Sequential Detection of Superoxide Anion and Hydrogen Polysulfides under Hypoxic Stress via a Spectral-Response-Separated Fluorescent Probe Functioned with a Nitrobenzene Derivative
Min Gao（高敏）,†,‡ Xia Zhang（张霞）,†,‡ Yue Wang（王悦）,†,‡ Qingluan Liu,§ Fabiao Yu（于法标）,*,∥ Yan Huang（黄严）,† Caifeng Ding,*,⊥and Lingxin Chen（陈令新）*,†,#
†CAS Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
∥Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China
§The Third Division of Clinical Medicine, China Medical University, Shenyang 110122, China
⊥Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
‡University of Chinese Academy of Sciences, Beijing 100049, China
#Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China

https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b01189#

Anal. Chem.2019XXXXXXXXXX-XXX

Publication Date:May 15, 2019

ABSTRACT: Chronic hypoxic stress disrupts the intracellular redox homeostasis, leads to a series of physiological dysfunction, and finally results in many diseases including cancer and inflammatory and cardiovascular diseases. The intracellular redox status is related to the homeostasis between reactive oxygen species (ROS) and cellular antioxidant species. Superoxide anion (O2•−) is considered to be a precursor of ROS. As a member of reactive sulfur species, hydrogen polysulfides (H2Sn) are a class of antioxidants in cells, which act as an important regulator for the intracellular redox state. Therefore, trapping the cross-talk of O2•− and H2Sn is a benefit for further understanding the physiological and pathological effects. Herein, we conceive a fluorescent probe HCy-ONO for sequential detection of O2•− and H2Sn in cells and in mouse models. Based on a tandem reaction, the probe HCy-ONO can be used to detect O2 •− and H2Sn in different fluorescence collection windows without spectral overlap interference with limits of detection 90 and 100 nM, respectively. The strategy affords high sensitivity and selectivity for our detection in living cell models under continuous hypoxic and intermittent hypoxic conditions, revealing the reason for ischemia-reperfusion injury. Moreover, the probe can distinguish the inflamed tissue from normal tissue in acute peritonitis mouse model. Finally, our probe is successfully applied for imaging of O2•− and H2Sn in the SH-SY5Y tumor-bearing mouse model, which is helpful to elucidate the physiological and pathological processes. These data demonstrated that different hypoxic status lead to different concentrations between H2Sn and O2•−.

Scheme 1. Structure and Proposed Reaction Mechanism of HCy-ONO for O2•− and H2Sn Detection

Figure 1. Properties of the HCy-ONO and Cy-ONO probes (10 mM HEPES, pH 7.4). (a) Fluorescence spectra of HCy-ONO (10 μM) upon addition of O2•− (0−20 μM). (b) The linear relationship between the fluorescent intensity and O2•− concentrations. (c) Fluorescence spectra of Cy-ONO upon addition of Na2S4 (0−100 μM). The excess O2•− was cleared by ascorbic acid. (d) The linear relationship between the fluorescent intensity and H2Sn concentrations (0−100 μM).

 Figure 2. Fluorescent images (a), flow cytometry analysis (b), and apoptosis analysis (c) of SH-SY5Y during hypoxic condition (1% O2) from 0 to 50 min. The cells were placed in 1% O2 condition for 0, 10, 20, 30, 40, and 50 min. (d, e) Quantitation of the mean fluorescent intensity in (b) and  apoptosis rate in (c) by flow cytometry assay. Fluorescent images (f), flow cytometry analysis (g), and apoptosis analysis (h) of SH-SY5Y during intermittent hypoxia. (i, j) Quantitation of the mean fluorescent intensity in (g) and apoptosis rate in (h) by flow cytometry assay. Prior to imaging, all the cells were treated with HCy-ONO (1 μM) for 10 min, and then washed with PBS for three times. Fluorescent signal collection windows were from 750 to 850 nm for channel I and from 600 to 700 nm for channel II. Apoptosis analysis: (Q1) necrotic, (Q2) late apoptosis, (Q3) alive cell, and (Q4) early apoptosis.

Figure 3. (a) Imaging of O2•− and H2Sn in the peritoneal cavity of BALB/c mice. All experimental groups were given an intraperitoneal injection of HCy-ONO (1 μM, 100 μL of 1:99 DMSO/saline v/v) for 30 min before in vivo imaging (Group a). The mice in Group b were injected intraperitoneal with PMA (100 nM, 100 mL in 1:9 acetonitrile/saline v/v) for 30 min. The mice in Group c were given an intraperitoneal cavity injection with Na2S4 (50 μM, 100 μL in saline) for 30 min after the same treatment with Group b. The mice in Group d were performed as indicated in Group b, then injected with LPS (10 mg/mL, 100 mL in 1:9 acetonitrile/saline v/v) for 12 h. The fluorescent images constructed from emission intensities collected window, channel I: 750−850 nm, λex = 730 nm; channel II: 600−700 nm, λex = 500 nm. (b) Total photon flux from entire peritoneal cavity of the mice in Figure 3a was quantified. Data are presented as mean ± SD (n = 5). (c) The details of the acute peritonitis mouse models were obtained from the camera. (d) X-ray imaging in peritoneal cavity of the BALB/c mice. (e) Fluorescent intensity changes of HCy-ONO in acute peritonitis mouse model. The mice were injection of HCy-ONO (1 μM, 100 μL of 1:99 DMSO/saline v/v) for 30 min prior to
in vivo imaging. (f) Fluorescent intensity changes of HCy-ONO in small intestine of the acute peritonitis mouse model. (g, h) Total photon flux from entire peritoneal cavity of the mice in Figure 3e and small intestine in Figure 3f were quantified. Data are presented as mean ± SD (n = 5).

Figure 4. (a) Time-dependent fluorescent images in the SH-SY5Y tumor-bearing mouse model. The mouse was intratumoral injected with HCy-ONO (1 μM, 50 μL in 1:99 DMSO/saline v/v). (b) Total  number of photons from tumor region in Figure 4a was qualified. Data are presented as mean ± SD (n = 5). (c) In vivo imaging of different tumor sizes in the SH-SY5Y tumor-bearing mouse model using HCy-ONO. (d) Fluorescence images of isolated organs. (e) Total number of photons from the tumor region in Figure 4c was qualified. Data are presented as mean ± SD (n = 5). The fluorescent images constructed from emission intensities collected window, channel I: 750−850 nm, λex = 730 nm; channel II: 600−700 nm, λex = 500 nm. Volume = length × width2 × 0.5.