Evaluation of suitability and detection range of fluorescent dye-loaded nanoliposomes for sensitive and rapid sensing of wide ranging osmolarities.

Abstract

Measurement of osmolarity is critical for optimizing bioprocesses including antibody production and detecting pathologies. Thus, rapid, sensitive, and in situ sensing of osmolarity is desirable. This study aims to develop and assess the suitability of calcein- and sulforhodamine-loaded nanoliposomes for ratiometric sensing of osmolarity by fluorescence spectroscopy and evaluate the range of detection. The detection is based on concentration-dependent self-quenching of calcein fluorescence (sensor dye at 6-15 mM) and concentration-independent fluorescence of sulforhodamine (reference dye) due to osmotic shrinkage of the nanoliposomes when exposed to hyperosmotic solutions. Using mathematical modeling, 6 mM calcein loading was found to be optimal to sense osmolarity between 300 and 3000 mOsM. Calcein (6 mM)- and sulforhodamine (2 mM)-loaded nanoliposomes were produced by thin-film hydration and serial extrusion. The nanoliposomes were unilamellar, spherical (108 ± 9 nm), and uniform in size (polydispersity index [PDI] 0.12 ± 0.04). Their shrinkage induced by exposure to hyperosmotic solutions led to rapid self-quenching of calcein fluorescence (F_Green), but no effect on sulforhodamine fluorescence (F_Red) was observed. F_Green/F_Red decreased linearly with increasing osmolarity, obeying Boyle van't Hoff's relationship, thus proving that the nanoliposomes are osmosensitive. A calibration curve was generated to compute osmolarity based on F_Green/F_Red measurements. As a proof-of-concept, dynamic changes in osmolarity in a yeast-based fermentation process was demonstrated. Thus, the nanoliposomes have great potential as sensors to rapidly and sensitively measure wide-ranging osmolarities.