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Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy
journal contribution
posted on 2015-07-21, 00:00 authored by Jonas Schartner, Nina Hoeck, Jörn Güldenhaupt, Laven Mavarani, Andreas Nabers, Klaus Gerwert, Carsten KöttingProtein 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.