49. Long-Term Quantitatively Imaging Intracellular Chloride Concentration Using a Core-/Shell-Structured Nanosensor and Time-Domain Dual-Lifetime Referencing Method

ABSTRACT: Luminescence lifetime based nanosensors for chloride ion were designed by incorporating a luminescent ruthenium dye (Ru(1,10-phenanthroline)3)) inside silica nanoparticles, and chemically labelling their outer surface with chloride ion sensitive fluorescent dyes (N,N'-bis(carboxypropyl)-9,9'-biacridine). The nanosensor surface was further functionalized with positively-charged amino groups to facilitate intracellular uptake via endocytosis and target lysosomes. The nanosensors have an average diameter of 52 nm, and are monodispersed in aqueous solutions. Due to the long lifetime of the reference ruthenium dye, sensor response can be analyzed using the time-domain dual lifetime referencing (td-DLR) approach. The use of pulsed excitation in td-DLR rather than intense continuous illumination in ratiometric measurement greatly prevents dye from photobleaching, which significantly improves measurement stability and reproducibility for long-term monitoring. At optimum condition, the sensor can measure chloride concentration in the range of 0-200 mM with a large ratiometric signal change from 140.9 to 40.2. Combined with our custom-built microscopic td-DLR system, variations of intracellular chloride concentration in lysosome were imaged quantitatively with high spatial resolution and accuracy.

KEYWORDS: fluorescence, ion-selective, nanoparticle, lifetime, imaging