https://onlinelibrary.wiley.com/doi/10.1002/adhm.

201601190

The authors reported a nanoreactor consisting of liposome-decorated silica particles encapsulating

the enzyme glucose oxidase which is able to convert glucose into d-glucano-1,5-lactone and
hydrogen peroxide (H2O2). The intracellular enzymatic activity of the nanoreactors was indirectly
measured by monitoring the decrease in cell viability due to the H2O2 production

https://www.tandfonline.com/doi/full/10.1081/BIO-120028669

Quantification of the Encapsulation Efficiency

AChE activity is measured using the Ellman reaction (Ellman et
al., Citation[[1961]]). AChE catalyzes the hydrolysis of acetylthiocholine,
producing acetate and thiocholine. Thiocholine is then able to react with di-
thionitrobenzoic acid, producing a thiocholine coupled to a thio-nitro-benzoic
acid by a disulfide bond and a thio-nitro-benzoate. This molecule is detectable
by O.D. measurements at 412 nm with an ε = 13,600 M−1cm−1. AChE activity is
equal to the absorbance variation plotted vs. time.

To determine the fraction of encapsulated AChE, the solution was incubated in

a solution of the proteolytic enzyme pronase. Pronase does not pass through
the liposomal bilayer and therefore the encapsulated AChE is protected from
enzymatic digestion. Free enzyme and enzyme bound to the external surface
of the liposome are inactivated according to first order kinetics. The proportion
of encapsulated enzyme was estimated by recording the remaining activity
with 1 mM acetylthiocholine in the presence of 0.1% Triton X-100. Mixed lipid–
detergent micelles were found to be reversible inhibitors of AchE. Minor
inhibition kinetics of AChE by POPS and eggPC were recorded and the results
shown in this paper take those inhibitions into account. The full activity of
AChE was considered after correction by its rate of inhibition in the conditions
of each experiment.

We chose to encapsulate Drosophila melanogaster acetylcholinesterase

(AChE) as a reporter enzyme because of the availability of an easy and
accurate functional assay. Moreover, this type of enzyme is highly interesting
as a biosensor to detect insecticide residues (Villatte et al., Citation[[1998]]).
Here we demonstrate that 40% of the initial active enzyme can be
encapsulated in its fully functional state.
O.D. measurument: optical density