26710239

PMC5051691

Mutually reactive, fluorogenic molecules are presented as a simple and novel technique for in-solution biosensing. The hypothesis behind this work was that aggregating droplets 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 droplets but mix once aggregated. First, the hydrophobic cyanine dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) was reduced with sodium borohydride to form a nonfluorescent analog (HDiI). Hydrophobic quinone derivatives were then screened as oxidizing agents, and it was found that p-fluoranil oxidized nonfluorescent HDiI back to fluorescent DiI. Next, HDiI and p-fluoranil were loaded into NEOBEE oil nanodroplets of average diameter 600 nm that were stabilized by a monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol (PEG), and DSPE-PEG-biotin. Addition of streptavidin caused aggregation of droplets and the appearance of red fluorescent aggregates within 30 min. Next, Nanoparticle Tracking Analysis was used to record the fluorescence of the droplets and their aggregates. By integrating the fluorescence emission of the tracked droplets, streptavidin could be detected down to 100 fM. Finally, the droplets were reformulated to sense for vascular endothelial growth factor (VEGF), a biomarker for tumor metastasis. Using anti-VEGF aptamers attached to DSPE-PEG incorporated into the nanodroplet monolayer, VEGF could also be detected down to 100 fM.

CDC Public Access

R00 CA153935/CA/NCI NIH HHS/United States
S10 RR026641/RR/NCRR NIH HHS/United States
R21 EB018034/EB/NIBIB NIH HHS/United States
DP2EB020401/DP/NCCDPHP CDC HHS/United States
DP2 EB020401/EB/NIBIB NIH HHS/United States
R00CA153935/CA/NCI NIH HHS/United States
S10RR026641/RR/NCRR NIH HHS/United States
R21EB018034/EB/NIBIB NIH HHS/United States