Controlled Initiation of Enzymatic Reactions in Micrometer-Sized Biomimetic Compartments

Abstract

We present a technique to initiate chemical reactions involving few reactants inside micrometer-scale biomimetic vesicles (10(-12) to 10(-15) L) integral to three-dimensional surfactant networks. The shape of these networks is under dynamic control, allowing for transfer and mixing of two or several reactants at will. Specifically, two nanotube-connected vesicles were filled with reactants (substrate and enzyme, respectively) by microinjection. Initially, the vesicles are far apart and any diffusive mixing (on relevant experimental time scales) between the contents of the separated vesicles is hindered because of the narrow diameter and long axial extension of the nanotube. To initiate a reaction, the vesicles were brought close together, the nanotube was consumed by the vesicles and at a critical distance, the nanotube-vesicle junctions were dilated leading to formation of one spherical reactor, and hence mixing of the contents. We demonstrate the concept using a model enzymatic reaction, which yields a fluorescent product (two-step hydrolysis of fluorescein diphosphate by alkaline phosphatase), where product formation was measured as a function of time using a FRAP fluorescence microscopy protocol. By comparing the enzymatic activity with bulk measurements, the enzyme concentration inside the vesicle could be determined. Reactions could be followed for systems having as few as approximately 15 enzyme molecules confined to a reactor vesicle. To describe the experiments we use a simple diffusion-controlled reaction model and solve it using a survival probability approach. The agreement with experiment is qualitative, but the model describes the trends well. It is shown that the model correctly predicts (i) single-exponential decay after a few seconds, and (ii) that the substrate decay constant depends on the number of enzymes and geometry of reaction container. The numerical correction factor Lambda is introduced in order to ensure semiquantitative agreement between experiment and theory. It was shown that this numerical factor depends weakly on vesicle radius and number of enzymes, thus it is sufficient to determine this factor only once in a single calibration measurement.

Generation of phospholipid vesicle-nanotube networks and transport of molecules therein

“…Vesicle-nanotube networks are uniquely shapeable and have been functionalized and tailored to provide insights into microscale phenomena, such as local confinement of reactants and catalysts to initiate and follow the progress of enzymatic reactions 16,31 . Membrane protein integration was demonstrated 64 , as was the interconnection and interaction with biological cells and active surfaces 17,43,44 .…”

“…9a,b). Figure 9c shows an example of a branched network on the first daughter vesicle, which is created either directly from the daughter vesicle or, alternatively, by translating a vesicle along an existing nanotube 16,31 . Once-used needles can be reused for further injections, as long as sufficient backfilled solution remains and the tip is not damaged or contaminated.…”

“…The networks are fabricated from individual phospholipid molecules by a combination of selforganization processes, which lead to spherical bilayer membrane compartments, and subsequent forced shape transition operations, which transform selected membrane regions into nanotubes and new liposomes. The networks are useful as spatially separated, yet interconnected, chemical reactors of differentiated contents, allowing the study of membrane proteins and (bio)chemical reactions on biologically relevant size scales 16,17 .…”

Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways

“…There have also been elegant methods to form networked vesicle nanoreactors, with nanotubes facilitating reagent transfer between regions [21][22][23] . However, these require manual manipulation of individual vesicles using expensive apparatus, and are therefore unsuitable for scale-up and for potential use as functional devices.…”

Diffusive transport in networks built of containers and tubes

“…The walls are particle impenetrable ∂ n ρ( r, t) = 0 (2) where ∂ n ≡n · ∇, andn is the unit vector perpendicular to the wall. The total number of particles is a conserved quantity.…”