; D019075:Electrophoresis, Capillary / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D011108:Polymers / Q000737:chemistry*
D011108:Polymers
Adhesion forces; Atomic force microscopy; Capillary electrophoresis; Recognition imaging; SMIL coating