A FRET‐Based Near‐Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria

Abstract

We report a near‐infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near‐infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18‐fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.

Recent research progress of red-emitting/near-infrared fluorescent probes for biothiols

“…Liu et al , in 2019, reported FRET-based ratiometric red-emitting fluorescent probe 11 which is based on the strategy of combining rhodamine with coumarin as a whole fluorophore. 44 Probe 11 used coumarin as the energy donor, rhodamine as the energy receptor, piperazine as the spacer linker and trifluoromethylphenyl thiester as the recognition group of biothiols. In the PBS/EtOH (V/V = 7/3, pH = 7.4) mixed system, the fluorescence emission of probe 11 was decreased at 467 nm (the emission wavelength of the coumarin part) and increased at 645 nm (the emission wavelength of rhodamine part) due to the effect of FRET.…”

Ratiometric Detection of Glutathione Based on Disulfide Linkage Rupture between a FRET Coumarin Donor and a Rhodamine Acceptor

“…Many fluorescent probes for biothiol detection have been developed with different sensing mechanisms [3b,4] . These include photo‐induced electron transfers, [4a,b,j,5] addition reactions with selectivity of glutathione over cysteine, [6] removal of a heavy atom fluorescence quenching effect by halogen nucleophilic substitution, [7] intramolecular charge transfer, [8] and FRET [4b,c,9] . Among these different sensing approaches, FRET‐based ratiometric fluorescent probes possess unique advantages with self‐calibration capabilities [9,10] .…”

“…These include photo‐induced electron transfers, [4a,b,j,5] addition reactions with selectivity of glutathione over cysteine, [6] removal of a heavy atom fluorescence quenching effect by halogen nucleophilic substitution, [7] intramolecular charge transfer, [8] and FRET [4b,c,9] . Among these different sensing approaches, FRET‐based ratiometric fluorescent probes possess unique advantages with self‐calibration capabilities [9,10] . Systematic errors, which are characteristic of intensity‐based fluorescent probes resulting from fluctuations in the excitation light source, sample heterogeneity, uneven dye distribution, variations, and compartmental localization, do not occur with ratiometric probes [10,11] .…”

Recognition of Thiols in Living Cells and Zebrafish Using an Imidazo[1,5-α]pyridine-Derivative Indicator

“…Given the advantages of the real-time and nondestructive detection on the living biosamples, fluorescence sensors have attracted considerable attention as effective molecular-recognition tools in vivo [18,19,20]. While numerous fluorescent probes were designed specifically for thiols assay [21,22,23,24,25,26,27,28,29,30,31], most of them respond toward thiols with small Stokes shifts (Table S1). It’s known that fluorescence probes with small Stokes shifts are usually associated with a decrease in sensitivity that results from the overlap of excitation and emission spectra.…”