A well-defined surface architecture is essential to generate water-dispersible UCNPs that are long-term stable and enable a wealth of bioanalytical applications.

Photon upconverting nanoparticles convert near-infrared into visible light (anti-Stokes emission), which strongly reduces the background of autofluorescence and light scattering in biological materials. Hexagonal NaYF(4) nanocrystals doped with Yb(3+) as the sensitizer and Er(3+)/Ho(3+)/Tm(3+) as the activator display at least two emission lines that respond differently to temperature changes. The ratio of the main emission line intensities enables a self-referenced optical readout of the temperature in the physiologically relevant range from 20 to 45 °C. Upconverting nanoparticles of the type NaYF(4):Yb, Er covered by an inactive shell of NaYF(4) are bright and allow for resolving temperature differences of less than 0.5 °C in the physiological range. The optical readout of this nanoparticle-based thermometer offers many options for imaging the two-dimensional distribution of temperature.

Photon-upconverting nanoparticles (UCNPs) have attracted much interest as a new class of luminescent label for the background-free detection in bioanalytical applications. UCNPs and other nanoparticles are commonly coated with a silica shell to improve their dispersibility and chemical stability in aqueous buffer and to incorporate functional groups for subsequent bioconjugation steps. The process of silica coating, however, is difficult to control without suitable analytical and preparative methods. Here, we have introduced agarose gel electrophoresis for the analysis and purification of silica-coated UCNPs. The silica shell can be doped with a fluorescent dye for direct detection in the gel without influencing the structure or electrophoretic mobility of the nanoparticles. The preparation of a bare silica shell by reverse microemulsion resulted in individual nanoparticles but also distinct aggregates that could be separated and isolated from the agarose gel. In contrast, the preparation of an ultrathin carboxylated silica shell yielded non-aggregated UCNPs only that could be directly used for protein conjugation. Agarose gel electrophoresis has also facilitated an efficient separation of protein-UCNP conjugates from excess reagents.