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A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0−9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.
Bioanalytical, diagnostic, and security applications require the fast and sensitive determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats.[1,2] Ideal candidates for spectral encoding and multiplexing schemes are luminescent nanocrystals like semiconductor quantum dots (QDs), particularly Cd-containing II/VI QDs with their narrow and symmetric emission bands. With the availability of relatively simple and inexpensive instrumentation for time-resolved fluorescence measurements, similar strategies utilizing the compound-specific parameter fluorescence lifetime or fluorescence decay kinetics become increasingly attractive.[3-5] The potential of different types of QDs like II/VI, III/V and Cd-free ternary QDs such as AgInS (AIS) QDs for lifetime-based encoding and multiplexing has been, however, barely utilized, although the lifetimes of these nanocrystals cover a time windows which is barely accessible with other fluorophores. Here we present a brief insight into the photophysics of AIS QDs and show the potential of dye- and QD-encoded beads for lifetime-based encoding and detection schemes in conjunction with flow cytometry and fluorescence lifetime imaging microscopy