Highly Sensitive Laser Scanning of Photon-Upconverting Nanoparticles on a Macroscopic Scale.

An upconversion laser scanner has been optimized to exploit the advantages of photon-upconverting nanoparticles (UCNPs) for background-free imaging on a macroscopic scale. A collimated 980 nm laser beam afforded high local excitation densities to account for the nonlinear luminescence response of UCNPs. As few as 2000 nanoparticles were detectable, and the linear dynamic range covered more than 5 orders of magnitude, which is essentially impossible by using conventional fluorescent dyes. UCNPs covered by a dye-doped silica shell were separated by agarose gel electrophoresis and scanned by a conventional fluorescence scanner as well as the upconversion scanner. Both optical labels could be detected independently. Finally, upconversion images of lateral flow test strips were recorded to facilitate the sensitive and quantitative detection of disease markers. A marker for the parasitic worm Schistosoma was used in this study.

Surface modification and characterization of photon-upconverting nanoparticles for bioanalytical applications.

Photon-upconverting nanoparticles (UCNPs) can be excited by near-infrared light and emit visible light (anti-Stokes emission) which prevents autofluorescence and light scattering of biological samples. The potential for background-free imaging has attracted wide interest in UCNPs in recent years. Small and homogeneous lanthanide-doped UCNPs that display high upconversion efficiency have typically been synthesized in organic solvents. Bioanalytical applications, however, require a subsequent phase transfer to aqueous solutions. Hence, the surface properties of UCNPs must be well designed and characterized to grant both a stable aqueous colloidal dispersion and the ability to conjugate biomolecules and other ligands on the nanoparticle surface. In this review, we introduce various routes for the surface modification of UCNPs and critically discuss their advantages and disadvantages. The last part covers various analytical methods that enable a thorough examination of the progress and success of the surface functionalization.

Electrophoretic characterization and purification of silica-coated photon-upconverting nanoparticles and their bioconjugates.

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.

Photon upconverting nanoparticles for luminescent sensing of temperature.

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.