Date of Award
Doctor of Philosophy (PhD)
Andrew P. Goodwin
Mark A. Borden
Daniel K. Schwartz
Nanodroplets (NDs) are liquid-in-liquid dispersions of ~100-800 nm size range that are often stabilized by a shell of lipids, polymer, proteins, or surfactants. NDs have been explored for a variety of biomedical applications, mostly involving drug formulation and delivery. However, the unique properties of encapsulated liquids, and the effects of interfacial chemistry on these properties, makes NDs potentially powerful candidates for new biosensing technologies. This dissertation explores different oil-in-water or fluorocarbon-in-water ND systems for in-solution sensing of biomarkers as both a platform for diagnostic assays and as a precursor to in vivo biosensing. Nanodroplets, because of their size, provide a large total surface area for analytes, making the assay both sensitive and fast compared to standard assay methods like ELISA. The type of response of the droplet to a stimulus also depends on the internal phase. This work includes NDs with three types of core materials: vegetable oil (fluorescent response), perfluorocarbon (PFC, acoustic response), and a thermotropic liquid crystal (LC, orientational response under polarized light). Each of these ND types is stabilized by a shell consisting of a primary saturated phospholipid and a lipopolymer, where the lipid shell is not only responsible for providing stability over reasonable time scales but also for recognizing analytes and causing a hierarchical change in the internal phase.
The first part of this study examined how ND response to a stimulus changes based on droplet aggregation. This was achieved by functionalizing the lipid monolayer shell of the NDs with aptamers or small molecules capable of specifically associating with dimeric or tetrameric analyte proteins. Through this process, vegetable oil NDs – doped with either a deactivated dye or an activating agent – were able to come together in the presence of a specific analyte, undergo content mixing, and generate a unique fluorescent signal. Similarly, PFC NDs were able to generate a higher acoustic response on aggregation. Streptavidin, as a proof-of-concept protein, and vascular endothelial growth factor, as a practical biomarker, were detected using droplet aggregation down to picomolar levels in bulk solution in 15-30 min.
The second part of this study focused on the effects of lipid monolayer phase separation and disruption on ND response. PFC NDs were found to have a heterogeneous monolayer, and acoustic response increased when unsaturated lipids were incorporated into the monolayer. Liquid crystal NDs were observed to transition from radial to bipolar orientations upon increasing the saturated lipid chain length above C16. Disruption of a gel phase C18 monolayer transitioned the orientation from bipolar back to radial. Using these observations for perfluorocarbon and LC droplets, a biologically relevant enzyme, phospholipase A2, was detected down to clinically relevant nanomolar levels in 15-30 min: the enzyme cleaves the lipid molecules in the monolayer and disrupts its organization, generating a higher acoustic contrast for PFC NDs and an alternate orientation for internal phase molecules in LC NDs.
In summary, the current study details the effects of the ND lipid monolayer composition, phase separation, geometry, and functionalization on the internal phase response to stimuli, thus providing a framework for testing the potential of fluorocarbon, hydrocarbon, and liquid crystal nanodroplets as in-solution biosensors.
Chattaraj, Rajarshi, "Engineering the Phospholipid Monolayer on Fluorocarbon, Hydrocarbon, and Liquid Crystal Nanodroplets for Applications in Biosensing" (2018). Mechanical Engineering Graduate Theses & Dissertations. 174.