The discovery and elucidation of the structural and fluorescent properties of the green fluorescent protein (GFP) have revolutionized our understanding of biological systems. Through GFP mutagenesis and the discovery of GFP-like proteins many fluorescent proteins (FP) were developed, with fluorescence ranging from blue to far-red and widespread applications in biotechnology, such as dynamic localization in living cells, protein-protein interaction, catalytic activity, biosensors and so forth. One of the most useful application of FP is the fluorescence resonance energy transfer (FRET), where proteins with overlapping emission and excitation spectra are bonded sufficiently close to enhance the spatial and temporal resolution in biological imaging. Despite the good fluorescent properties, FP suffer from severe photobleaching, hindering its applications in FRET. An interesting approach to overcome this issue is the conjugation of FP with compounds with good optical properties, where the FP can act as an emission sensitizing agent. Lanthanide compounds (Ln3+) are well-known for having sharp emission bands, long emission lifetimes and excitation bands in the UV, visible and near-infrared spectral regions, which are suitable for the overlapping with FP emission spectra. Although the unique optical properties, the increasing use of Ln3+ in many technological applications requires new strategies to improve its efficiency. In this way, we herein report an unprecedented route to enhance europium (III) nanoparticles (Eu3+ NP) emission through bioconjugation with FP. The proteins Azurite (enhanced blue variant of GFP) and EGFP (enhanced GFP) were attached to citrate stabilized europium-doped yttrium vanadate nanoparticles (YVO4:Eu3+) surface using maleimide-thiol coupling. Under UV excitation, Azurite and EGFP emission energy is non-radiatively transferred to Eu3+ 5D0 emitting level, increasing both emission lifetime and intensity of Eu NP3+. The bioconjugation of EGFP and Azurite yields a 2.5 and 4.3-fold Eu3+ NP emission increase, respectively.

Fluorescent proteins as emission sensitizing agents in europium nanoparticles bioconjugates

255th National Meeting (2018)

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