Synthesis and evaluation of self-calibrating ratiometric viscosity sensors

Abstract

We describe the design, synthesis and fluorescent profile of a family of self-calibrating dyes that provide ratiometric measurements of fluid viscosity. The design is based on covalently linking a primary fluorophore (reference) that displays a viscosity-independent fluorescence emission with a secondary fluorophore (sensor) that exhibits a viscosity-sensitive fluorescence emission. Characterization of fluorescent properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. The chemical structures of both fluorophores and the linker length have been evaluated in order to optimize the overall brightness and sensitivity of the viscosity measurements. We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model.

Molecular rotors as luminescence sensors of local viscosity and viscous flow in solutions and organized systems

“…Quite promising are systems in which the rotor molecule is covalently bonded to the reference fluorophore molecule. 53,62,63 A method that involves recording the changes in the fluorescence lifetime of the molecular rotor as a function of viscosity was proposed. 64 The full local viscosity distribution map in a heterogeneous environment is obtained by the fluorescence lifetime imaging (FLIM), which is suitable for measuring the fluorescence lifetime (independent of the fluorophore concentration but fairly sensitive to parameters of the environment) with high spatial and temporal signal resolution.…”

Self-calibrating viscosity probes: Design and subcellular localization

“…For the more lipophilic ratiometric dye, we attached the same coumarin donor to an amino-thiophene rotor via a polymethylene linker of five carbons. 21c,23 To further increase the overall lipophilicity we substituted the piperidine motif of the rotor with a more fatty dihexylamine subunit.…”

“…Commercially available aldehyde 17 was converted to the dihexylamine derivative 18 in 83% yield. 28 Knövenagel condensation of 18 with β-cyanoester 21 21c gave compound 22 in very good yield as a single stereoisomer. 19 Deprotection of the primary amine 22 by treatment with trifluoroacetic acid, followed by coupling with N -succinimidyl-7-methoxycoumarin-3-carboxylate ( 11 ), yielded dye 24 in 84% yield.…”

“…21 Here we expand this design to the synthesis and study of self-calibrating dyes with different solubility profiles for potential applications in cell imaging. We envisioned that, by virtue of their solubility profiles, these new compounds could be localized in different parts of cells allowing studies either in the cytoplasm (hydrophilic dye) or the cell membrane (hydrophobic dye).…”

Ratiometric mechanosensitive fluorescent dyes: design and applications

“…5, 6, or 8 carbon atoms) that permit lower energy transfer and thus, higher emission from the reference dye. 61 Since long linkers may lead to folding of the dye system, rigidifying the linker with unsaturated chains or rings ( e.g. 19 ) may be beneficial In fact, the balance of reference and rotor peak between 19 and 22 is similar.…”