Fluorescence lifetime can be used to discriminate between two fluorophores emitting at the same wavelength - even between two molecules of the same species located in different chemical or physical environments. Fluorophores experience altered emission lifetimes when incorporated into and liberated from macromolecules or molecular aggregates; with this in mind, we considered the possibility of creating a responsive self-aggregating fluorescent probe that would exhibit a new lifetime upon exposure to a desired chemical trigger. We developed a model compound by conjugating fluorescein isothiocyanate (FITC) to the known self-assembling scaffold consisting of cysteine and cyanobenzothiazole (CBT) to create CBT-FITC. The CBT group was “caged” with a disulfide bond to make its aggregation responsive to reducing conditions. We exposed the test compound CBT-FITC to a reducing agent, tris(2-carboxyethyl)phosphine (TCEP), and spectroscopically examined the lifetime changes resulting from its reduction-triggered aggregation both in solution and in live cells.<br/>CBT-FITC incubated with TCEP in PBS buffer was observed to generate nanoaggregates, while no such structures were detected from the compound alone in PBS. Steady-state absorbance and emission spectra revealed the emergence of new peaks corresponding to the oligomerized CBT group, and inhomogeneous broadening of FITC absorbance and emission peaks at higher probe concentration, indicating perturbation of the local FITC environment.<br/>Time-resolved emission spectra revealed a novel two-component lifetime for FITC in the aggregated CBT-FITC and an average lifetime of 3.79 ns, down from 3.96 ns for the monomer CBT-FITC. When CBT-FITC was incubated with TCEP in the presence of CT26 cells, fluorescence lifetime imaging microscopy (FLIM) measured an average lifetime of 2.92 ns, while the inactive compound reported a much slower lifetime (4.32 ns). Together, these results indicate that aggregation can alter lifetime to a detectable extent, revealing a possible approach for designing responsive self-aggregating lifetime imaging probes.

Responsive fluorophore aggregation provides contrast for lifetime imaging in cells

2019 Spring National Meeting

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