Lanthanide-doped nanoparticles (LnNPs) have become an important class of fluorophores for advanced biosensing and bioimaging. LnNPs that are photosensitized by surface-attached antenna ligands can possess exceptional brightness. However, their functional bioconjugation remains an important challenge for their translation into bioanalytical applications. To solve this problem, we designed a ligand that can be simultaneously applied as efficient light harvesting antenna for Tb surface ions and strong linker of biomolecules to the LnNPs surfaces. To demonstrate generic applicability of the photosensitized TbNP-bioconjugates, we applied them in two prototypical applications for biosensing and bioimaging. First, in-solution biorecognition was shown by time-resolved Förster resonance energy transfer (FRET) between streptavidin-functionalized TbNPs to biotinylated dyes (ATTO 610). Second, in situ detection of ligand-receptor binding on cells was accomplished with TbNP-antibody (Matuzumab) conjugates that could specifically bind to transmembrane epidermal growth factor receptors (EGFR). High specificity and sensitivity were demonstrated by time-gated imaging of EGFR on both strongly (A431) and weakly (HeLa and Cos7) EGFR-expressing cell lines, whereas non-expressing cell lines (NIH3T3) and EGFR-passivated A431 cells did not show any signals. Despite the relatively large size of TbNP-antibody conjugates, they could be internalized by A431 cells upon binding to extracellular EGFR, which showed their potential as bright and stable luminescence markers for intracellular signaling.
Keywords: FRET; bio-labeling; lanthanide nanoparticles; luminescence microscopy; time-resolved detection.
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