SERS nanosensors that report pH of endocytic compartments during FcεRI transit.

Recently, the development of an IgE receptor (FcεRI)-targeted, pH-sensitive, surface-enhanced Raman spectroscopy (SERS) nanosensor has been demonstrated by Nowak-Lovato and Rector (Appl Spectrosc 63:387-395, 2009). The targeted nanosensor enables spatial and temporal pH measurements as internalized receptors progress through endosomal compartments in live cells. Trafficking of receptor-bound nanosensors was compared at physiological temperature (37 °C) versus room temperature (25 °C). As expected, we observed markedly slower progression of receptors through low-pH endocytic compartments at the lower temperature. We also demonstrate the utility of the nanosensors to measure directly changes in the pH of intracellular compartments after treatment with bafilomycin or amiloride. We report an increase in endosome compartment pH after treatment with bafilomycin, an H(+) ATPase pump inhibitor. Decreased endosomal luminal pH was measured in cells treated with amiloride, an inhibitor of Na(+)/H(+) exchange. The decrease in amiloride-treated cells was transient, followed by a recovery period of approximately 15-20 min to restore endosomal pH. These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors.

Targeted surface-enhanced Raman scattering nanosensors for whole-cell pH imagery.

In this paper the ability to develop a gold-core, silver-shell surface-enhanced Raman scattering (SERS) nanosensor that has both a targeting component as well as a sensing component is demonstrated. The nanosensor is capable of targeting through the FcepsilonRI receptor-mediated endocytic pathway due to the adsorption of 2,4-epsilon-dinitrophenol-L-lysine (DNP) ligand on the nanoparticle surface. The nanosensor is also sensitive to pH changes in the endocytic vesicle within the pH range of 4.5-7.5 by 4-mercaptopyridine (4-MPy) adsorbed to the particle surface. The targeting and sensing moieties do not significantly interfere with each other's function. The sensing component of the nanosensor is calibrated with in vivo measurements of rat basophil leukemia (RBL-2H3) cells in standard buffer solutions using the ionophore nigericin, which serves to equilibrate the external [H(+)] concentration with that of the cell compartments. The targeting of the nanosensor is verified with a beta-hexosaminidase assay. It is also demonstrated that the targeted nanosensor is capable of making accurate cellular pH measurements in RBL-2H3 cells, out to ninety minutes after targeted nanosensor addition. Whole-cell, time-lapse, hyperspectral image cubes demonstrating endocytic vesicular pH changes during FcepsilonRI receptor mediated endocytosis are demonstrated.