Photon upconverting nanoparticles (UCNPs) are promising reporters for supersensitive bioaffinity assays because they can be detected completely without autofluorescence background. This is enabled by anti-Stokes shifted luminescence where low-energy infrared radiation is converted to high-energy emission at visible wavelengths. Because the excitation and emission are at an optical window for biomaterials, UCNPs can be detected even in challenging sample materials such as whole blood. However, wider use of UCNPs in bioaffinity assays is limited due to certain challenges. Recently, there has been ongoing studies on UCNP structural integrity and luminescence in water and the effect of surface modification to biomolecule interactions, which should still be studied further in order to use the full potential of UCNPs in bioaffinity assays.

The aim of this thesis was to study the applicability of UCNP reporters in sensitive homogeneous and heterogeneous bioaffinity assays. The applicability of UCNPs to homogeneous assays was demonstrated by introducing Eu3+ chelate as an acceptor in an upconversion resonance energy transfer based assay. The Eu3+ chelate’s intrinsic luminescence lifetime was longer than that of the UCNP donor and therefore the sensitized acceptor emission could be measured even after the donor’s luminescence had decayed resulting in higher signal-to-background ratios. Additionally, the potential of UCNPs as reporters for cross-correlation spectroscopy was demonstrated with a homogeneous sandwich immunoassay. The assay was based on simultaneous detection of two differently emitting UCNPs brought together by binding of an analyte. UCNPs would be suitable reporters for cross-correlation spectroscopy applications where traditional fluorescence reporters suffer from autofluorescence.

One of the major factors limiting the immunoassay sensitivity is non-specific binding of UCNP conjugates. In this thesis, the non-specific binding of poly(acrylic acid) (PAA)-functionalized UCNP conjugates was reduced significantly in heterogeneous sandwich immunoassays by adding free PAA to the buffer during reporter incubation. The free PAA most likely blocked those areas in solid support where the UCNP conjugate might have bound non-specifically. The reduction in non-specific binding enabled detection of three and half times lower analyte concentration demonstrating that high sensitivity assays can be achieved with UCNP reporters.

In addition, the ion dissolution from fluoride-based UCNPs and its effect on luminescence and structural integrity of nanoparticles was studied in water-based solutions. The ions were found to dissolve until solubility equilibrium was achieved and the fluoride ions were observed to have major impact on the dissolution. The UCNPs disintegrated completely in highly diluted suspensions resulting in the disappearance of luminescence. The ion dissolution was prevented by adding free fluoride ions to the solution, which was an important observation in order to use diluted UCNP concentrations in bioaffinity assays. The study also demonstrated the requirement for surface capping that would protect the UCNPs from environmental effects.