Development of a novel near-infrared fluorescence light-up probe with a large Stokes shift for sensing of cysteine in aqueous solution, living cells and zebrafish

Xiaoyu Zhang^a, Heng Liu【刘恒】^a,c,∗∗, Yingying Ma^a, Wangbo Qu^a, Hanping He^a, Xiuhua Zhang^a,

Shengfu Wang^a, Qi Sun^b,∗∗∗, Fabiao Yu【于法标】^c,∗

a Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan, 430062, China

b Key Laboratory for Green Chemical Process of Ministry of Education and School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, 430205, China

c Institute of Functional Materials and Molecular Imaging, Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, College of Clinical Medicine, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China

Dyes and Pigments Volume 171, December 2019, 107722

https://doi.org/10.1016/j.dyepig.2019.107722

Abstract

As an important sulfur-containing amino acid, aberrant levels of cysteine are closely related with an array of dieases. Although many Cys-specific fluorescent probes have been designed, most of them wouldn't be able to detect Cys in buffer solution, which limits the application of the probe in biological system. In this work, a novel near-infrared fluorescent probe Cys-WR with a large Stokes shift (about 110 nm) was developed for the detection of Cys by conjugating the fluorophore WR-4 with crotonate moiety. The probe exhibited off-on response to Cys with wide linear range of 0–100 μM, as well as the color of the probe solution changes from deep pink to brown observed by naked eyes. Moreover, the probe showed little cytotoxicity. Imaging of Cys using this probe in living A549 cells and zebrafish had also been well demonstrated.

Fig. 1. UV–vis absorption (A) and fluorescence spectra (B) of probe Cys-WR (10 μM) upon gradual addition of various amounts of Cys (0–300 μM). Inset (A, B): photographs of the probe without or with Cys under room light or UV light. (C) Fluorescence intensities at 653 nm of the probe as a function of the concentration of Cys. (D) Fluorescence intensities at 653 nm of the probe in the absence or presence of 200 μM of Cys under different pH values. (E) Fluorescence response of the probe toward Cys and different interfering analytes. (F) The selectivity of Cys-WR (10 μM) toward different interfering analytes. From left to right: 200 μM for Thr, Tyr, Val, Ser, Phe, Met, Leu, Arg, His, Pro, Ala, Gly and Ile; 100 μM for Fe3+, Al3+, Ca2+, Mg2+, S2−, S2O32−, SO42−; 50 μM for Hcy; 1 mM for GSH; 200 μM for Cys.

Scheme 1. Proposed sensing mechanism of Cys-WR toward Cys.

Fig. 2. Fluorescence images of A549 cells incubated with Cys-WR. (A1-A3) cells incubated with Cys-WR (10 μM) for 30 min; (B1-B3) NEM (200 μM, 60 min) pretreated cells incubated with Cys-WR (10 μM) for 30 min; (C1-C3) NEM (200 μM, 60 min) pretreated cells incubated with Cys-WR (10 μM) for 30 min and followed by treatment with Cys (100 μM) for 30 min. (D) Relative fluorescence intensity of the corresponding fluorescence images (B1-B3). Values represent mean standard error (n = 3). Scale bar: 40 μm.

Fig. 3. Fluorescence images of zebrafish. (A1-A3) Zebrafish incubated with Cys-WR (10 μM); (B1-B3) NEM (50 μM, 60 min) pretreated zebrafish incubated with Cys-WR (10 μM) for 30 min; (C1-C3) NEM (50 μM, 60 min) pretreated zebrafish incubated with Cys-WR (10 μM) for 30 min and followed by treatment with Cys (100 μM) for 60 min. (D) Relative fluorescence intensity of the corresponding fluorescence images (B1-B3). Values represent mean standard error (n = 3). Scale bar: 200 μm.

Acknowledgement

This work was supported by the National Natural Science Foundation of China(NSFC.21602051, 21775162, 21804102), Talent Program of Hainan Medical University (Grants XRC180006), and Hundred-Talent Program (Hainan 2018).