Long-wavelength long-lifetime luminophores

Abstract

We describe a new approach to making luminophores that display long emission wavelengths, long decay times, and high quantum yields. These luminophores are covalently linked pairs with a long-lifetime resonance-energy-transfer donor and a long-wavelength acceptor. The donor was a ruthenium (Ru) metal-ligand complex. The acceptor was the Texas Red. The donor and acceptor were covalently linked by polyproline spacers. The long-lifetime donor results in a long-lived component in the acceptor decay, which is due to RET. Importantly, the quantum yield of the luminophores approaches that of the higher quantum yield acceptor, rather than the lower quantum yield typical of metal-ligand complexes. The emission maxima and decay time of such tandem luminophores can be readily adjusted by selection of the donor, acceptor, and distance between them. Luminophores with these useful spectral properties can also be donor-acceptor pairs brought into close proximity by some biochemical association reaction. Luminophores with long-wavelength emission and long lifetimes can have numerous applications in biophysics, clinical diagnostics, DNA analysis, and drug discovery.

Figure 1.

Effect of energy transfer efficiency on the total quantum yield.

Figure 2.

Simulated time-dependent decays of the donor and acceptor, each alone and in a D–A pair. For these simulations, ${\tau }_{\text{D}}^0=1000\text{ns}$, and ${\tau }_{\text{A}}^0=10\text{ns}$.

Figure 3.

Emission spectra of the Ru–(pro)₆ donor (D) the TR acceptor (A) and the covalently linked pair (D–A) in aqueous buffer.

Figure 4.

Absorption (top) and excitation (bottom) spectra of Ru–(pro)₆(D), TR(A), and Ru–(pro)₆-TR (D–A) in aqueous buffer.

Figure 5.

Ratio of the absorption spectra (top) and emission spectra (bottom) of the D–A pair divided by that of the acceptor in aqueous buffer.

Figure 6.

Frequency domain intensity decays of the donor alone (D), acceptor alone (A), and the covalently linked D-pro₆-A pair in aqueous buffer (top) and in propylene glycol (bottom).

Figure 7.

Frequency domain intensity decays of the donor alone (D), acceptor alone (A), and the covalently linked D-pro₈-cys-A pair in the aqueous buffer (top) and in propylene glycol (bottom).

Figure 8.

Reconstructed time-domain intensity decays of the donor alone (D), acceptor alone (A), and the covalently linked pair (D–A) in water (top) and in propylene glycol (bottom). The solid line, τ_DA, is for D-pro₆-A and the dashed–dotted (–•–•–) line, τ_DA, is for D-pro₈-A.

Scheme 1.. Chemical Structure of a Ru MLC Covalently Linked to Texas Red (D–A)^a

^a The donor-alone control had the sulfhydryl group blocked with iodoacetamide. The acceptor alone was the peptide without the MLC group.

Scheme 2.. Jablonski Diagram for an Irreversible Excited-State Process