Sensitive and selective fluorescent probe for hypochlorite in 100% aqueous solution and its application for lysosome-targetable cell imaging

Sensitive and selective fluorescent probe for hypochlorite in 100% aqueous solution and its application for lysosome-targetable cell imaging

Journal Pre-proof Sensitive and selective fluorescent probe for hypochlorite in 100% aqueous solution and its application for lysosometargetable cell ...

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Journal Pre-proof Sensitive and selective fluorescent probe for hypochlorite in 100% aqueous solution and its application for lysosometargetable cell imaging

Liang-Liang Gao, Wan-Wan Wang, Wei-Na Wu, Yuan Wang, Xiao-Lei Zhao, Yun-Chang Fan, Hui-Jun Li, Zhi-Hong Xu PII:

S1386-1425(20)30087-1

DOI:

https://doi.org/10.1016/j.saa.2020.118110

Reference:

SAA 118110

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

13 November 2019

Revised date:

23 January 2020

Accepted date:

23 January 2020

Please cite this article as: L.-L. Gao, W.-W. Wang, W.-N. Wu, et al., Sensitive and selective fluorescent probe for hypochlorite in 100% aqueous solution and its application for lysosome-targetable cell imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118110

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

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Sensitive and selective fluorescent probe for hypochlorite in 100% aqueous solution and its application for lysosome-targetable cell imaging Liang-Liang Gaoa,1, Wan-Wan Wanga,1, Wei-Na Wua,*, Yuan Wanga,*, Xiao-Lei Zhaoa, Yun-Chang Fana, Hui-Jun Lia, Zhi-Hong Xub,c,* a

College of Chemistry and Chemical Engineering, Henan Key Laboratory of Coal Green

Key Laboratory of Chemo/Biosensing and Detection, School of Chemistry and Chemical

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b

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Conversion, Henan Polytechnic University, Jiaozuo 454000, PR China

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, 450052, PR

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c

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Engineering, Xuchang University, 461000, PR China

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These authors are equally contributed to this work.

Corresponding author. Tel.: +86 391 3987818; Fax: +86 391 3987811; e-mail: [email protected]

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*

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China.

Abstract:

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(W.-N. Wu); [email protected] (Y. Wang); [email protected] (Z.-H. Xu).

A morpholine-functionalized pyrrole-cyanine probe was synthesized via a simple condensation reaction in high yield. This probe exhibits high selectivity toward ClO− on fluorescence and UV–vis spectra in neat aqueous solution. The strong green emission of the probe solution was quenched and the yellow color faded immediately upon the addition of ClO−. The detection limit of the probe for ClO− was 0.165 M. The mechanism of hypochlorite-induced C=C breakage was supposed on the basis of EIS-MS, NMR, and density functional theory (DFT) calculation. Finally, the probe was utilized to image ClO− in lysosomes of living cells.

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Keywords: Cell imaging; fluorescent probe; hypochlorite; lysosome target; water soluble. 1. Introduction Hypochlorous acid (HClO)/hypochlorite (ClO−) that is synthesized from the chloride ions and H2O2 catalyzed by enzyme myeloperoxidase, has been regarded as one of the most important reactive oxygen species in living organisms [1]. As a strong oxidative agent, ClO− plays essential roles in many cellular

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processes and acts as an effective antibacterial species [2]. ClO− is also widely used as a water cleaning

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agent and a well-known universal disinfectant in our daily life [3]. However, abnormal concentrations

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of ClO− may cause several diseases, such as arthritis, nephropathy, atherosclerosis, and even cancer [4].

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Therefore, the detection of ClO− in environmental and biological samples is of greatest importance.

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Fluorescent probes are an indispensable tool for the detection of biological agents because of their high sensitivity and selectivity [1]. Taking the strong oxidation of hypochlorite into consideration,

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many successful examples equipped with different reactive groups as recognition sites have been

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developed, such as S [5-13], Se [14], double bond [15-20], amine [21, 22], imine [23], hydrazine [24-28], and hydroximic acid [29]. However, the applications of some probes in biological field are restricted by several drawbacks, such as poor water solubility, long response time, narrow working pH window, and harsh reaction conditions or tedious synthetic procedures [30]. The ClO− level has a tight relationship with redox balance in lysosomes [31], and excessive or misplaced ClO− could induce apoptosis of cells through the rupture of lysosomes [11]. In this regard, designing a water-soluble fluorescent probe for the detection of lysosomal ClO− with fast response would be particularly meaningful [32]. Since lysosome is an acidic organelle (pH 4.0–5.5), good tolerance and stability to H+ for the probe is strongly recommended [10].

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Our previous work reported a lysosome-targeting, ratiometric fluorescent probe for Cu2+ using pyrrole aldehyde as the linker of 4-(2-aminoethyl)morpholine [33]. Considering the designing principle of hypochlorite-induced breakage of C=C bonds [20], we fabricate probe 1 by the reaction between this pyrrole

compound

and

3-ethyl-2-methyl-1,3-benzothiazol-3-ium

iodide

(Scheme

1).

The

as-synthesized probe not only exhibits a excellent water solubility but also incorporates a

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lysosome-targeting group. Furthermore, the probe works in a wide pH range of 1.0–11.0 with a very

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fast response (< 30 s) and has been successfully applied for the trace detection of ClO− in lysosomes of

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living cells.

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Insert Scheme 1.

2.1 Synthesis

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2. Results and Discussion

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Probe 1 was synthesized via the condensation of 3-ethyl-2-methyl-1,3-benzothiazol-3-ium iodide

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and 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-morpholin-4-yl-ethyl)-amide [33] in accordance with a previously described method [34]. Its structure was carefully characterized by 1H NMR,

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C NMR, and ESI-MS spectroscopy. Detailed methods and procedures are presented in S1,

ESI. 2.2 Spectroscopic response of 1 to ClO− Probe 1 is well-soluble in water. Thus, its detection to ClO− was investigated in a neat citric acid-sodium citrate buffer solution (20 mM, pH 4.0). As shown in Fig. 1a, free probe 1 (10 M) displayed an obvious absorption band at 465 nm because of intramolecular charge transfer (ICT) [20]. When it was excited using this wavelength, a strong emission peak at 520 nm could be observed (Fig.

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1b), and the fluorescence quantum Φ was determined to be 0.28 by using fluorescein as the standard [35]. After the addition of ClO− (10 eq.), the hypochromic effect on the absorbance peak occurred, accompanied with a remarkable fluorescence quenching response (Φ=0.05). However, other anions (AcO−, Br−, Cl−, ClO4−, CN−, F−, H2PO4−, HPO4−, I−, PO43−, S2−, and SO32−) and ROS (H2O2, 1O2, ·OH, NO, O2−) only triggered negligible spectral changes. In addition, the above mentioned analytes did not

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lead to any apparent interference in emission of probe 1 in the detection of ClO− (Fig. S1, ESI). These

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results indicate that probe 1 has excellent selectivity for ClO− in aqueous solution.

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Insert Figure 1.

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2.3 Spectral Titrations

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A UV titration experiment was first conducted to evaluate the sensing sensitivity of probe 1 toward ClO−. Results showed that the absorbance peak of 1 at 468 nm gradually decreased and finally

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blue-shifted to 430 nm upon the addition of ClO− up to 10 eq (Fig. 2a). The linear response covered the

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ClO− concentration range of 22.5–50.0 M. In addition, the isosbestic point at 374 nm clearly supports the decomposition reaction (side infra) of probe 1 in the presence of ClO− [20]. Fluorescence titration experiments were also conducted to ascertain the sensitivity of probe 1 to ClO−. With increasing amount of ClO−, the emission intensity at 520 nm of probe 1 gradually decreased and showed a good linearity in a ClO− concentration range of 10.0-32.0 M (Fig. 2b). The detection limit of 1 for ClO− was determined to be 0.165 M from the equation of 3/k [14], which is comparable with some previously reported ClO− fluorescent probes [30]. An important feature of the probe 1 is its capability for sensing ClO− in 100% aqueous medium, indicating that probe 1 is a potential tool to monitor ClO− in live cells.

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Insert Figure 2. 2.4 Kinetic assay and effect of environmental factors The value of pH varies remarkably in different cell organelles. In general, lysosomes maintain an acidic pH of ∼5.0 in normal cells while even lower than 4.0 in cancer cells [36]. Therefore, the pH dependence of the probe 1 must be verified to extend its application in biological systems. As presented

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in Fig. S2, ESI, the great intensity differences at 520 nm between 1 and 1+ClO− remain constant from

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pH 1.0 to 11.0, which enables its imaging in acidic cell organelles, such as lysosomes.

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As an important fundamental parameter for a reaction-based probe, the time-course fluorescence

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responses of 1 (10 μM) toward ClO− were measured (Fig. S3, ESI). Results showed that the reaction

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between 1 and ClO− could finish within 30 s, which makes it realizable for the real-time detection of

(Table S2, ESI).

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2.5 Sensing mechanism

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ClO−. This feature of the probe 1 is absent in some reported lysosome-locating fluorescent ClO− probes

The mixture of 1+ClO− was characterized by ESI-MS analysis to prove the recognition mechanism of 1 to ClO−. As illustrated in Fig. 3, the peaks at 439.2271 and 220.1192 assigned to [1+H]+ and [1+2H]2+ of 1 disappeared in the spectrum of 1+ClO−, clearly indicating the accomplishment of the reaction. Simultaneously, the generation of peaks at 193.0619 and 280.1464 confirmed the existence of compounds 1a and 1b in the reaction products. Figs. S4 and S5, ESI illustrated the difference of 1H NMR between 1+ClO− and 1. The characteristic peaks of CH=CH of 1 disappeared in the spectrum of 1+ClO−, clearly indicating the C=C breakage process. In addition, the corresponding peaks of 1a (eg. 9.48 ppm for CH=O, 3.29-3.30 for CH2, and 2.38 and 2.26 for CH3) and 1b (eg. 8.37 ppm for CH=O,

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Journal Pre-proof 4.24-4.27 for CH2, and 1.27-1.29 for CH3) could be observed in the spectrum of 1+ClO−. Taking comprehensive consideration of previous literatures [30], we inferred the C=C breakage mechanism as illustrated in Scheme 2. Insert Figure 3. Insert Scheme 2.

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We also performed density functional theory calculations with the Becke-3-Lee-Yang-Parr (B3LYP)

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exchange function to justify the above observations [37]. As shown in Fig. 4, the HOMO of 1 was

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mainly located in the morpholine group, whereas the LUMO dispersed over the pyrrole and

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benzothiazol moieties. The well delocalization of the electron cloud density in the LUMO clearly

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indicated the active ICT process in free probe 1, suggesting the strong fluorescence emission of the probe. In addition, the simulated spectral data in Table S1, ESI showed that the maximum emission of

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1 was centred at 507 nm, which should be assigned to the anti-Kasha’s rule transition from S2 to S0

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with a large oscillator strength factor of 1.60. In the cases of 1a and 1b, the fluorescence emission peaks were calculated to be 490 and 497 nm, with the oscillator strength factor as low as 0.05 and 0.04, respectively, clearly indicating the weak emission of the products after the addition of ClO−. The HOMO-LUMO energy gap of 1 (2.64 eV) was lower than those of 1a (4.07 eV) and 1b (3.76 eV), which was in agreement with the blue-shift of maximum absorption (from 468 nm to 430 nm) of probe 1 with the treatment of ClO−. All facts are in accordance with the experimental results. Insert Figure 4. On the other hand, the charge distributions of bridged C=C bond were -0.127 and -0.052 (Fig. S6, ESI), respectively, which are consistent with the high electron density of this C=C bond as shown in

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Journal Pre-proof the 3D isosurface LUMO diagrams of 1. That is, the electrophilic reagent Cl+ derived from ClO− preferred to attack the bridged C=C bond and subsequently induced its breakage, thus the ICT process of the probe was inhibited, consequently resulting in a weak fluorescence emission. 2.6 Fluorescence imaging experiments of 1 in living cells Prior to the bioimaging applications in living cells, the cytotoxicity of probe 1 was estimated in

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living HeLa cells by MTT assays. A cell viability percentage of 82.5% was observed at 40 M

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concentration (Fig. S7, ESI), suggesting the low cytotoxicity of probe 1 to living cells. Then, HeLa

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cells were costained with 1 and LysoTracker Red (a commercial mitochondrial tracker) to evaluate the

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subcellular-targeting capability of probe 1. The images of green and red channels merged well with an

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overlap coefficient of 0.83 (Fig. 5), manifesting that probe 1 can stain lysosomes of the living cells. Insert Figure 5.

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Moreover, fluorescence imaging experiments of the probe 1 were performed (Fig. 6). Incubated with

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1 (10 μM) at 37 °C for 30 min, the HeLa cells exhibited a strong fluorescence in the green channel, which quickly became dark after treatment with ClO− ions (30 μM), indicating that probe 1 could have a potential application to determine lysosomal ClO− in live cells. Insert Figure 6. 3. Conclusion We developed a simple cyanine-based probe for the detection of ClO−. Due to its excellent water solubility, wide working pH range, fast response process, and existence of lysosome-targeting group, the as-synthesized probe was successfully used to image ClO− in the lysosomes of live cells. Acknowledgements

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This work was supported in part by the National Natural Science Foundation of China (No. 21907023 and 21001040), the Joint Program for Fostering Talents of National Natural Science Foundation of China and Henan Province (U1304202 and U1604124), the Science and Technology Department of Henan Province (182300410183), the Education Department of Henan Province (19A150001 and 17A150011), the Fundamental Research Funds for the Universities of Henan

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Province (NSFRF180323), Foundation of Henan Polytechnic University (T2018-3 and J2015-4).

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Figure captions Scheme 1. Synthetic route of probe 1. Scheme 2. The proposed reaction mechanism of 1 with ClO−. Fig. 1 Absorption (a) and fluorescence (b) spectra of 10 M probe 1 in citric acid-sodium citrate buffer solution (20 mM, pH = 4.0) with 10 eq. of anions (AcO−, Br−, Cl−, ClO−, ClO4−, CN−, F−, H2PO4−,

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Journal Pre-proof HPO4−, I−, PO43−, S2−, and SO32−) and ROS species (H2O2, 1O2, ·OH, NO, O2−) and blank. The excitation wavelength was 465 nm. Fig. 2 Absorption (a) and fluorescence (b) spectra of 10 M probe 1 upon the addition of ClO− (0–10 eq.) in citric acid-sodium citrate buffer solution (20 mM, pH = 4.0). The insets show the absorbance at 468 nm and fluorescence intensity at 520 nm as a function of ClO− concentration with linear

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Fig. 3 ESI-MS spectra of 1 with and without ClO− in CH3OH.

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relationship, respectively. The excitation wavelength was 465 nm.

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Fig. 4 Energy-minimized structures and HOMO/LUMO of 1, 1a, and 1b by DFT calculation.

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Fig. 5 Bright field and fluorescence images of HeLa cells stained with 10M of probe 1 and Lyso

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Tracker Red: (a) from green channel; (b) from the red channel (lysosomes staining); (c) an overlay green and red channels; (d) bright field image; (e) an overlay of bright field, green, and red channels. (f)

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and the green channel of 1.

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Intensity profile of the linear region of interest across the HeLa cell co-stained with Lyso Tracker Red

Fig. 6 Confocal fluorescence images of Hela cells: Confocal fluorescence (a), brightfield (b), and overlay (c) images of HeLa cells incubated with 10 M of 1 for 30 min at 37℃; Confocal fluorescence (d), brightfield (e), and overlay (f) images of HeLa cells incubated with 10 M of 1 for 30 min at 37℃ and then incubated with 30 M ClO− for another 30 min at 37℃. Scheme 1

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Scheme 2

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Credit author statement Liang-Liang Gao: Formal analysis, Writing - original draft. Wan-Wan Wang: Investigation, Writing - original draft. Wei-Na Wu: Writing - review & editing, Supervision. Yuan Wang: Writing - review & editing. Xiao-Lei Zhao: Formal analysis. Yun-Chang Fan: Investigation. Hui-Jun Li: Formal

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analysis. Zhi-Hong Xu: Funding acquisition.

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Declaration of Competing Interest We declare that we have no conflict of interest.

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Graphical abstract

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Journal Pre-proof Highlights A morpholine functionalized pyrrole-cyanine probe has been developed. The probe could detect ClO− in 100% aqueous solution on both fluorescence and UV–vis spectra.

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The probe has been applied for imaging lysosomal ClO− in living cells.

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