Here we address an important roadblock that prevents the use of bright fluorescent nanoparticles as individual ratiometric sensors: the possible variation of fluorescence spectra between individual nanoparticles. Ratiometric measurements using florescent dyes have shown their utility in measuring the spatial distribution of temperature, acidity, and concentration of various ions. However, the dyes have a serious limitation in their use as sensors; namely, their fluorescent spectra can change due to interactions with the surrounding dye. Encapsulation of the d, e in a porous material can solve this issue. Recently, we demonstrated the use of ultrabright nanoporous silica nanoparticles (UNSNP) to measure temperature and acidity. The particles have at least two kinds of encapsulated dyes. Ultrahigh brightness of the particles allows measuring of the signal of interest at the single particle level. However, it raises the problem of spectral variation between particles, which is impossible to control at the nanoscale. Here, we study spectral variations between the UNSNP which have two different encapsulated dyes: rhodamine R6G and RB. The dyes can be used to measure temperature. We synthesized these particles using three different ratios of the dyes. We measured the spectra of individual nanoparticles and compared them with simulations. We observed a rather small variation of fluorescence spectra between individual UNSNP, and the spectra were in very good agreement with the results of our simulations. Thus, one can conclude that individual UNSNP can be used as effective ratiometric sensors.
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