Research paperA novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2S) in nucleolus region and its application for H2S detection in zebrafish and live mice
Introduction
Hydrogen sulfide (H2S) has been recognized as one of the most important gaseous signaling molecules that plays important roles in physiological activities in recent years [1], [2]. For example, H2S is involved in aging and age ralated deseases and organs injury [3]. The genomic stability can be affected by H2S concentration and high concentration of H2S in nucleus and nucleolus regions may cause DNA damage [4]. Enzymatically generated endogenous H2S can be produced by enzymes such as 3-mercaptopyruvate sulfurtransferase, cystathionine β-synthase (CBS) and cystathionine gamma-lyase [5], [6], [7]. Investigations have demonstrated that higher concentration of H2S than its normal level in physiological environment is involved in lesion of cells and organs [8]. Inhalation of H2S may lead to respiratory depression and neural paralysis with intense neurotoxin and mucous membrane irritation [9], [10], [11]. Misregulation of H2S is related with diverse physiological diseases, including neurodegenerative disease, Down’s syndrome, angiocardiopathy, diabetes and liver cirrhosis [12], [13], [14], [15], [16]. Hence, detection of H2S in cells and live organisms is of great importance for exploring the biological role of H2S in molecular level [17].
Traditionally, many methods have been established for H2S detection [18], [19], such as gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), adsorption-desorption, electrochemical methods [20] and fluorescence detection. Among them, fluorescence detection is one of the most powerful methods for H2S analysis in biological samples, such as intracellular environment, tissue and live organisms for the noninvasive and high temporal-spatial resolution property [21], [22], [23], [24], [25]. Many fluorescent probes based on traditional dyes such as fluorescein, rhodamine, bodipy and naphthalene derivatives were developed and applied for H2S imaging in cellular and subcellular environment [26], [27], [28], [29], [30], [31], [32], [33]. However, fluorescence detection of H2S in nucleolus region is still a big challenge due to the protective obstacle of nucleus membrane.
Compared with one-photon excitation methods, two-photon fluorescence imaging technique owned many advantages such as long wavelength excitation [34], deep tissue penetration and three-dimensional imaging ability. Most of the two-photon fluorescent probes owns comparatively short emission wavelength, the maxima fluorescence signals is usually below 620 nm [35], [36], [37]. The shorter emission wavelength may lead to a series of problems in its biological application, such as tissue auto-fluorescence interference and weak tissue penetration ability of the emission light. Thus, development of two-photon probes with longer emission wavelength is urgent for improving the tissue penetration ability and extending the application in tissue imaging experiment [38]. Herein, we report the first example of red light emissive two-photon probe for H2S in nucleolus region and its application in live animals imaging. The probe was constructed through extending the conjugated system of traditional two-photon platform such as naphthalene and coumarin (Fig. 1). The probe was designed (Fig. 1C) and the photophysical properties in the absence or presence of H2S in aqueous solution and the fluorescence imaging of H2S in cells and live animals were investigated. Interestingly, the fluorescent probe was found to aggregate in the nucleolus region of cells and can detect H2S near the nucleolus region. The probe was also applied for fluorescence imaging of H2S in zebrafish and live mice model.
Section snippets
Materials and instruments
All the reagents (purity over 99.9%) were purchased from commercial source (Sigma Aldrich, China) and used without further purification unless specified. The solvents were dried and purified by distillation according to standard procedures before use. All reactions were performed under nitrogen protection and monitored by thin-layer chromatography (TLC). The products were purified by silica gel (200–300 mesh) column chromatography. TLC plates and silica gels were purchased form Qingdao Ocean
Photophysical properties of 1-H2S
The photophysical properties of 1-H2S in solution were explored. 1-H2S exhibits an intense absorption band at around 556 nm and a weak fluorescence peak at about 635 nm in PBS (pH 7.4) and DMSO (8:2) buffer (Fig. 2a and Fig. S1). As shown by Fig. S2, 1-H2S shows a stable fluorescence emission when irradiated by 580 nm light for 4 h or heated the test solution to 80 °C, which indicated a good photo-stability and thermal-stability of 1-H2S in solution. In absorption spectra (Fig. S1), in the presence
Conclusions
Most of the two-photon fluorescent dyes have emission wavelength at comparatively short wavelength region. Development of two-photon dyes with emission in red light region is attractive for increasing their application area in biological imaging, which can eliminate interference from strong tissue auto-fluorescence and increase tissue penetration ability. Herein, a novel two-photon fluorescent probe 1-H2S for H2S with red light emission was constructed through extending the conjugated system of
Acknowledgements
This work was financially supported by NSFC (61605060, 21672083, 21472067), Natural Science Foundation of Shandong Province (ZR2015PB016), Taishan Scholar Foundation (TS 201511041) and the startup fund of University of Jinan (309-10004, 160100137).
Keyin Liu obtained his Ph. D. degree from Fudan University in 2014. Currently, he is a lecturer in Institute of Fluorescent Probes for Biological Imaging in University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications.
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Keyin Liu obtained his Ph. D. degree from Fudan University in 2014. Currently, he is a lecturer in Institute of Fluorescent Probes for Biological Imaging in University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications.
Chuang Liu obtained his B.S. degree at Langfang Teachers University in 2017. Currently, he is a graduate student under the supervision of Professor Weiying Lin in the Institute of Fluorescent Probes for Biological Imaging, University of Jinan. His research interests focus on the design and synthesis of fluorescent probes.
Huiming Shang obtained his B.S. degree at Dezhou University for Nationalities in 2014. Currently, he is a graduate student under the supervision of Professor Weiying Lin in the Institute of Fluorescent Probes for Biological Imaging, University of Jinan. His research interests focus on the design and synthesis of fluorescent probes.
Mingguang Ren received his Ph.D. degree from University of Science and Technology of China in 2011. Now, he is an associate professor in Institute of Fluorescent Probes for Biological Imaging at University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications.
Weiying Lin received his Ph.D. from the University of Kansas in 2000. After completing postdoctoral research at Massachusetts Institute of Technology, in 2005, he joined the faculty at Hunan University. Subsequently, he moved to the University of Jinan as the Dean and the Distinguished Professor of the Institute of Fluorescent Probes for Biological Imaging. His research interests cover the interdisciplinary areas of molecular recognition, photochemistry, materials chemistry, analytical chemistry, and chemical biology.