Johanna Vuojola

Lanthanides represent the chemical elements from lanthanum to lutetium. They intrinsically exhibit some very exciting photophysical properties, which can be further enhanced by incorporating the lanthanide ion into organic or inorganic sensitizing structures. A very popular approach is to conjugate the lanthanide ion to an organic chromophore structure forming lanthanide chelates. Another approach, which has quickly gained interest, is to incorporate the lanthanide ions into nanoparticle structures, thus attaining improved specific activity and a large surface area for biomolecule immobilization. Lanthanide-based reporters, when properly shielded from the quenching effects of water, usually express strong luminescence emission, multiple narrow emission lines covering a wide wavelength range, and exceptionally long excited state lifetimes enabling time-gated luminescence detection. Because of these properties, lanthanide-based reporters have found widespread applications in various fields of life. This review focuses on the field of bioanalytical applications. Luminescent lanthanide reporters and assay formats utilizing these reporters pave the way for increasingly sensitive, simple, and easily automated bioanalytical applications.

The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb³⁺-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb³⁺-LBP–GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases.

Förster resonance energy-transfer (FRET) is a powerful and widely applied bioanalytical tool. According to the definition of FRET by Förster, for energy-transfer to take place, a substantial spectral overlap between the donor emission and acceptor excitation spectra is required. Recently also a phenomenon termed nonoverlapping FRET (nFRET) has been reported. The nFRET phenomenon is based on energy-transfer between a lanthanide chelate donor and a spectrally nonoverlapping acceptor and thus obviously differs from the conventional FRET, but the mechanism of nFRET and resulting implications to assay design have not been thoroughly examined. In this work, a homogeneous DNA-hybridization assay was constructed to study the distance and temperature dependency of both nFRET and conventional FRET. Capture oligonucleotides were labeled at the 5′-end with a Eu(III)-chelate, and these conjugates hybridized to complementary tracer oligonucleotides labeled with an organic fluorophore at various distances from the 3′-end. The distance dependency was studied with a fluorometer utilizing time-resolution, and the temperature dependency was studied using a frequency-domain (FD) luminometer. Results demonstrated a difference in both the distance and temperature dependency between conventional FRET and nFRET. On the basis of our measurements, we propose that in nFRET thermal excitation occurs from the lowest radiative state of the ion to a higher excited state that is either ionic or associated with a ligand-to-metal charge-transfer state.

Förster resonance energy transfer (FRET) is a powerful tool in studying biomolecular interactions. Intrinsically fluorescent lanthanide chelates are increasingly used as FRET donors due to their long emission lifetime that enables the use of time resolution. Fluorescent proteins, on the other hand, owe their popularity to the intrinsic luminescent properties, facilitating their use as fusion proteins. In this investigation, two energy transfer pairs, terbium(III) chelate with green fluorescent protein (GFP) and europium(III) chelate with yellow fluorescent protein (YFP), were studied by expressing the fluorescent protein acceptor as a fusion protein together with Rab21 GTPase. GTP-conjugated lanthanide chelates were used as donor conjugates. In contrast to conventional FRET observed with the Tb³⁺−GFP pair, a phenomenon called nonoverlapping FRET (nFRET) was observed with the Eu³⁺−YFP pair. In nFRET, the sensitized emission of the acceptor was measured at shorter wavelength than where the emission of the donor was observed. Regardless of the lower signal levels, nFRET resulted in a substantially higher signal-to-background ratio. Conventional FRET from sensitized acceptor yielded a single apparent fluorescence emission lifetime, while with nFRET two lifetimes were observed. The lanthanide chelates together with fluorescent proteins enable a straightforward and sensitive assay technology in nFRET applications.