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Title Enzyme immobilization on protein-resistant PNIPAAm brushes: Impact of biotin linker length on enzyme amount and catalytic activity
Date 02.11.2018
Number 55118
Abstract Thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) brushes with terminal click functionality can be used to selectively immobilize enzymes. Exploiting their inherent protein-repellent and thus non-fouling properties, surfaces with specific bioactivity can be created in this way. This report describes the functionalization of alkyne-PNIPAAm brushes with two biotin linkers of different poly(ethylene glycol) (PEG) spacer length via click chemistry and the subsequent immobilization of streptavidin-conjugated horseradish peroxidase (SA-HRP) by the strong interaction between biotin and streptavidin. Spectroscopic ellipsometry is used to quantify the biotin and the SA-HRP amount on the PNIPAAm brushes. Enzyme activities are determined by UV-vis spectroscopy. A better accessibility to the alkyne-functionalized chain ends for the short biotin-PEG3 linker leads to a higher biotin amount on these PNIPAAm brushes, which in turn results in higher SA-HRP amounts on biotin-PEG3-modified brushes in the swollen (20·°C) and collapsed state (37·°C) compared to biotin-PEG23-modified ones. For both linkers, an increased immobilization temperature leads to higher SA-HRP amounts due to an enhanced reaction kinetics and mobility. All immobilized SA-HRP amounts are in range of 81–98% monolayer coverage, except for SA-HRP immobilized on biotin-PEG23-modified PNIPAAm brushes at 20·°C, exhibiting only 43% monolayer coverage. A high mobility of the biotin linker combined with a low surface loading of SA-HRP is found to be beneficial for the activity of SA-HRP. Hence, the highest specific activity as measured by HRP-catalyzed 3,3´,5,5´-tetramethylbenzidine (TMB) oxidation is found for a low SA-HRP amount on biotin-PEG23-modified brushes.
Publisher Colloids and Surfaces B: Biointerfaces
Identifier
Citation Colloids and Surfaces B: Biointerfaces 171 (2018) 351-357
DOI https://doi.org/10.1016/j.colsurfb.2018.07.047
Authors Rosenthal, A. ; Rauch, S. ; Eichhorn, K.-J. ; Stamm, M. ; Uhlmann, P.
Tags polymer brushes enzymes biocatalysis click chemistry

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