Effects of geometrical confinement in membrane pores onenzyme-based layer-by-layer assemblies

tMicro- and nanoporous systems incorporating bioactive molecules, such as enzymes, are very promisingsupports for biocatalysis. Here, we investigate the influence of geometrical confinement on the layer-by-layer (LbL) assembly of enzyme-based thin films, using the polyionic couple (chitosan/ß-lactamase)n. Thinfilms with different number of layers were prepared on flat silicon wafers and within cylindrical submi-cron pores of polycarbonate membranes to determine the impact of the confinement of macromoleculeson: (i) the LbL film growth, (ii) the enzyme loading, and (iii) the biocatalytic efficiency. Solid-state NMRis employed to estimate the amount of enzyme loaded in the different types of LbL films, and the enzymeactivity is determined by the study of the kinetics of nitrocefin hydrolysis. Film growth and loading ofenzyme occur faster in the confined medium, until pores reach saturation. Moreover, when LbL filmsare grown within nanopores, the weight fraction of enzyme is very high and remains constant alongthe build-up. Conversely, the relative amount of enzyme in flat films significantly decreases with thenumber of layers due to the partial exchange during the growth. Finally, our study emphasizes that theimmobilization of enzymes through LbL assembly in confined media can lead to very active surfaces witha restricted number of LbL cycle