Surface interaction of well-defined, concentrated poly(2-hydroxyethyl methacrylate) brushes with proteins

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Abstract

The interaction of concentrated polymer brushes with proteins was chromatographically investigated. By the use of surface-initiated atom transfer radical polymerization, a low-polydispersity poly(2-hydroxyethyl methacrylate) (PHEMA) was densely grafted onto the inner surfaces of silica monoliths with mesopores of about 50 and 80 nm in mean size. The graft density reached 0.4–0.5 chains/nm2. The 80-nm-mesopore monolithic column with the concentrated PHEMA brush was characterized through the elution of low-polydispersity pullulans with different molecular weights, clearly showing two modes of size exclusion, that is, one by the mesopores and the other by the brush phase. The latter mode gave a sharp separation with a critical molecular weight (size-exclusion limit) of about 1000. This molecular size of pullulan was comparable to the distance between the nearest-neighbor graft points. The elution behaviors of five proteins of different sizes (bovine serum thyroglobulin, bovine serum immunoglobulin G, bovine serum albumin, horse heart myoglobin, and bovine serum aprotinin) were studied with this PHEMA-grafted column. The smallest protein, aprotinin, with a pullulan-reduced molecular weight slightly larger than the critical value of 1000, was eluted much behind the corresponding pullulan, and this indicated that it barely got into the brush layer, suffering from a strong affinity interaction within the brush. On the other hand, the other four larger proteins were eluted at the same elution volumes as the equivalent pullulans, and this meant that they were perfectly excluded from the brush layer and separated only in the size-exclusion mode by the mesopores without an affinity interaction with the brush surface. This excellent inertness of the concentrated brush in the interaction with the large proteins should afford the system long-term stability against biofouling. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4795–4803, 2007

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