Real-Time Monitoring and Control of Nanoparticle Formation.

Methods capable of controlling synthesis at the level of an individual nanoparticle are a key step toward improved reproducibility and scalability in engineering complex nanomaterials. To address this, we combine the spatially patterned activation of the photoreductant sodium pyruvate with interferometric scattering microscopy to achieve fast, label-free monitoring and control of hundreds of gold nanoparticles in real time. Individual particle growth kinetics are well-described by a two-step nucleation-autocatalysis model but with a distribution of individual rate constants that change with reaction conditions.

Measuring Encapsulation Efficiency in Cell-Mimicking Giant Unilamellar Vesicles.

One of the main drivers within the field of bottom-up synthetic biology is to develop artificial chemical machines, perhaps even living systems, that have programmable functionality. Numerous toolkits exist to generate giant unilamellar vesicle-based artificial cells. However, methods able to quantitatively measure their molecular constituents upon formation is an underdeveloped area. We report an artificial cell quality control (AC/QC) protocol using a microfluidic-based single-molecule approach, enabling the absolute quantification of encapsulated biomolecules. While the measured average encapsulation efficiency was 11.4 ± 6.8%, the AC/QC method allowed us to determine encapsulation efficiencies per vesicle, which varied significantly from 2.4 to 41%. We show that it is possible to achieve a desired concentration of biomolecule within each vesicle by commensurate compensation of its concentration in the seed emulsion. However, the variability in encapsulation efficiency suggests caution is necessary when using such vesicles as simplified biological models or standards.

Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers.

Single-channel recording from pore-forming toxins (PFTs) provides a clear and direct molecular readout of toxin action. However to complete any mechanistic understanding of PFT behavior, this functional kinetic readout must be linked to the underlying changes in toxin structure, binding, conformation, or stoichiometry. Here we review how single-molecule imaging methods might be used to further our understanding of PFTs, and provide detailed practical guidance on the use of droplet interface bilayers as a method capable of examining both single-molecule fluorescence and single-channel electrical signals from PFTs.