A sensitive probe for amyloid fibril detection with strong fluorescence and early response

Abstract

We synthesized a new probe, 4-[2-(2-naphthyl)-(E)-ethenyl]-benzyl(triphenyl)phosphonium bromide (NEB), to detect the formation of amyloid fibrils of bovine insulin. The fluorescence intensity of NEB in the presence of insulin fibrils was 30 times higher than that before fibrillation, with the fluorescence quantum yield increased from 2.5% to 78%. In comparison with the commercially available probe, thioflavin T (ThT), NEB exhibits a 10 times stronger fluorescence and a shorter identification lag phase for detecting insulin fibrillation, indicating a higher sensitivity in detection of insulin oligomers and fibrils.

Fig. 1. Chemical structure of NEB.

Fig. 2. (A) Normalized absorption (red) and emission (black) spectra of NEB alone in ethanol solution. (B) Emission spectra of NEB in different ratios of ethanol: glycerol. 100 : 0 (black), 80 : 20 (red), 60 : 40 (blue), 50 : 50 (pink) 40 : 60 (green), 20 : 80 (dark blue), inset: plot of the emission intensity for each solution versus percentage glycerol present.

Fig. 3. Emission spectra of (A) NEB (2 μM) and (B) ThT (2 μM) dissolved in water (black) and mature fibrils solution (red), respectively. λ_ex = 325 nm (for NEB), 410 nm (for ThT). Photographs of mixtures of NEB and ThT with fibrillar forms of insulin taken under UV illumination.

Fig. 4. Fluorescence decay profiles of NEB (A) and ThT (B) in water solution (black) and mature fibrils solution (red), respectively.

Fig. 5. (A) The emission spectra of ThT bound with insulin amyloid fibrils (black) before and (red) after the addition of NEB, λ_ex = 410 nm. (B) The emission spectra of ThT bound with insulin amyloid fibrils after addition of NEB at different excitation wavelength, λ_ex = 310 nm (blue), λ_ex = 410 nm (red).

Fig. 6. (A) Emission intensity of NEB (black, 2 μM) and ThT (red, 2 μM) monitoring insulin (5 μM) fibrillation, λ_ex = 325 nm (for NEB), 410 nm (for ThT). NEB and ThT monitoring insulin fibrillation at the same time, the emission of intensity of NEB (blue, λ_ex = 310 nm), ThT (green, λ_ex = 310 nm) and ThT (pink, λ_ex = 410 nm). (B) The enlarged part of insulin fibrillation from t = 0 to 7 h.