Polymer nanoparticles (PNP) are increasingly used as tools in (bio)analytics. Typical applications of PNP include carriers for drugs, carriers for dye molecules for signal amplification in optical assays, nanosensors and targeted probes for bioimaging studies. These applications in life sciences impose stringent requirements on particle size, size distribution, morphology, colloidal stability, biocompatibility, optical properties, and ease of surface functionalization with, for example, targeting ligands, sensor molecules, and linkers. PNP, including the polystyrene particles (PSP) which are in the focus of this work, are non-fluorescent by nature but can be made fluorescent with the aid of luminophores such as organic dyes. Fluorescent PNP can be obtained by coupling reactive fluorophores to the surface groups of the PNP or by encapsulation of fluorophores into the PNP matrix. The latter approach is particularly attractive due to its versatility since the encapsulation into preformed PNP does not require luminophores with reactive functional groups (FGs), but rather only requires hydrophobic luminophores. Moreover, the fluorophores are protected from the potentially fluorescence quenching environment surrounding the PNP matrix and the reactive groups on the PNP surface can be exclusively used for the attachment of targeting ligands. Dye loading of PNP typically requires a dye-specific optimization of the loading concentration with respect to signal strength as conventional dyes commonly form barely emissive or even non-fluorescent aggregates at high loading concentrations. Exceptions exist, which are dyes with propeller-like groups that show aggregation-induced emission (AIE). These dyes can be loaded with high concentrations into PNP without detrimental fluorescence quenching effects and can even exhibit fluorescence and hence signal amplification upon aggregation. Due to these attractive properties, AIE dyes for use in PNP were investigated. The spectroscopic properties of different AIE dye derivatives were systematically studied in organic solvents, solvent−water mixtures, and in the solid state. Dyes with optimal performance were entrapped in PSP. The staining of PSP with these AIE dyes resulted in a considerable increase in the dye fluorescence quantum yield and lifetime, reflecting the combined influence of the restricted molecular motion and the reduced polarity of the dye microenvironment. Functionalization of undoped and dye loaded PNP with, for example, targeting ligands requires knowledge of the chemical nature and total amount of the surface groups as well as the amount of surface FGs accessible for coupling reactions such as the conjugation of biomolecules. These numbers can differ considerably depending on factors including particle morphology and sterical constraints. Ideal methods for surface group quantification should be robust, reliable, and fast. Moreover, they should not require expensive instrumentation and should be versatile to enable the characterization of a broad variety of particle systems independent of their optical properties, including systems that scatter or include systems with encoded dyes. For this respect, we studied a variety of optical assays for the quantification of carboxy and amino surface groups commercial and custom-made particles with varying surface group densities. We performed these studies using both conventional reporters such as fluorescein derivatives, Fluram, or IR 797 as well as synthetically customized cleavable labels. Special emphasis was dedicated to the development of a platform of cleavable and multimodal labels which consist of a suitable reactive group such as NHS-esters or amine, a quantitatively cleavable linker such as disulfide, and an optically active moiety such as 2-thiopyridone for optical assays. Conventional reporters are measured when bound to the particle surface, which renders the resulting optical signals prone to distortions by scattering and interferences from encoding dyes. In contrast, these cleavable labels can be detected photometrically or fluorometrically in the supernatant after quantitative cleavage of the linker. Moreover, the linker unit is designed in such a way that the products of the cleaved linkers remaining at the particle surface can also be detected optically. In addition, the presence of a heteroatom such as sulfur, nitrogen or fluorine in the reporter and/or the linker can be detected by an analytical method relying on a different measurement principle. This allows for straightforward validation by method comparison with, for instance, ICP-OES. Thereby, FGs on a broad variety of different particles such as PNP, silica nanoparticles (NPs), and metal particles can also be quantified.
