The impact of the spatial confinement of polystyrene-block-poly(acrylic acid) (PS-b-PAA) block copolymer (BCP) vesicles on the reactivity of encapsulated bovine pancreas trypsin is studied. Enzymes, as well as small molecules, are encapsulated with loading efficiencies up to 30% in BCP vesicles with variable internal volumes between 0.014 aL (internal vesicle diameter, din = 30 nm) and 8 aL (din = 250 nm), obtained by manipulating the vesicle preparation conditions. The kinetics of the trypsin-catalyzed reaction of a fluorogenic substrate inside and outside the vesicles is quantitatively estimated using fluorescence spectroscopic analyses in conjunction with the use of NaNO2 as selective quencher for non-encapsulated fluorophores. The values of the catalytic turnover number obtained for reactions in differently sized nanoscale reactors show a significant increase (up to ≈5×) with decreasing BCP vesicle volume, while the values of the Michaelis–Menten constant decrease. The observed increase in enzyme efficiency by two orders of magnitude compared to bulk solution is attributed to an enhanced rate of enzyme–substrate and molecule–wall collisions inside the nanosized reactors, as predicted in the literature on the basis of Monte Carlo simulations.
- fluorogenic substrates
- Block copolymers