Flavone-based ESIPT ratiometric chemodosimeter for detection of cysteine in living cells

Abstract

We have designed and synthesized a novel ratiometric fluorescent chemodosimeter MHF-based ESIPT process for specific detection of cysteine among the biological thiols. The probe MHF shows very weak blue fluorescence under UV excitation. Upon addition of cysteine (Cys), the reaction of Cys with MHF induces acrylate hydrolysis, thereby enabling the ESIPT process to shift the weak blue emission to a strong green emission with about 20-fold enhancement. We utilized (1)H NMR spectra to elucidate the fluorescence sensing mechanism. Moreover, the cellular imaging experiment indicated the MHF possessed excellent selectivity, low cytotoxicity, and desirable cell permeability for biological applications.

Scheme 1. (a) Chemical Structures of MHF along with the Proposed Sensing Mechanism; (b) Schematic Representation of ESIPT Process of HF

Figure 1

Time-dependent (a) absorption spectral changes and (b) absorbance changes (λ = 285 nm and 350 nm) of MHF (10μM) in the present of 100 μM Cys in MeCN-H₂O (1:1, v/v) solution with 10 mM HEPES buffer.

Figure 2

Time-dependent (a) fluorescence spectral changes and (b) fluorescence intensities (λ=510 nm) of MHF (10 μM) in the present of 100 μM Cys in MeCN-H₂O (1:1, v/v) solution with 10 mM HEPES buffer. (c) Fluorescence spectra changes and (d) fluorescence intensity changes (λ=510 nm) of 10 μM MHF in the presence of increasing concentrations of Cys (final concentration: 0, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1 mM) in MeCN-H₂O (1:1, v/v) solution with 10 mM HEPES buffer. Each spectrum was recorded after 60 min.

Figure 3

¹H NMR spectrum of MHF in d6-DMSO, and the resulting spectrum after addition of 1 equiv. Cys in D₂O for 5 min, 1 h, and 5 h. The starred “*” signals are attributed to DMSO and water solvents.

Figure 4

The fluorescence intensities (λ = 510 nm) of 10 μM MHF upon addition of 100 μM physiological important amino acids (Glu, Asp, His, Arg, Lys, Gln, Asn, Tyr, Thr, Ser, Cys, Gly, Met, Trp, Phe, Pro, Ile, Leu, Val, and Ala) in MeCN-H₂O (1:1, v/v) solution with 10 mM HEPES buffer.

Figure 5

The fluorescence intensities (λ = 510 nm) of 10 μM MHF upon addition of 100 μM biologically important thiols (Cys, GSH, Hcy, and NaHS) in MeCN-H₂O (1:1, v/v) solution with 10 mM HEPES buffer.

Figure 6

Fluorescence microscopy images of hMSCs. (a) Bright-field image, and fluorescence images in (c) blue and (e) green channel after hMSCs were pre-treated with 100 μM NEM for 30 min, and then incubated with 20 μM MHF for 1 h. (b) Bright-field image, fluorescence images in (d) blue and (f) green channel after hMSCs being incubated with 20 μM MHF for 1 h at 37°C.