The applicability of the three-step Förster resonance energy transfer (FRET) to detection of insulin amyloid fibrils was evaluated, using the chromophore system, containing Thioflavin T (ThT), 4-dimethylaminochalcone (DMC), and two squaraine dyes, referred to here as SQ1 and SQ4. The mediator chromophore DMC was found to enhance the fluorescence intensity of the terminal acceptor, SQ1, excited at 440 nm (at the absorption maximum of the principal donor, ThT), in fibrillar insulin compared to the system without DMC, providing the evidence for the cascade energy transfer in the chain ThT→DMC→SQ4→SQ1. Furthermore, the resulting Stokes shift in the four-chromophore system was 240 nm, as compared to 45 nm for the fibril-bound ThT, suggesting that higher signal-to-noise ratio is the advantage of amyloid fibril detection by multistep FRET. The maximum efficiencies of energy transfer in the insulin fibrils estimated from the quenching of the donor fluorescence in the presence of acceptor for the donor-acceptor pairs ThT-DMC, DMC-SQ4 and SQ4-SQ1 were 40%, 60% and 30% respectively, while negligible FRET occurred in the non-fibrillized protein. The most pronounced differences between fibrillar and non-fibrillized insulin were observed in the 3D fluorescence spectra. Specifically, two intensive spots centered at the emission wavelengths ~ 650 nm (SQ4) and ~ 685 nm (SQ1) were revealed at the excitation wavelength ~ 440 nm in the 3D patterns of insulin amyloid aggregates. In contrast, in the case of the non-fibrillized protein, the barely noticeable spots centered at the same wavelengths, as well as higher fluorescence intensities at the excitation above 550 nm were observed, suggesting the predominant impact of the direct excitation of SQ1 and SQ4 on their fluorescence responses. The inter-chromophore distances calculated from the experimental values of the energy transfer efficiency assuming the isotropic rotation of the dyes, were found to be 2.4, 4.5 and 4.3 nm for the ThT-DMC, DMC-SQ4 and SQ4-SQ1 pairs, respectively, revealing the different fibril binding sites for the examined dyes. The quantum-chemical calculations and simple docking studies provided evidence for the SQ1, SQ4 and ThT, DMC binding to the wet and dry interface of the insulin amyloid protofilament, respectively. The dye-protein complexes are likely to be stabilized by the hydrophobic, van der Waals, aromatic and electrostatic interactions. In summary, the above technique based on the multistep FRET can be employed for the identification and characterization of amyloid fibrils in vitro along with the classical ThT assay, allowing the increase of the amyloid detection sensitivity and lowering the probability of the pseudo-positive result. The applicability of the multistep FRET for amyloid visualization in vivo can be also tested by the involvement of the near-infrared fluorescent dyes to the cascade.

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The three-step Förster resonance energy transfer (FRET) within the cascade of four dyes, including the classical amyloid marker Thioflavin T as a primary donor, two jumper dyes, benzanthrone ABM and squaraine SQ4, and terminal acceptor SQ1, was tested as a possible tool for detection and characterization of insulin amyloid fibrils. The results obtained confirm the occurrence of highly efficient multistep FRET (msFRET) in the chromophore ensemble in the presence of insulin fibrils formed at elevated temperature under pH 2 (InsF1) or pH 7.4, 0.15 M NaCl (InsF2), while negligible FRET efficiencies were obtained for the control unfibrillized protein, suggesting the specificity of msFRET to cross-β-sheet architecture characteristic of amyloid fibrils. Specifically, the efficiencies of FRET for the donor-acceptor pairs ThT-ABM, ABM-SQ4 and SQ4-SQ1 at maximum acceptor concentrations (~0.4 µM – 1.6 µM) were estimated to be 86%/94%, 48%/34% and 66%/32%, respectively, in the presence of InsF1/InsF2. The most significant differences between InsF1/InsF2 and the control protein were observed for the donor-acceptor pair ThT-ABM, suggesting that ABM is the key mediator in the whole process of msFRET. Assuming the isotropic rotation of the fluorophores, the average donor-acceptor distances were estimated in the presence of InsF1, yielding the values 1.3 nm, 5.3 nm, and 3.9 nm for the ThT-ABM, ABM-SQ4 and SQ4-SQ1 pairs, respectively. The obtained distances are indicative of different fibril binding sites for the chromophores in the insulin fibrils, although due to their high specificity to the fibrillar structure, the dyes are most likely to localize in the surface grooves of β-sheets running along the main axis of amyloid fibril. Remarkably, the differences in the insulin amyloid morphology can be clearly distinguished using msFRET. As evidenced from TEM, InsF2 were thinner, shorter and contained amorphous aggregates, as compared to InsF1. Thus, different amyloid formation pathways under neutral and acidic pH resulted in the changes in the dye affinity for to the fibril binding sites, and, as a consequence, in the distinct msFRET efficiencies, especially for the pair SQ4-SQ1. The ability of ThT to serve as an efficient amplifier for the two near-infrared dyes, SQ4 and SQ1, with the benzanthrone fluorophore ABM as a jumper dye, allows detection of fibrillar insulin in the optical window of the biological samples, with the Stokes shift of the four-chromophore being ca. 240 nm. The proposed msFRET-based approach can be employed not only for insulin amyloid detection but also for distinguishing between different amyloid fibril morphologies and gaining further insights into the mechanisms involved in the development of the injection-localized insulin amyloidosis.