Upconversion nanoparticles as the basis for versatile intracellular luminescence bioassays

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The intention of the proposed work is to learn to build multi-functional nanoparticle (NP) platforms for intracellular analysis of dynamic processes, and for interventions in cellular processes. The unique optical resonance energy transfer properties of luminescent NPs allow for local interrogation of selective biomolecular interactions at a distance scale confined to the surface of the NP, and the luminescence can also be used for induction of photo-reactions that can release a cargo or activate a bioprobe associated with the NP. NPs are made to luminescence by irradiation with a low-power laser. Excitation wavelengths towards the infrared would be well suited for penetrating into complex biological samples and would reduce background scatter and autofluorescence, but typically offer insufficient energy to stimulate electronic excitation to achieve the desired optical interrogation processes. A desire to optically switch on the functionality of bioprobes or to release a cargo conjugated to NPs also is limited by insufficient energy at long wavelengths for activation of photo-reactions. An elegant approach to convert near-infrared (NIR) excitation radiation into ultraviolet (UV) and visible light can be achieved by an upconversion process in which two or more photons are absorbed and then combined. This happens within upconversion nanoparticles (UCNPs) containing lanthanides. UCNPs can serve as donors to transfer energy to proximal acceptors via luminescence resonance energy transfer (LRET), providing a transduction method that is sensitive only to interactions at molecular distances, and affording opportunity to drive photo-cleavage reactions on the surface of UCNPs. The research work will address issues that must be overcome before such technology will be effective for intracellular studies, including: Project 1 - Core-shell-shell UCNP designs that can stabilize high luminescence intensity at desired wavelengths while concurrently managing the spatial proximity of conjugates for LRET. Project 2 - Improvement of luminescence intensity from UCNPs by increasing the electric field strength associated with excitation using resonance-coupled gold nanorods assembled with UCNPs. Project 3 - Improving colloidal stability of UCNPs in biologically-relevant solutions with coatings of multi-dentate phosphonate-based ligands in combination with polyethylene glycol. Project 4 - Photo-reactions within polymeric coatings caused by UV emission from UCNPs to release cargo or activate bioprobes on-demand. The long-term goal is to produce a versatile tool for intracellular applications. The short-term objectives of the projects are to learn how to build UCNPs of higher emissivity and to coat these UCNPs with assemblies that can concurrently perform multiple tasks.