Cherkouk Charaf, Rebohle Lars, Lenk Jens, Keller Adrian, Ou Xin, Laube Markus, Neuber Christin, Haase-Kohn Cathleen, Skorupa Wolfgang, Pietzsch Jens
Technische Universität Bergakademie Freiberg, Institute of Experimental Physics, Freiberg, Germany.
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Ion Beam Physics and Materials Research, Dresden, Germany.
Clin Hemorheol Microcirc. 2015;61(3):523-39. doi: 10.3233/CH-151950.
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.
文献中广泛报道了用镍-次氮基三乙酸（Ni²⁺-NTA）作为自组装单分子层（SAM）功能化的金表面，用于固定组氨酸（His）标签的生物分子。然而，对微流控系统和生物传感器的需求不断增加，使得越来越多地利用硅技术，该技术提供了专用的玻璃表面，并在很大程度上实现了成本和资源的节约。在此，我们提出了一种在经Ni²⁺-NTA层功能化的玻璃表面上可控定向固定His标签蛋白的新方法。例如，通过扫描电子显微镜（SEM）研究了His6标签的晚期糖基化终产物可溶性受体（sRAGE）固定在Ni²⁺-NTA层上后的蛋白质图案形态，并与sRAGE在氨基SAM上的非定向固定进行了比较。此外，我们借助峰值力轻敲原子力显微镜（PF-AFM）证明了固定化的sRAGE与三种结构不同的配体S100A12、S-100A4和糖化低密度脂蛋白（glycLDL）之间的相互作用。我们通过差分高度测量清楚地分辨出了不同蛋白质-配体取向。
