Poster Presentation 2014 International Biophysics Congress

Mass-production, characterization, functionalization and imaging of nanoruby photoluminescent probes towards imaging of GPCR-signaling events, with single-particle precision (#424)

Varun K.A. Sreenivasan 1 , Wan Aizuddin Wan Razali 1 , Carlo Bradac 1 , Ekaterina A. Grebenik 1 , Marina Santiago 1 , Ewa M. Goldys 1 , Mark Connor 1 , Andrei Zvyagin 1
  1. Macquarie University, North Ryde, NSW, Australia

Cell signaling studies are hindered by the challenge of simultaneously visualizing more than one pathway downstream of receptor activation. We introduce newly developed nanorubies, characterized with excellent biocompatibility, ready surface functionalization, colloidal stability, strong photoluminescence, high photostability, narrow emission spectra and long emission lifetime, to simultaneously visualize opioid-triggered cellular hyperpolarization and ligand-receptor internalization.

Biocompatible nanoruby particles were synthesized from bulk ruby crystals using a high-energy ball milling technique. Particle separation and chemical etching procedures were established in similar alumina nanocrystals, which resulted in nanoalumina with a median size of 25 and 50 nm, based on TEM and DLS analyses, respectively, with 96% purity based on EDS. As a step towards nanoruby-opioid conjugation, nanoalumina surface was biofunctionalized with amine groups, replacing the pristine hydroxyl groups, to enable direction covalent conjugation of carboxyl-bearing opioid ligands. The –NH2 terminal groups were stable for several days of testing, monitored by a positive shift in the zetapotential by >20 mV and absorption peaks in the infrared spectrum at 1490, 2795-2991, 1330 and 1490 nm, corresponding to primary amine, methyl groups, C-Si and C-N bonds. Fluorescence imaging of single nanorubies was confirmed by correlative imaging with atomic force microscopy. Single particles of size ≈30 nm were visualized with diffraction limited resolution, over an area of 100 m x 100 m with ≈1 s imaging time. Single nanorubies appeared conspicuous in highly fluorescent cellular and hyperpolarization-sensitive dye-rich environment, made possible by time-gated photoluminescence detection scheme. These results open up the possibility of ultrafast, simultaneous, single-particle sensitive imaging of two linked cell-signaling events that follow opioid:opioid-receptor binding, namely, hyperpolarization and ligand internalization.