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Protein interactions in covalently attached dextran layers

1999; Elsevier BV; Volume: 13; Issue: 6 Linguagem: Inglês

10.1016/s0927-7765(99)00046-6

1873-4367

Jacob Piehler, Andreas Brecht, T. Hehl, Günter Gauglitz,

Microfluidic and Bio-sensing Technologies

Protein interactions with polymeric carbohydrates play an important role for application in chromatography, biomaterials and biophysics. In this study, we present a detailed morphological and functional characterization of covalently side-bound dextran layers by spectroscopic ellipsometry (SE) and reflectometric interference spectroscopy (RIfS). The surface chemistry was monitored step-by-step by ellipsometric characterization of the surface loading. Dextrans of various molecular masses (10–2000 kD) were immobilized leading to surface loadings between 3 and 8 ng mm−2. The refractive indices of the covalently attached dextran layers under atmospheric conditions (nD=1.51) were very close to the refractive index of a spin-coated dextran layer (nD=1.52) indicating dense and homogeneous coverage achieved by the coupling chemistry. Under buffer solution, refractive indices between 1.34 and 1.365 and thicknesses between 20 and 40 nm of these dextran layers were determined. A dextran concentration in the hydrated layers of 0.05–0.21 g cm−3 was estimated from the refractive index. The density and the thickness of the hydrated layers increased with molecular mass of the dextran. Non-specific binding was strongly reduced by the dextran layers and decreased with increasing thickness and density of the layer. Specific antibody binding to haptens immobilized in the dextran layer lead to an increase of both the density and the thickness of the layers. Time resolved detection by RIfS indicated significant decrease of protein mobility in the dextran layer. From these results we conclude that the functional properties of dextran layers with respect to protein interactions are determined by their effective pore-size, which is controlled by the number of bonds, the surface loading and the concentration of charged groups in the polymer.