Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 26;12(25):15861-15869.
doi: 10.1039/d2ra01048h. eCollection 2022 May 23.

Sequential detection of hypochlorous acid and sulfur dioxide derivatives by a red-emitting fluorescent probe and bioimaging applications in vitro and in vivo

Jianhua Liu  1   2 , Haoyuan Yin  1 , Zhuye Shang  1 , Pengli Gu  3 , Guangjie He  3 , Qingtao Meng  1 , Run Zhang  4 , Zhiqiang Zhang  1
Affiliations

Sequential detection of hypochlorous acid and sulfur dioxide derivatives by a red-emitting fluorescent probe and bioimaging applications in vitro and in vivo

Jianhua Liu et al. RSC Adv. .

Abstract

Hypochlorous acid (HOCl) and sulfur dioxide derivatives (SO3 2-/HSO3 -) play critical roles in complex signal transduction and oxidation pathways. Therefore, it is meaningful and valuable to detect both HOCl and SO2 derivatives in biosystems by a fluorescence imaging assay. In this work, we developed a red-emitting fluorescent probe (DP) by the condensation of malononitrile and phenothiazine derivatives through a C[double bond, length as m-dash]C double bond. DP was designed with a donor-π-acceptor (D-π-A) structure, which enables absorption and emission in the long wavelength region. In the presence of HOCl, specific oxidation of the thioether of phenothiazine in DP to a sulfoxide derivative (DP[double bond, length as m-dash]O) occurs, resulting in a hypochromic shift (572 nm to 482 nm) of the absorption spectra and "OFF-ON" response of the maximum emission at 608 nm. After the activation of the C[double bond, length as m-dash]C double bond by oxidation, DP[double bond, length as m-dash]O reacts specifically with SO3 2-/HSO3 - via a 1,4-nucleophilic addition reaction leading to a decrease in the intensity of the absorption and emission spectra, which enabled the realization of sequential detection of HOCl and SO3 2-/HSO3 - by a single fluorescent probe. The detection limits of DP for HOCl and SO3 2-/HSO3 - were calculated to be 81.3 nM and 70.8 nM/65.1 nm, respectively. The results of fluorescence microscopic imaging indicated that DP shows potential for the detection of intracellular HOCl and SO3 2-/HSO3 -. Using adult zebrafish and nude mice as live animal models, DP was successfully used for the fluorescence imaging of HOCl and SO3 2-/HSO3 - in vivo.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts to declare.

Figures

Scheme 1
Scheme 1. Proposed sensing mechanism of DP for the sequential detection of HOCl and SO2 derivatives (SO32−/HSO3−).
Fig. 1
Fig. 1. (A) Absorption spectra of probe DP (10 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4) upon addition of various analytes (60 μM). (B) UV-vis absorption spectra of probe DP (10 μM) in the presence of different amounts of HOCl (0–60 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). Inset: absorbance ratio of probe DP at 480 nm and 574 nm as a function of HOCl concentration. Colorimetric changes of probe DP (a) in the absence and (b) in the presence of HOCl (60 μM). (C) Colorimetric changes of probe DP (10 μM) in the presence of various analytes (60 μM): (1) free DP, (2) HOCl, (3) Br−, (4) F−, (5) Cl−, (6) P2O74−, (7) PO43−, (8) H2PO4−, (9) Cys, (10) GSH, (11) Hcy, (12) S2−, (13) SO32−, (14) HSO3−, (15) H2O2, (16) 1O2, (17) OH−, (18) CH3COO−, (19) HCO3−, (20) NO3−, (21) ONOO−, (22) NO2−, (23) NO3−, (24) HSO4−, (25) SO42−, (26) OH˙ and (27) O2−.
Fig. 2
Fig. 2. (A) Fluorescence spectra of probe DP (10 μM) in the presence of different amounts of HOCl (0–60 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). Inset: (a) fluorescence intensities of probe DP at 608 nm as a function of HOCl concentration. (b) Fluorescence color changes of probe DP (I) in the absence and (II) in the presence of HOCl. (c) Linear relationship between the fluorescence intensity of DP (5 μM) at 608 nm and HOCl concentration (3.2–35 μM). (B) Changes in the fluorescence intensity of probe DP (10 μM) towards HOCl (60 μM) in the presence of various competing analytes (60 μM): (1) blank, (2). Br−, (3) F−, (4) Cl−, (5) P2O74−, (6) PO43−, (7) H2PO4−, (8) 1O2, (9) OH−, (10) CH3COO−, (11) HCO3−, (12) NO3−, (13) ONOO−, (14) NO2−, (15) HSO4−, (16) SO42−, (17) OH˙, (18) O2−, (19) HOCl. (C) Fluorescence colour images of probe DP (10 μM) in the presence of various analytes (60 μM) under UV light of 365 nm. (1) Free DP. (2) HOCl, (3) Br−, (4) F−, (5) Cl−, (6) P2O74−, (7) PO43−, (8) H2PO4−, (9) Cys, (10) GSH, (11) Hcy, (12) S2−, (13) SO32−, (14) HSO3−, (15) H2O2, (16) 1O2, (17) OH−, (18) CH3COO−, (19) HCO3−, (20) NO3−, (21) ONOO−, (22) NO2−, (23) NO3−, (24) HSO4−, (25) SO42−, (26) OH˙ and (27) O2−. (D) Time-profile fluorescence enhancement at 608 nm of probe DP in the presence of (a) 0 μM, (b) 20 μM, (c) 25 μM, (d) 35 μM and (e) 50 μM HOCl in PBS aqueous solutions. Excitation was performed at 480 nm.
Fig. 3
Fig. 3. UV-vis absorption spectra of DPO (10 μM) in the presence of different amounts of (A) HSO3− (0–60 μM) and (B) SO32− (0–40 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). Inset: colorimetric changes of DPO (a) in the absence and (b) in the presence of HSO3− (60 μM) and SO32− (40 μM). Absorbances of DPO at 480 nm as a function of HSO3−/SO32− concentrations. (C) UV-vis absorption spectra of DPO (10 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4) in the presence of various analytes (60 μM). (D) Colorimetric changes of DPO (10 μM) in the presence of various analytes (60 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). (1) free DPO. (2) HSO3−, (3) SO32−, (4) F−, (5) Cl−, (6) P2O74−, (7) PO43−, (8) H2PO4−, (9) Cys, (10) GSH, (11) Hcy, (12) S2−, (13) HSO4−, (14) SO42−, (15) H2O2, (16) 1O2, (17) OH−, (18) CH3COO−, (19) HCO3−, (20) NO3−, (21) ONOO−, (22) NO2−, (23) Br−, (24) OH.
Fig. 4
Fig. 4. (A) Fluorescence spectra of DPO (10 μM) in the presence of different amounts of (A) HSO3− (0–60 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). Inset: (a) fluorescence intensities of DPO as a function of HSO3− concentrations. (b) Fluorescence color photos of DPO (10 μM) in the presence of HSO3− (60 μM) under UV light. (c) Linear relationship between the fluorescence intensity of DPO (5 μM) and HSO3− concentration (0–22 μM). (B) Fluorescence spectra of DPO (10 μM) in the presence of different amounts of SO32− (0–40 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). Inset: (a) fluorescence intensities of DPO as a function of SO32− concentrations. (b) Fluorescence color photos of DPO (10 μM) in the presence of SO32− (40 μM) under UV light. (c) Linear relationship between the fluorescence intensity of DPO (5 μM) and SO32− concentration (0–23 μM). (C) Fluorescence spectra of DPO (10 μM) in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4) in the presence of all kinds of analytes (60 μM). (D) Fluorescence color images of DPO (10 μM) in the presence of various analytes (60 μM) under UV light in PBS aqueous solutions (DMF : H2O = 1 : 9, v/v, 20 mM, pH = 7.4). (1) Free DPO. (2) HSO3−, (3) SO32−, (4) F−, (5) Cl−, (6) P2O74−, (7) PO43−, (8) H2PO4−, (9) Cys, (10) GSH, (11) Hcy, (12) S2−, (13) HSO4−, (14) SO42−, (15) H2O2, (16) 1O, (17) OH−, (18) CH3COO−, (19) HCO3−, (20) NO3−, (21) ONOO−, (22) NO2−, (23) Br−, (24) OH. The intensities were recorded at 608 nm, and excitation was performed at 480 nm.
Fig. 5
Fig. 5. Sequential fluorescence imaging of HOCl and SO32−/HSO3− in live HeLa cells. (a–c) HeLa cells treated with DP (2 μM) for 20 min. (d–f) HeLa cells pre-treated with HOCl (10 μM) and then incubated with DP (2 μM) for further 25 min. (g–i) A549 cells pre-treated with HOCl (10 μM) and then incubated with DP (2 μM) for 25 min, and finally incubated with SO32−/HSO3− (10 μM) for further 30 min. The images were acquired using a confocal microscope at 465 nm excitation and 610 nm emission. Scale bar = 100 μm.
Fig. 6
Fig. 6. Fluorescence imaging of exogenous HOCl in live mice. (1) Nude mouse; (2) DP (20 μM, 125 μL) was subcutaneously injected into the mouse; (3) followed by the injection of 25 μL HOCl (0.1 mM) to this area. Images were then recorded at different time points: (4) 5 min, (5) 10 min, (6) 15 min, (7) 20 min and (8) 25 min. The mouse was imaged using an excitation filter (465 nm) and an emission filter (610 nm).
Fig. 7
Fig. 7. Fluorescence imaging SO32−/HSO3− intake in mice. (A) (1) Control group; (2) 20 μM (125 μL) DPO was subcutaneously injected into the left hind limbs of the mouse; then SO32− (0.1 mM) was injected into the same areas of interest: (3) 5 min, (4) 10 min, (5) 15 min, (6) 20 min, (7) 25 min and (8) 30 min post injection. (C) (1) Control group; (2) 20 μM (125 μL) DPO was subcutaneously injected into the left hind limbs of the mouse; then HSO3− (0.1 mM) was injected into the same areas of interest for (3) 5 min, (4) 10 min, (5) 20 min and (6) 30 min post injection. (B) and (D) Mean fluorescence intensity of interested areas at different time points shown in (A) and (C). The mice were imaged with an excitation filter at 465 nm and an emission filter at 610 nm.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Zhang Y. Zuo Y. Yang T. Gou Z. Wang X. Lin W. Analyst. 2019;144:5075–5080. doi: 10.1039/C9AN00844F. - DOI - PubMed
    1. Cui Y. Lu Z. Bai L. Shi Z. Zhao W. Zhao B. Eur. J. Cancer. 2007;43:2590–2601. doi: 10.1016/j.ejca.2007.08.015. - DOI - PubMed
    1. Valko M. Leibfritz D. Moncol J. Cronin M. T. D. Mazur M. Telser J. Int. J. Biochem. Cell Biol. 2007;39:44–84. doi: 10.1016/j.biocel.2006.07.001. - DOI - PubMed
    1. Kettle A. J. Winterbourn C. C. Redox Rep. 1997;3:3–15. doi: 10.1080/13510002.1997.11747085. - DOI - PubMed
    1. Roos D. Winterbourn C. C. Science. 2002;296:669–671. doi: 10.1126/science.1071271. - DOI - PubMed