From Antenna to Assay: Lessons Learned in Lanthanide Luminescence

Ligand-sensitized luminescent lanthanide(III) complexes are of considerable current interest due to their unique photophysical properties (micro- to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts), which make them well suited to serve as labels in fluorescence-based bioassays. The long-lived Ln(III) emission can be temporally resolved from scattered light and background fluorescence, resulting in vastly enhanced measurement sensitivity. One of the challenges in this field is the design of sensitizing ligands that provide highly emissive Ln(III) complexes that also possess sufficient stability and aqueous solubility required for practical applications. In this account we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time Resolved Fluorescence (HTRF) technology, the requirements and current use of which will be briefly discussed. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms as well as using multi-chromophore chelates to increase molar absorptivity compared to earlier examples that utilize a single pendant antenna chromophore. We have found that ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ca. 60%. Solution thermodynamic studies have indicated that these complexes are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM-chromophore, in conjunction with time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong circularly polarized luminescence (CPL) activity. To efficiently sensitize Eu(III) emission, we have utilized ligands based on the 1-hydroxypyridin-2-one (1,2-HOPO) chelate, which are remarkable since they combine both excellent photophysical properties in addition to exceptional aqueous stabilities. A more compete understanding of this chromophore has been achieved by combining low temperature phosphorescence measurements with the same TD-DFT approach used with the IAM system. Also, Eu(III) complexes with strong CPL activity have been obtained through preparation of chiral 1,2-HOPO ligands. Using the unique spectroscopic properties of Eu(III), we have also undertaken the kinetic analysis of radiative and non-radiative decay pathways for a series of complexes, which has highlighted the importance of the metal ion symmetry on the ensuing photophysical properties. Lastly, the commercial development of a Tb-IAM compound that offers improved performance in the common HTRF platform and has the potential to vastly improve the sensitivity of measurements carried out using this technique is presented