title: Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy.
abstract: Protein immobilization studied by attenuated total reflection Fourier transform infrared (ATR-FT-IR) difference spectroscopy is an emerging field enabling the study of proteins at atomic detail. Gold or glass surfaces are frequently used for protein immobilization. Here, we present an alternative method for protein immobilization on germanium. Because of its high refractive index and broad spectral window germanium is the best material for ATR-FT-IR spectroscopy of thin layers. So far, this technique was mainly used for protein monolayers, which lead to a limited signal-to-noise ratio. Further, undesired protein-protein interactions can occur in a dense layer. Here, the germanium surface was functionalized with thiols and stepwise a dextran brush was generated. Each step was monitored by ATR-FT-IR spectroscopy. We compared a 70 kDa dextran with a 500 kDa dextran regarding the binding properties. All surfaces were characterized by atomic force microscopy, revealing thicknesses between 40 and 110 nm. To analyze the capability of our system we utilized N-Ras on mono-NTA (nitrilotriacetic acid) functionalized dextran, and the amount of immobilized Ras corresponded to several monolayers. The protein stability and loading capacity was further improved by means of tris-NTA for immobilization. Small-molecule-induced changes were revealed with an over 3 times higher signal-to-noise ratio compared to monolayers. This improvement may allow the observation of very small and so far hidden changes in proteins upon stimulus. Furthermore, we immobilized green fluorescent protein (GFP) and mCherry simultaneously enabling an analysis of the surface by fluorescence microscopy. The absence of a Förster resonance energy transfer (FRET) signal demonstrated a large protein-protein distance, indicating an even distribution of the protein within the dextran.
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Schartner
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Hoeck
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Güldenhaupt
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Laven
authors.initials: L
authors.lastname: Mavarani
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nabers
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University, 44801 Bochum, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
mesh_terms: D005591:Chemical Fractionation; D003911:Dextrans / Q000737:chemistry / Q000302:isolation & purification*; D005857:Germanium / Q000737:chemistry* / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D015394:Molecular Structure; D010316:Particle Size; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D000097573:Red Fluorescent Protein
chemical_list: D003911:Dextrans; D005857:Germanium; D049452:Green Fluorescent Proteins; D055767:Immobilized Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein