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 Fc<i>ε</i>RI receptor-mediated endocytic pathway due to the adsorption of 2,4-<i>ε</i>-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 <i>in vivo</i> measurements of rat basophil leukemia (RBL-2H3) cells in standard buffer solutions using the ionophore nigericin, which serves to equilibrate the external [H<sup>+</sup>] concentration with that of the cell compartments. The targeting of the nanosensor is verified with a β-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 Fc<i>ε</i>RI receptor mediated endocytosis are demonstrated.
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