Controlled immobilization of His-tagged proteins for protein-ligand interaction experiments using Ni²⁺-NTA layer on glass surfaces

Article type: Research Article

Authors: Cherkouk, Charaf^{a; b; *} | Rebohle, Lars^b | Lenk, Jens^c | Keller, Adrian^b | Ou, Xin^b | Laube, Markus^c | Neuber, Christin^c | Haase-Kohn, Cathleen^c | Skorupa, Wolfgang^b | Pietzsch, Jens^{c; d; *}

Affiliations: [a] Technische Universität Bergakademie Freiberg, Institute of Experimental Physics, Freiberg, Germany | [b] Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany | [c] Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany | [d] Technische Universität Dresden, Department of Chemistry and Food Chemistry, Dresden, Germany

Correspondence: [*] Corresponding author: Charaf Cherkouk, Technische Universität Bergakademie Freiberg, Institute of Experimental Physics, Freiberg, Germany. Tel.: +49 3731 419147; E-mail: cherkouk@mailserver.tu-freiberg.de.

Correspondence: [*] Corresponding author: Jens Pietzsch, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany. Tel.: +49 351 2602622; E-mail: j.pietzsch@hzdr.de.

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.

Keywords: His-tagged proteins, glycated low density lipoproteins, microfluidic systems and biosensors, S100 proteins, self-assembled monolayers, soluble receptor for advanced glycation endproducts

DOI: 10.3233/CH-151950

Journal: Clinical Hemorheology and Microcirculation, vol. 61, no. 3, pp. 523-539, 2015