Enzymatic reactivity in geometrically confined spaces

ENVR 150

Zhen Wang, zwang3@nd.edu and Paul W. Bohn, pbohn@nd.edu. Department of Chemical and Biomolecular Engineering, University of Notre Dame, 182 Fitzpatrick Hall, Notre Dame, IN 46556
Study of macromolecular reactivity in confined environments is quite challenging and very few reports are available so far. The confined environment may affect the molecular recognition event because the access to active sites may be restricted. Our work is aimed at comparing the reactivity of biomolecules in confined spaces, such as nanopores, with that in free solution. Single cylindrical nanopores are created by focused ion beam (FIB) milling of a polymer membrane consisting of a layer of co-poly(methyl methacrylate-glycidyl methacrylate) (PMMA-GMA) sandwiched between two layers of PMMA. Horseradish peroxidase (HRP) was immobilized on the surface of PMMA/PGMA membrane. A PDMS microchannel was sealed to the membrane, then laser-induced fluorescence (LIF) was employed to monitor the enzymatic oxidation of amplex red by hydrogen peroxide. Immobilized HRP shows very high activity, reflected by the much higher maximum reaction rate (1.28 µM/sec) than the free-solution value (0.032 µM/s).