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. 2021 Sep 24;11(50):31656-31662.
doi: 10.1039/d1ra06224g. eCollection 2021 Sep 21.

Quinoline-based fluorescent probe for the detection and monitoring of hypochlorous acid in a rheumatoid arthritis model

Xinyi Yang  1 , Yue Wang  1 , Zhuye Shang  1 , Zexi Zhang  2 , Haijun Chi  3 , Zhiqiang Zhang  3 , Run Zhang  2 , Qingtao Meng  1
Affiliations

Quinoline-based fluorescent probe for the detection and monitoring of hypochlorous acid in a rheumatoid arthritis model

Xinyi Yang et al. RSC Adv. .

Abstract

The development of effective bioanalytical methods for the visualization of hypochlorous acid (HOCl) in situ in rheumatoid arthritis (RA) directly contributes to better understanding the roles of HOCl in this disease. In this work, a new quinoline-based fluorescence probe (HQ) has been developed for the detection and visualization of a HOCl-mediated inflammatory response in a RA model. HQ possesses a donor-π-acceptor (D-π-A) structure that was designed by conjugating p-hydroxybenzaldehyde (electron donor) and 1-ethyl-4-methylquinolinium iodide (electron acceptor) through a C[double bond, length as m-dash]C double bond. In the presence of HOCl, oxidation of phenol to benzoquinone led to the red-shift (93 nm) of the adsorption and intense quenching of the fluorescence emission. The proposed response reaction mechanism was verified by high performance liquid chromatography (HPLC) and high-resolution mass spectroscopy (HRMS) titration analysis. The remarkable color changes of the HQ solution from pale yellow to pink enabled the application of HQ-stained chromatography plates for the "naked-eye" detection of HOCl in real-world water samples. HQ featured high selectivity and sensitivity (6.5 nM), fast response time (<25 s) to HOCl, reliability at different pH (3.0 to 11.5) and low cytotoxicity. HQ's application in biological systems was then demonstrated by the monitoring of HOCl-mediated treatment response to RA. This work thus provided a new tool for the detection and imaging of HOCl in inflammatory disorders.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Scheme 1
Scheme 1. (A) The proposed sensing mechanism of HQ to HOCl. (B) Schematic illustration of the application in the monitoring of HOCl-mediated RA model: (a) mice only. (b) Both left and right hind limbs were stimulated with λ-carrageenan and (c) followed by incubation with HQ. (d) The both left and right hind limbs pre-stimulated with λ-carrageenan, followed by administration with MTX (a standard therapeutic drug for RA) into the right leg and PBS into the left leg of a mouse, and finally stimulated with HQ in the both hind limbs.
Fig. 1
Fig. 1. Absorption spectra of HQ (10 μM) in the presence of increasing amounts of HOCl (0–2 mM) in PBS aqueous buffer (DMSO : H2O = 1 : 9, 20 mM, pH = 7.4). Inset: (A) absorbance ratio at 512 nm and 419 nm as a function of the concentrations of HOCl. (B) Colorimetric response of HQ in the (a) absence and (b) presence of HOCl.
Fig. 2
Fig. 2. (A) Absorption spectra and (B) color changes of HQ (10 μM) upon the addition of various analytes (1.5 mM) in PBS aqueous buffer (DMSO : H2O = 1 : 9, 20 mM, pH = 7.4). (B) (1) Blank, (2) HOCl, (3) SO32−, (4) HSO3−, (5) SO42−, (6) HSO4−, (7) ONOO−, (8) 1O2, (9) HCO3−, (10) H2PO4−, (11) P2O74−, (12) PO42−, (13) S2−, (14) Br−, (15) Cl−, (16) F−, (17) NO2−, (18) NO3−, (19) OH−, (20) H2O2, (21) Pi, (22) CH3COO−, (23) Hcy, (24) Cys and (25) GSH.
Fig. 3
Fig. 3. (A) Fluorescence responses of HQ (10 μM) towards various amount of HOCl (0–2.0 mM) in PBS aqueous buffer (DMSO : H2O = 1 : 9, 20 mM, pH = 7.4). Inset: fluorescence intensity of HQ at 550 nm as a function of HOCl. (B) The linearity between the fluorescence intensity at 550 nm and increasing HOCl concentrations (0.33 mM). Excitation was performed at 450 nm.
Fig. 4
Fig. 4. (A) Fluorescence emission and (B) fluorescence color responses of HQ (10 μM) towards various bioactive analytes (1.5 mM) in PBS aqueous buffer (DMSO : H2O = 1 : 9, 20 mM, pH = 7.4). (C) Fluorescence intensities of HQ (10 μM) at 550 nm in PBS aqueous buffer (DMSO : H2O = 1 : 9, 20 mM, pH = 7.4) towards HOCl in the presence of diverse coexisting competitive analytes. (1) Blank, (2) SO32−, (3) HSO3−, (4) SO42−, (5) HSO4−, (6) ONOO−, (7) 1O2, (8) HCO3−, (9) H2PO4−, (10) P2O74−, (11) PO42−, (12) S2−, (13) Br−, (14) Cl−, (15) F−, (16) NO2−, (17) NO3−, (18) OH−, (19) H2O2, (20) Pi, (21) CH3COO−, (22) Hcy, (23) Cys, (24) GSH and (25) HOCl. Excitation was performed at 450 nm.
Fig. 5
Fig. 5. (A) Time course of fluorescence response of HQ (10 μM) upon the addition of HOCl at the concentration of (a) 0.5 mM, (b) 1.0 mM and (c) 1.5 mM in PBS buffer (DMSO : H2O = 1 : 9, 20 mM, pH = 7.4). (B) Influence of pH on the fluorescence emission of HQ (10 μM) in the absence and presence of HOCl. Excitation was performed at 450 nm, and emission was collected at 560 nm.
Fig. 6
Fig. 6. Fluorescence imaging of endogenous HOCl using HQ in live nude mice. (1) Control group 1 (mice only), (2) left leg of the mouse was given a skin-pop injection of LPS (5 μg mL−1, 80 μL) for 5 h, followed by the injection of HQ (20 μM, 125 μL) into the same area of the left leg. The images were then recorded at different times (3) 0.5 min, (4) 1 min, (5) 1.5 min, (6) 2 min, and (7) 2.5 min. (8) Mean fluorescence intensity analysis of HOCl in the left and right leg of the mouse at different times shown in (1–7). The right leg was injected with HQ (20 μM, 125 μL) only as the control group 2. Nude mice were imaged using an excitation filter (465 nm) and emission filter (570 nm).
Fig. 7
Fig. 7. Visualization of HOCl-mediated inflammatory response in RA of mice using HQ. (1) Control group 1 (mouse only), (2) left hind limb was stimulated with λ-carrageenan (2 μg mL−1, 80 μL) in PBS for 4 h, (3) HQ (20 μM, 125 μL) was injected into the same area of left hind limb. (4) Mean fluorescence intensity analysis of HOCl in the left and right leg of the mouse at different times shown in (1–3). The right hind limb was injected with HQ (20 μM, 125 μL) only as the control group 2. Nude mice were imaged using an excitation filter (465 nm) and emission filter (570 nm).
Fig. 8
Fig. 8. Monitoring of HOCl-mediated RA treatment response using HQ. (1) Control group 1 (mouse only), (2) both left and right hind limbs were stimulated with λ-carrageenan (2 μg mL−1, 80 μL) in PBS for 4 h, (3) left hind limb was administrated with MTX (20 μg in 20 μL PBS) for another 6 hour, (4) HQ (20 μM, 125 μL) was injected locally into both right and left hind limbs. (5) Mean fluorescence intensity analysis of HOCl in the left and right leg of the mouse at different times shown in (1–4). The right hind limb administrated without MTX as the control group 2. Nude mice were imaged using an excitation filter (465 nm) and emission filter (570 nm).
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