CONICET | Buscador de Institutos y Recursos Humanos

This work was aimed at designing an enzyme-basedbiosensor. So, Langmuir films from bovine erythrocyte membranes (LFBEM)were prepared and transferred to alkylated glasses (Langmuir-Blodgett films, LBBEM).Epifluorescence Microscopy (EFM) and Brewster Angle Microscopy (BAM) wereperformed on LFBEM. Additionally, EFM and Atomic Force Microscopy(AFM) were performed on LBBEM. The LBBEM was used as theenzyme source for measuring the activity of Bovine Erythrocyte Acetylcholinesterase (BEA).While the rheological behavior of LFBEM wascompatible with an expanded monolayer throughout the entire isotherm, in EFMand BAM images, it exhibited a marked topographic heterogeneity which wasassociated to coexisting fluid domains. Remarkably, in BAM images at p ³ 30 mN/m irregular dark regions with reflectivityvalues similar to the clean interface were found, suggesting the presence ofcracks in the film.EFM images of LBBEM roughly conserved thetopography of the original LFBEM but with less heterogeneity. The AFM images of LBBEM showed somestructures with < 60 nm height, which resembled closed vesicles and whentransferred at 35mN/m (a bilayer equilibrium surface pressure) it exhibited a ~4mm wide depressed regions of ~5 nm depth typical of thephase coexistence. Taken together, EFM, BAMand AFM images suggest that over the air-water interface, as well as over thesilanized glass substrate, the surface is mostly covered by a monolayer with afew particles dispersed. It is worth tonote that BEA present in LBBEM could retain its catalytic activity alongseveral days of storage and maintained the expected kinetic behavior in thepresence of some known enzyme modulators.In these systems, the use of natural membranes offerscompositional and structural complexity allowing the study of various phenomenaof biophysical and cellular interest and facilitates, the building up ofbiosensors based on the activity of membrane bound enzymes preserving theprotein´s natural environment.