Mimicry of peroxidase by immobilization of hemin on N-isopropylacrylamide-based hydrogel

Abstract

To mimic the peroxidase activity of horseradish peroxidase (HRP), hemin was chemically attached, through its carbon–carbon double bond, to N,N′-methylenebisacrylamide (MBA)-cross-linked N- isopropylacrylamide (NIPAAm) hydrogel to form a hemin-based mimetic enzyme [poly(NIPAAm)/MBA/hemin]. The newly synthesised mimetic enzyme exhibits a lower critical solution temperature (LCST) of 39.5 °C in neutral solution. It precipitates out of the aqueous solution as the temperature is raised to and above the LCST and redissolves when it is cooled below the LCST. This behavior is reversible. The characteristics of this mimetic enzyme were systematically studied with H₂O₂ as oxidizing agent and p-hydroxyphenylacetic acid as fluorogenic substrate. The results showed that it may be used as a novel substitute for peroxidase. The mimetic enzyme showed an optimum reaction pH of about 9.8, which is lower than that in hemin-catalyzed reaction systems where the optimum reaction pH is about 11. The activity of the mimetic enzyme is higher than that of hemin. The LCST behavior of the newly developed mimetic enzyme made its preparation, purification and further chemical modification easier and faster. The detection limits for H₂O₂ and poly(NIPAAm)/MBA/hemin are 1.5×10^–8 and 2.5×10^–9 mol l^–1, respectively.

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