title: Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.
abstract: A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.
authors.affiliation: Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden, The Netherlands; Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040, Madrid, Spain. Electronic address: emorenog@ucm.es.
authors.forename: Estefanía
authors.initials: E
authors.lastname: Moreno-Gordaliza
authors.affiliation: Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden, The Netherlands; Department of Molecular Cancer Research, Universitair Medisch Centrum Utrecht, Wilhelmina Kinder Ziekenhuis, Lundlaan 6, 3584, EA Utrecht, The Netherlands.
authors.forename: Edwin C A
authors.initials: EC
authors.lastname: Stigter
authors.affiliation: Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden, The Netherlands.
authors.forename: Petrus W
authors.initials: PW
authors.lastname: Lindenburg
authors.affiliation: Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hankemeier
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D057625:Isotachophoresis; D007779:Lactobacillus acidophilus / Q000737:chemistry; D008074:Lipoproteins / Q000097:blood; D015203:Reproducibility of Results; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins
keywords: Capillary coating; Capillary electrophoresis; Isotachophoresis; Lipoproteins; Silanization; Surface layer proteins