Evaluating spectral overlap with the degree of quenching in UCP luminescence energy transfer systems

The use of organic-based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic-based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limit their effectiveness and longevity both in vitro and in vivo. As an alternative, lanthanide-doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and FRET sensing; they exhibit high photo- and chemical stability and utilise near infrared (nIR) excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd₂SO₄:Yb:Er (PTIR545) and Gd₂SO₄:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.