Enzyme immobilization on protein-resistant PNIPAAm brushes: impact of biotin linker length on enzyme amount and catalytic activity.

Graphical abstract Highlights • Non-fouling surfaces can be equipped with specific bioactivity. • Terminal click functionality useful for covalent attachment of enzymes on brushes. • Control of immobilized amount by spacer length and brush conformation. • Control of activity by spacer length and immobilized amount. 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-PEG 3 linker leads to a higher biotin amount on these PNIPAAm brushes, which in turn results in higher SA‑HRP amounts on biotin-PEG 3 -modified brushes in the swollen (20 °C) and collapsed state (37 °C) compared to biotin-PEG 23 -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-PEG 23 -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-PEG 23 -modified brushes. [ABSTRACT FROM AUTHOR]