Upconversion nanoparticles as the basis for versatile intracellular luminescence bioassays

拟议工作的目的是学习建立多功能纳米颗粒(NP)平台，用于动态过程的细胞内分析和细胞过程的干预。发光纳米颗粒独特的光学共振能量转移特性允许在局限于纳米颗粒表面的距离尺度上对选择性生物分子相互作用进行局部询问，并且发光还可以用于诱导光反应，从而可以释放货物或激活与纳米颗粒相关联的生物探针。*纳米粒子是通过低功率激光照射而发光的。红外激发波长将非常适合穿透复杂的生物样品，并将减少背景散射和自体荧光，但通常提供的能量不足以刺激电子激发，以实现所需的光学询问过程。光学开启生物探针的功能或释放与纳米粒子结合的货物的愿望也受到激活光反应的长波能量不足的限制。一种将近红外(NIR)激发辐射转换为紫外线(UV)和可见光的巧妙方法，可以通过上转换过程实现，在该过程中，两个或更多个光子被吸收，然后合并。这发生在含有稀土元素的上转换纳米颗粒(UCNP)中。UCNPs可以作为供体，通过发光共振能量转移(LRET)将能量转移到近端受体，提供了一种只对分子距离上的相互作用敏感的转导方法，并提供了在UCNPs表面进行光裂解反应的机会。研究工作将解决在这种技术用于细胞内研究之前必须克服的问题，包括：*项目1核-壳-壳UCNP设计，该设计可以将高发光强度稳定在所需的波长，同时管理LRET的共轭物的空间接近。*项目2通过使用与UCNP组装的共振耦合金纳米棒增加与激发相关的电场强度来提高UCNP的发光强度。*项目3通过结合聚乙二醇包覆多齿膦酸基配体来提高UCNP在生物相关溶液中的胶体稳定性。*项目4通过UCNP发出的紫外线辐射引起聚合物涂层内的光反应，按需释放货物或激活生物探针。*长期目标是生产一种用于细胞内应用的通用工具。这些项目的短期目标是学习如何建造发射率更高的UCNP，并在这些UCNP上覆盖可以同时执行多项任务的组件。

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.*****