title: Controlled immobilization of His-tagged proteins for protein-ligand interaction experiments using Ni²⁺-NTA layer on glass surfaces.
abstract: Gold surfaces functionalized with nickel-nitrilotriacetic acid (Ni²⁺-NTA) as self-assembled monolayers (SAM) to immobilize histidine (His)-tagged biomolecules are broadly reported in the literature. However, the increasing demand of using microfluidic systems and biosensors takes more and more advantage on silicon technology which provides dedicated glass surfaces and substantially allows cost and resource savings. Here we present a novel method for the controlled oriented immobilization of His-tagged proteins on glass surfaces functionalized with a Ni²⁺-NTA layer. Exemplarily, the protein pattern morphology after immobilization on the Ni²⁺-NTA layer of His6-tagged soluble receptor for advanced glycation endproducts (sRAGE) was investigated and compared to non-oriented immobilization of sRAGE on amino SAM by using scanning electron microscopy (SEM). Moreover, we demonstrated interaction of immobilized sRAGE with three structurally different ligands, S100A12, S100A4, and glycated low density lipoproteins (glycLDL), by means of peak-force tapping atomic force microscopy (PF-AFM). We showed a clear discrimination of different protein-ligand orientations by differential height measurements.
authors.affiliation: Technische Universität Bergakademie Freiberg, Institute of Experimental Physics, Freiberg, Germany.|Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany.
authors.forename: Charaf
authors.initials: C
authors.lastname: Cherkouk
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Rebohle
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Lenk
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Keller
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany.
authors.forename: Xin
authors.initials: X
authors.lastname: Ou
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Laube
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Neuber
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.
authors.forename: Cathleen
authors.initials: C
authors.lastname: Haase-Kohn
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Skorupa
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.|Technische Universität Dresden, Department of Chemistry and Food Chemistry, Dresden, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Pietzsch
mesh_terms: D015374:Biosensing Techniques; D005898:Glass; D006639:Histidine / Q000235:genetics*; D008024:Ligands; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics*; D009942:Organometallic Compounds / Q000378:metabolism*; D009418:S100 Proteins
chemical_list: C471213:His-His-His-His-His-His; D008024:Ligands; D009842:Oligopeptides; D009942:Organometallic Compounds; D009418:S100 Proteins; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009571:Nitrilotriacetic Acid
keywords: His-tagged proteins; S100 proteins; glycated low density lipoproteins; microfluidic systems and biosensors; self-assembled monolayers; soluble receptor for advanced glycation endproducts