Mutually-reactive, fluorogenic molecules have been explored as a simple and novel technique for in-solution biosensing. The hypothesis behind this work was that aggregating colloidal nanoemulsions into close proximity would cause rapid mixing of their contents. To take advantage of this effect, a novel pair of fluorogenic redox molecules were designed to remain in lipid-stabilized oil nanodroplets but mix once aggregated. First, the hydrophobic cyanine dye DiI was reduced with sodium borohydride to form a non-fluorescent hydrocyanine analog (HDiI). Hydrophobic quinone derivatives were then screened as oxidizing agents, and it was found that p-fluoranil oxidized non-fluorescent HDiI back to fluorescent DiI. Next, HDiI and p-fluoranil were loaded into non-toxic NEOBEE oil nanodroplets of average diameter ~600 nm that were stabilized by a monolayer of phospholipids and polymer-lipids, namely, DPPC, DSPE-PEG, and DSPE-PEG-biotin. For proof-of-concept studies, streptavidin was added leading to aggregation of droplets and content mixing, causing the appearance of red fluorescent aggregates within 30 min. Absence of streptavidin provided little or no aggregation or fluorescnece. Next, utilizing a sensitive Nanoparticle Tracking Analysis method (NanoSight, Malvern), the fluorescence of the droplets and their aggregates were recorded. By integrating the fluorescence emission of the tracked droplets, streptavidin could be detected down to a concentration of 100 fM. Subsequently, to apply this technique to a biologically relevant analyte, the droplets were reformulated to sense for Vascular Endothelial Growth Factor (VEGF), a biomarker for tumor metastasis. Using modified anti-VEGF aptamers attached to DSPE-PEG incorporated into the nanodroplet monolayer, VEGF could also be similarly detected down to 100 fM. This is a bulk sensing mechanism with only one complementary antibody/aptamer required, and with no washing steps involved, in contrast to most currently available sandwich assays. In future studies, the work described here will be expanded for biosesning of more complex analytes via antibody interactions.

In-solution biosensing via aggregation of nanodroplets containing mutually reactive, fluorogenic hydrocyanine/quinone reporter molecules

251st National Meeting (2016)

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