Surface characterization of Langmuir-Blodgett films from bovine erythrocyte membranes (BEM)

IVAN FELSZTYNA; ANAHI V. TURINA; MARÍA A. PERILLO; EDUARDO M. CLOP

Congreso; Primeras Jornadas virtuales SAB 2020; 2020

This work was aimed at designing an enzyme-based biosensor. So, Langmuir films frombovine erythrocyte membranes (LFBEM) were prepared and transferred to alkylatedglasses (Langmuir-Blodgett films, LBBEM). Epifluorescence Microscopy (EFM) and BrewsterAngle Microscopy (BAM) were performed on LFBEM. Additionally, EFM and Atomic ForceMicroscopy (AFM) were performed on LBBEM. The LBBEM was used as the enzyme sourcefor measuring the activity of Bovine Erythrocyte Acetylcholinesterase (BEA).While the rheological behavior of LFBEM was compatible with an expanded monolayerthroughout the entire isotherm, in EFM and BAM images, it exhibited a markedtopographic heterogeneity which was associated to coexisting fluid domains.Remarkably, in BAM images at π ≥ 30 mN/m irregular dark regions with reflectivityvalues similar to the clean interface were found, suggesting the presence of cracks in thefilm.EFM images of LBBEM roughly conserved the topography of the original LFBEM but withless heterogeneity. The AFM images of LBBEM showed some structures with < 60 nmheight, which resembled closed vesicles and when transferred at 35 mN/m (a bilayerequilibrium surface pressure) it exhibited a ~4μm wide depressed regions of ~5 ηmdepth typical of the phase coexistence.Taken together, EFM, BAM and AFM images suggest that over the air-water interface, aswell as over the silanized glass substrate, the surface is mostly covered by a monolayerwith a few particles dispersed. It is worth to note that BEA present in LBBEM could retainits catalytic activity along several days of storage and maintained the expected kineticbehavior in the presence of some known enzyme modulators.In these systems, the use of natural membranes offers compositional and structuralcomplexity allowing the study of various phenomena of biophysical and cellular interestand facilitates, the building up of biosensors based on the activity of membrane boundenzymes preserving the protein´s natural environment.