A selective colorimetric and fluorescent probe for the detection of ClO⁻ and its application in bioimaging

Abstract

Disperse Violet 26 is a commercially available fluorochrome used as an on–off fluorescent probe for the detection of ClO⁻, which is one of the biologically important reactive oxygen species (ROS), in HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0) with an excellent selectivity and sensitivity for ClO⁻ compared to other analytes.

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Hypochlorous acid (HOCl) is one of the biologically important reactive oxygen species (ROS).^1–4 Because of its sub-acidity, HOCl usually undergoes a spontaneous hydrolysis reaction in neutral solutions at pH 7.0, resulting in the formation of free ClO⁻. Biologically, the ClO⁻ ion is believed to be produced by hydrogen peroxide and chloride ions in activated neutrophils catalyzed by a heme-containing enzyme myeloperoxidase (MPO).^5,6 Hypochlorite plays an essential role in the immune system due to significant antibacterial properties.⁷ However, there is growing evidence that excessive hypochlorite can lead to tissue damage and diseases such as neuron degeneration,⁸ cancer,^9,10 cardiovascular diseases,¹¹ and arthritis.¹² This may be attributed to the fact that hypochlorite in physiological condition can react with DNA, RNA, fatty acids, cholesterol, and proteins.⁷ As a decolorizer and disinfector, hypochlorite is also widely used in our daily lives.¹³ Therefore, it is of vital practical significance to detect hypochlorite through highly sensitive and selective methods. There have been some successful quantification examples reported very recently based on colorimetric, luminescent, electrochemical, and chromatographic methods.^14–26 Even so, fluorescent probe detection is a promising method for the detection of hypochlorous acid because of the low cytotoxicity of fluorescent probes, which can realize detection in living cells.^27–29 For example, Wu et al. reported a BODIPY-based fluorescent probe (detection limit was 17.7 nM) which could be successfully applied to fluorescence imaging in RAW 264.7 cells.³⁰ Another fluorescein-based probe for the hypochlorite anion (detection limit was 40 nM) was synthesized by Yin et al., which was used for labeling in organisms.³¹ All the abovementioned probes are expensive and time-consuming due to certain organic synthesis processes.

In this work, a commercially available organic pigment, Disperse Violet 26 (abbreviated as DV26 afterwards, Scheme 1), was developed as a highly selective fluorescence probe for the hypochlorite ion compared to other anions. This probe works well at physiological pH and has a high selectivity and sensitivity for ClO⁻ compared to other analytes. These desirable attributes make the sensor suitable for the detection of ClO⁻.

Scheme 1 Structure of DV26.

The recognition ability of DV26 was investigated by UV-vis and fluorescence spectra. To verify the selectivity of the probe to hypochlorite, absorbance studies in aqueous solutions were carried out. The absorbance changes that DV26 undergoes upon the addition of various analytes are shown in Fig. 1. After adding 1000 μM of analytes, such as H₂O₂, ClO₂⁻, ONOO⁻, F⁻, ClO₃⁻, CN⁻, NO₂⁻, S²⁻, SCN⁻, MnO₄⁻, ClO₄⁻, CO₃²⁻, and P₂O₇⁴⁻, to the HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0) solution containing the probe (45 μM), after 5 min of contact, only ClO⁻ induced a decrease in the absorption peaks at 544 nm and 586 nm. Accordingly, the addition of ClO⁻ to the probe produces a colorimetric change from purple to colorless, which can be detected by the naked eye. Other analytes show negligible changes in absorbance spectra under the same conditions.

Fig. 1 UV-vis absorption spectra of probe (45 μM) in HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0) in the presence of 1000 μM analyte (H₂O₂, ClO₂⁻, ONOO⁻, F⁻, ClO₃⁻, CN⁻, NO₂⁻, S²⁻, SCN⁻, MnO₄⁻, ClO₄⁻, CO₃²⁻ and P₂O₇⁴⁻). Inset: a color change photograph for ClO⁻ and other analytes.

We also carried out titration experiments for ClO⁻. With an increasing ClO⁻ concentration (0–110 μM), a gradual decrease in the absorption peaks at 544 nm and 586 nm and a progressive increase of a new absorption band at around 300 nm, by addition of ClO⁻, were observed (Fig. 2). All these indicate the formation of a new species.

Fig. 2 Absorption spectra of the probe (45 μM) in the presence of various concentrations of ClO⁻ (0–110 μM) in HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0). Inset: a color change photograph for ClO⁻.

The fluorescence response of DV26 for ClO⁻ was also examined. After adding 500 μM of analytes, such as H₂O₂, ClO₂⁻, ONOO⁻, F⁻, ClO₃⁻, CN⁻, NO₂⁻, S²⁻, SCN⁻, MnO₄⁻, ClO₄⁻, CO₃²⁻, and P₂O₇⁴⁻, to the HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0) solution containing the probe, after 5 min of contact, only ClO⁻ induced a remarkable fluorescence decrease at 625 nm, which also resulted in a visual fluorescence change (from pink to colorless) under illumination with a 365 nm UV lamp. Other analytes show negligible changes in fluorescence spectra under the same conditions (Fig. 3 and S1, ESI†).

Fig. 3 Optical density graph of the probe (35 μM) at 625 nm upon the addition of several analytes (500 μM), after contact for 5 min, in HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0) (λ_ex = 520 nm, slit: 5/5 nm). Inset: a color change photograph for ClO⁻ and other analytes (H₂O₂, ClO₂⁻, ONOO⁻, F⁻, ClO₃⁻, CN⁻, NO₂⁻, S²⁻, SCN⁻, MnO₄⁻, ClO₄⁻, CO₃²⁻, and P₂O₇⁴⁻).

Moreover, we carried out a detailed investigation on the DV26 recognition of ClO⁻ on a fluorescence spectrometer. Fig. 4 shows a regular change in the fluorescence spectrum when the ClO⁻ solution was added to the CH₃CN–HEPES buffer (pH = 7, v/v = 1 : 1) containing the probe (35 μM). The probe is strongly fluorescent in the absence of ClO⁻; however, an increase in the ClO⁻ concentration caused a dramatic change in the fluorescence spectra. A significant decrease (23-fold) in the fluorescence intensity was observed at 625 nm.

Fig. 4 Fluorescence spectra of the probe (35 μM) in the presence of various concentrations of ClO⁻ (0–80 μM) in HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0) (λ_ex = 520 nm, slit: 5 nm/5 nm). Inset: a color change photograph for ClO⁻.

Time-dependent modulations in the fluorescence spectra of the probe were monitored in the presence of 10 equiv. of ClO⁻ (Fig. 5). The kinetic study showed that the reaction was completed within 4 min for ClO⁻, indicating that the probe reacts rapidly with ClO⁻ under the experimental conditions. This unprecedented fast response could provide the possibility of quantitative detection without any pretreatment of the samples.

Fig. 5 Reaction time profile of the probe with ClO⁻.

The pH range for the determination of ClO⁻ was also studied. Fig. S2 ESI† shows the fluorescence intensity obtained for the free probe and the probe-ClO⁻ at different pH values. It was obvious that the fluorescent signal of the probe in the CH₃CN–HEPES buffer (pH = 7.0, v/v = 1 : 1) changed in the pH range 2–13. When the solution pH is in the range 3–5 or 9–11, ClO⁻ induced a fluorescence intensity for the probe such that there was no quenching or only partial quenching. Therefore, the pH range of 6–8 is effective for this probe and neutral pH was used for further studies.

To investigate the detection limit of the probe for ClO⁻, the probe (35 μM) was treated with various concentrations of ClO⁻ (0–80 μM) and the fluorescence intensity at 625 nm was plotted as a function of the ClO⁻ concentration (Fig. S3, ESI†). The fluorescence intensity of the probe is linearly proportional to ClO⁻ concentrations, and the detection limit is 0.037 μM, based on the IUPAC definition 139 (C_DL = 3 Sb m⁻¹).³² The detection limit indicates that the commercially available fluorescence probe DV26 shows a specific sensitivity towards ClO⁻ that is comparable to other synthetic probes for ClO⁻ (Fig. S4, ESI†).^33–38

The reaction mechanism of the present system was investigated. We assumed that the color change and fluorescence quenching could be attributed to the oxidation of DV26 to its azo derivative. As it is known, aromatic amines may be oxidized to the corresponding azo compounds in the presence of an oxidant. We speculated that the mechanism was based on a specific reaction promoted by hypochlorite, namely, hypochlorite is a strong oxidant and can oxidize the amino group of DV26 to form the azo product. However, the C–N bond in the azo product is easily broken to form a radical, which could combine with a chlorine radical to form compound 2 (Scheme 2). To elucidate the detailed signal mechanism, ESI-MS analysis of the isolated product from the complete reaction mixture of the probe with ClO⁻ was carried out. The identification of stable products in the ESI-MS analysis made it possible to propose the signaling mechanism: a peak at m/z = 541.08 corresponding to [compound 2 + Na]⁺ is clearly observed (Fig. S5, ESI†). The reaction mechanism is different from other recognition mechanisms, such as oxidation reactions of p-methoxyphenol to benzoquinone,^33,34 utilizing the oxidative deoximation reaction of luminescent oxime,^35,36 oxidation reactions of benzidinediimine to dibenzoyl,³⁷ the oxidized ring opening reaction³⁸ e.g. This made it possible for organic complexes containing amino groups to be used for designing hypochlorite fluorescence probes.

Scheme 2 Mechanism of chemosensor.

The ability of the probe to detect ClO⁻ within living cells was also evaluated by laser confocal fluorescence imaging using a Leica TCS SP5 laser scanning microscope. The optical window at the yellow channel (600–700 nm) was chosen to be a signal output. As shown in Fig. 6a, under selective excitation at 488 nm, HepG2 cells incubated with 20 μM probe for 30 min at 37 °C showed pink fluorescence. In a further experiment, it was found that HepG2 cells displayed no fluorescence when the cells were first incubated with 20 μM of probe for 30 min at 37 °C and then incubated with 40 μM NaClO (Fig. 6c). These cell experiments show the good cell membrane permeability of the probe, and it can be used to mark ClO⁻ within living cells.

Fig. 6 Confocal fluorescence images in HepG2 cells. (a) Fluorescence image of HepG2 cells when adding DV26 (20 μM) and its bright field image (c); (b) fluorescence image of HepG2 cells incubated with 20 μM DV26 for 30 min at 37 °C, then incubated with 40 μM ClO⁻ for 30 min at 37 °C and its bright field image (d).

In summary, we have developed a colorimetric and fluorescent probe for the detection of ClO⁻ “fluorescence turn-off” compared with other analytes in aqueous solution. This probe is based on a commercially available and cheap luminescent dye DV26, which has a high selectivity and sensitivity for ClO⁻ compared with other analytes in HEPES : CH₃CN = 1 : 1 (v/v pH = 7.0). The probe displayed a dramatic change in fluorescence intensity and absorbance intensity when ClO⁻ was added to the system. Furthermore, the system was used for bioimaging. Therefore, this work will prove useful for the development of organic dyes or fluorescent dyes as chemosensors.

Acknowledgements

The work was supported by the National Natural Science Foundation of China (no. 21102086, 21472118), the Shanxi Province Science Foundation for Youths (no. 2012021009-4 and 2013011011-1), the Shanxi Province Foundation for Returnee (no. 2012-007), the Taiyuan Technology star special (no. 12024703), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, no. 2013802), talents Support Program of Shanxi Province (no. 2014401) and CAS Key Laboratory of Analytical Chemistry for Living Biosystems Open Foundation (no. ACL201304).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06435f

‡ Fangjun Huo and Jianfang Li contributed equally.

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