The surface modification of upconverting nanoparticles was a main focus of this thesis and a wide variety of different functionalities were introduced on the nanoparticle surface in the process. The growth of a silica shell with a preferably simultaneous silanization proved to be a convenient way to create both a hydrophilic surface crucial for bioanalytical applications and an accessible functionality for subsequent modification. The utilization of silanol, amine, carboxyl, or phosphonate groups yielded UCNPs with strongly differing stabilities in dispersion and surface charges. A strong aggregation was observed for both an amine modification and the non-modified silanol groups of a pure silica shell with a tendency enhancement for a reduced shell thickness. Furthermore, surface phosphonate groups led to the formation of aggregates with high uniformity in both size and shape. In contrast, an optimized modification process to introduce carboxylic acids on the surface yielded monodisperse UCNPs with a diameter of 13 nm that are efficiently stabilized in aqueous dispersion. The presence of these predominantly single nanoparticles in high abundance was confirmed both by dynamic light scattering and transmission electron microscopy studies. This high monodispersity was also realizable by a ligand exchange utilizing the polymer polyacrylic acid. The method applied relied on a prior exchange of the hydrophobic oleic acid with BF4- ions allowing a subsequent modification with the polymer with simplified process conditions and reduced expenditure of time.
Furthermore, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) was studied as a potential beneficial coupling reaction since it merged the advantages of the "Click chemistry" and "bioorthogonal reactions" concept. The associated alkyne and azide groups were bound to the nanoparticle surface by silanization of silica-coated UCNPs and the nanoparticles differed drastically in their dispersion stability. While an azide-functionalized surface exerted a certain degree of stabilization, alkyne functionalities led to a strong nanoparticle aggregation and precipitation in a very short time. However, both functional groups allowed an efficient and highly specific binding of their respective counterpart shown by an exemplary Click reaction with a fluorophore and also the important role of the copper(I) catalyst was demonstrated. The attachment of more complex molecules including biochemical functionalities or additionally stabilizing moieties revealed a good feasibility of a modification by the copper-catalyzed azide-alkyne cycloaddition. A simultaneous modification of the UCNPs with azide and carboxyl groups led to an enhanced nanoparticle stability compared to a pure azide functionalization.
Studies about the creation of a biomimetic nanoparticle surface comprised the encapsulation of UCNPs by virus capsid leading to the formation of virus-like particles (VLPs). After the process feasibility was confirmed to both dissemble and reassemble the capsid of a brome mosaic virus (BMV), small nanoparticles with an optimized surface were utilized to act as an artificial nucleation grain for self-assembly. These modified UCNPs met the three requirements for encapsulation: a hydrophilic surface to be dispersible in aqueous systems, an absolute diameter below 16 nm to fit in the capsid cavity of the BMV, and a negative surface charge to initiate the self-assembly of the capsomers similar to the viral RNA. In addition to the options described, citric acid was investigated as a potential surface ligand. While silanol and phosphonate groups were inappropriate to induce any kind of self-assembly, a form of unspecific interaction of the viral proteins with carboxylic acids was found both for covalent and non-covalent methods. An exception was citric acid, since it was prone to an irreversible removal from the UCNP surface during the VLP preparation process. However, for all nanoparticles with a modified surface no clear evidence of encapsulation was found as confirmed by both TEM imaging and immunogold staining.
The second research focus of this thesis was the development of a new imaging method of UCNPs. At first, different imaging parameters of the scan mode of a Hidex Plate Chameleon Multilabel Detection Platform with a 980 nm excitation source were studied and optimized for UCNPs of the type NaYF4: Yb3+, Er3+ or NaY4:Yb3+, Tm3+. A collecting time from 250 to 500 ms was sufficient to obtain an upconversion emission signal distinguishable from the background and to minimize laser-induced damages to the sample due to heating effects. Furthermore, both the overall emission of the UCNPs and the emission in a narrow wavelength domain were suitable as the detection signal for the image acquisition. The respective filter or filter combination additionally influenced the signal-to-noise ratio and thus the detection sensitivity. While the green emission of erbium-doped UCNPs was favorable in this regard, limitations of the detector sensitivity in the near infrared range hampered the utilization of the strong near infrared emission of thulium-doped UCNPs for scans with sensitive detection. The lateral resolution of the resulting scan images was reduced to 200 µm providing both a good resolution of luminescent structures and a good discrimination of signal and background. Since a lower scan point distance resulted in longer scan times without additional structural information, a lateral resolution of 200 µm was defined as the lower limit of the lateral resolution of the scan modus. Finally, a limit of detection of 1 ng was determined for both erbium- and thulium-doped UCNPs with high accuracy regardless of the emission utilized as the detection signal.
The applicability of this optimized process to real samples was demonstrated for both electrophoresis gels and lateral flow assays. The imaging of these gels and assays showed a high accuracy and reproducibility and allowed a good discrimination of the UCNP signal from the background and an illustration of differences in the UCNP concentrations or sample materials. Furthermore, the downconversion emission of fluorescein doped in the silica shell of the UCNPs allowed a comparison between the imaging methods based either on up- and downconversion. In addition to a high conformity of the images regarding the position and intensity of the gel bands the absence of any signal from the fluorophore in the upconversion scan images of the gels confirmed a high discriminability of both signal types. This enables a bimodal readout. Moreover, the evaluation of the lateral flow assays yielded a limit of detection of the exemplary analyte "Schistosoma circulating anodic antigen (CAA)" of 44 or 64 pg/mL for the wet and dry condition of the array, respectively. For these measurements the the overall emission of the erbium-doped UCNPs was utilized as the detection signal. Consequently, a high sensitivity of this new imaging method was evident compared to other instrument options.
Finally, the scan mode of the Chameleon reader was also utilized for studies about a potential enhancement of the upconversion emission by surface plasmon resonance of a gold surface. Both NaYF4: Yb3+, Er3+ or NaY4:Yb3+, Tm3+ were applied on a gold or silica surface of a commercial wafer with gold electrodes whose complete surfce was modified before with the same functionality to ensure the same chemical properties. The comparison of the emission intensities of UCNP on the different surface materials indicated a strong dependency of the enhancement effect on the emission wavelength. While the intensity of the green or near infrared emission of erbium- or thulium-doped UCNPs was increased by the gold surface, the overall emission of both UCNP types was reduced indicating a simultaneous quenching of the upconversion emission at other wavelengths
