An oxidative cleavage-based ratiometric fluorescent probe for hypochlorous acid detection and imaging

Abstract

A new fluorescent molecule of an imidazole derivative was synthesized through simple cyclization of a hetero-aromatic compound and successfully applied for the rapid and ratiometric sensing of HOCl with high sensitivity and selectivity. It has also been demonstrated for the bioimaging of HOCl using A549 cells.

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Reactive oxygen species (ROS), a class of important oxygen-containing molecules which mediate redox modifications of a variety of biomolecules, have received considerable attention over the past decades.¹ As one of the biologically important ROS in living systems, hypochlorous acid (HOCl) exhibits high reactivity and plays a significant role in many immune processess.² The endogenous HOCl origins from the peroxidation of chloride by hydrogen peroxide under the catalysis of the enzyme myeloperoxidase (MPO) which is mainly localized in neutrophils and monocytes.³ Excessive production of HOCl can lead to several diseases such as atherosclerosis, arthritis, neuron degeneration and cancer.⁴ On the other hand, HOCl is also widely used as an antimicrobial and bleaching agent in our daily lives.⁵ Therefore, developing highly sensitive and selective probes for detecting HOCl in biological and environmental samples is of great importance.

Toward this end, a number of fluorescent probes such as rhodamine,⁶ nanoparticles,⁷ cyanine,⁸ and BODIPY,⁹ have been reported for the detection of HOCl. Most of these probes are based on the change of a single fluorescence intensity.¹⁰ A limitation is that the single signal is easily interfered by various factors, such as the probe concentration, instrumentation and environmental conditions. Ratiometric fluorescence technique can efficiently overcome the above mentioned drawbacks and thus attract increasing interest in recent years.¹¹ Ratiometric fluorescent probes provide a built-in correction for environmental effects by measuring two or more different fluorescence intensities and improve the sensitivity at trace levels. Furthermore, ratiometric fluorescent probes exhibit color changes in the presence of analytes which can be used for the rapid and visual identification.

In this work, we reported a fluorescent ratiometric strategy for detecting HOCl in living systems using a novel fluorescent imidazole compound, 2-(pyridin-2-ylmethyl)imidazo[1,5-a]pyridine-3(2H)-thione (PIPT). Imidazole compounds possess optical properties in the visible wavelength range due to conjugated n and pi electrons, which are potential for many applications such as fluorescence probes, organic dyes, optoelectronics, and etc.¹² However, the synthesis of imidazole rings often involves usage of catalysis or extreme conditions.¹³ We now demonstrated a facile route for the synthesis of fluorescent molecule PIPT under mild conditions. This new probe PIPT showed good stability and high fluorescent quantum yield. Upon reaction with HOCl, the probe exhibited a immediate and ratiometric fluorescent response to HOCl with high selectivity and sensitivity. In addition, the probe was utilized to image HOCl in living cells and sense gaseous HOCl with PIPT-indicating fluorescent paper, indicating its potential in practical applications.

The designed probe PIPT was synthesized from the reaction of di-(2-picolyl)amine (DPA) with carbon disulfide (CS₂) in the presence of ammonium hydroxide (NH₃·H₂O), as outlined in Scheme 1. The structure of PIPT was confirmed by ¹H NMR, ¹³C NMR, and high resolution mass spectrometry (Fig. S1, S2, and S3†). The high resolution mass spectrum shows a peak at m/z = 242.07 with an isotope distribution which is in consistent with the simulated isotope distribution of the molecular formula [M + H]⁺.

Scheme 1 Procedure for the synthesis of the ratiometric fluorescence probe PIPT. The synthesis can be carried out under mild conditions and the yield is very high.

Spectral properties of PIPT were first examined. It is found that the probe displays three absorption peaks at 260, 300 and 375 nm (ε = 2100 M⁻¹ cm⁻¹, Fig. S4 and S5†), and a strong green fluorescence with an emission maximum at 505 nm (Fig. S4†). Its fluorescence quantum yield was determined to be Φ_F = 67.8% using fluorescein in 0.1 M NaOH (Φ_F = 95.0%) as a reference (Fig. S6†).

The fluorescence stability of PIPT was systematically investigated by consecutively irradiating the probe solution in PBS/EtOH (3 : 1). The experiment was performed using a 20 kW pulse Xenon discharge lamp as light source. It is clear that the fluorescence intensity has no apparent change after consecutive irradiation for 30 minutes, indicating the excellent stability of the probe which ensures the reliability for the measurement of HOCl (Fig. S7†).

The sensing ability of the ratiometric probe to HOCl was examined and the results were shown in Fig. 1. Clearly, addition of HOCl leads to an immediate and obvious change in the fluorescence of the probe. The emission at 505 nm gradually decreases, and a new blue-shifted emission band centered at 430 nm forms concomitantly. This large hypsochromic shift results in a ca.12-fold variation in the fluorescence intensity ratio (I₄₃₀/I₅₀₅) from 0.22 in the absence of HOCl to 2.71 in the presence of 7.0 μM of HOCl (Fig. 1a). Additionally, a definite isoemission point is observed at 450 nm indicating that only one new species is formed during the reaction. High resolution mass spectrometry and ¹H NMR analysis confirm the formation of the product PIPI (Fig. S8 and S9†). The high resolution mass spectrometry of the product exhibits a peak at m/z = 210.10. The isotope distribution is identical to the simulated isotope distribution of an ylide molecule formula [M]⁺ and the chemical shifts of the protons are in well assignment with the ylide structure. The quantum yield of PIPI is determined to be Φ_F = 19.5% using quinine sulfate in 0.1 M H₂SO₄ (Φ_F = 54.6%) as a reference (Fig. S10†). As the fluorescence intensity ratio increases, the fluorescence color of the probe solution changes continuously from green to blue, allowing the visualization of HOCl (Fig. 1b). Notably, a good linear correlation is observed between the fluorescence intensity ratio (I₄₃₀/I₅₀₅) and the concentration of HOCl, which can be used for the quantification of HOCl, and the limit of detection (LOD) is thus calculated to be as low as 0.27 μM based on the definition of three times the standard deviation of the blank signal (3σ). The sensitivity of the probe is higher and comparable with those of other fluorescence probes based on molecular backbones of anthracene (0.3 μM), coumarin (0.8 μM), or resorcinol (0.48 μM).¹⁴

Fig. 1 (a) Fluorescence spectra of the probe solution upon the addition of increasing concentrations of HOCl in PBS/EtOH (3 : 1) at pH 7.4. Inset: Stern–Volmer plot of fluorescence intensity of the ratiometric probe as a function of the concentration of HOCl. (I₄₃₀/I₅₀₅)₀ and (I₄₃₀/I₅₀₅) represent the ratio of the fluorescence intensities at 430 and 505 nm of PIPT in the absence and presence of HOCl, respectively. (b) The corresponding fluorescence colors of the probe in the presence of HOCl (0–7.0 μM). Images were taken under a UV lamp (365 nm).

As pH is an important factor in practical sensing, the pH-dependence of the probe was then tested to examine the effect of pH condition on HOCl detection. Results show that the free probe is stable over a wide pH range from 4.5 to 10.5 (Fig. S11†). Interestingly, the variation of pH also causes no significant effect on the fluorescence intensity of the probe in the presence of two concentrations of HOCl at 3.0 and 6.0 μM (Fig. S12†), suggesting that PIPT is a very practical fluorescent probe and can be used within a wide pH range.

Next, we investigated the selectivity of the probe to HOCl against other biologically important ROS, including hydrogen peroxide (H₂O₂), tert-butylhydroperoxide (TBHP), superoxide (O₂^−·), hydroxyl radical (˙OH), peroxynitrite (ONOO⁻), nitrite (NO₂⁻), and sulfide (S₂⁻). As shown in Fig. 2 and S13,† the ratiometric fluorescent response of PIPT only occurs upon the addition of HOCl. About 3-fold and 11-fold increments in the fluorescence intensity ratio (I₄₃₀/I₅₀₅) of the probe are observed at HOCl concentrations of 3.0 μM and 7.0 μM, respectively, accompanied by distinct fluorescence color changes which can be easily observed under a UV lamp (the inset of Fig. 2). However, 7.0 μM of other ROS produce no significant changes in the fluorescence of the probe, indicating the high selectivity of the fluorescent probe for HOCl.

Fig. 2 The selectivity of PIPT to various selected ROS in PBS/EtOH (3 : 1) at pH 7.4. The striped bars represent the emission intensity ratio (I₄₃₀/I₅₀₅) of the probe PIPT (1.0 μM) in the presence of 7.0 μM of various ROS, and the black bar represents the addition of 3.0 μM of HOCl. The emission intensities were recorded at 3 minutes after the ROS addition. PBS buffer solution (pH 7.4) was added with the same volume as ROS solution for control experiment. The fluorescent images in the inset visually show the corresponding color changes of the probe in the presence of various ROS. All the photos were taken under a 365 nm UV lamp.

Density function theory (DFT) theoretical calculation and UV-vis absorption titration were also carried out to understand the reaction mechanism. For the probe PIPT, the electron density of HOMO is mainly localized on the imidazopyridine-thione moiety and the LUMO is located over the whole molecule. While for PIPI, both the HOMO and LUMO are mainly distributed at the imidazopyridine moiety, suggesting a charge transfer process (Fig. S14†). Furthermore, the HOMO–LUMO energy gap of PIPT is smaller than that of PIPI, consistent with their spectral variation. The UV-vis spectral changes of PIPT in the absence and presence of HOCl also confirm their interaction. It can be seen in Fig. S15† that, as the amount of HOCl added increases, the two absorption bands at 300 nm and 375 nm gradually decrease and disappear when reaches an equilibrium. For comparison, the absorption spectral changes of the probe toward other ROS were also investigated. Hydroxyl radical was in situ generated by hydrogen peroxide and ferrous sulfate. As expect, no apparent changes are observed in the absorption spectra of PIPT upon the addition of those ROS except for hydroxyl radical experiment (Fig. S16†). The absorption spectral change of PIPT in the hydroxyl radical experiment could be attributed to the overlap of the absorption of ferric ion which was generated during the preparation of hydroxyl radical (Fig. S17†).

Therefore, an oxidative reaction mechanism can be proposed for the detection, as shown in Scheme 2. First, one molecule HOCl initially reacts with one C=S double bond through an electrophilic addition to form an intermediate, which quickly loses one chloride anion to generate the corresponding sulfenic acid.¹⁵ The intermediate is confirmed by mass spectroscopy analysis (Fig. S18†). The sulfur atom then attacks the oxygen center of the second HOCl molecule through nucleophilic reaction, leading to the production of the corresponding sulfinic acid. The sulfinic acid intermediate subsequently hydrolyzes to eliminate one molecule of H₂SO₃ as side product and finally produces an ylide product PIPI, which can be supported by high resolution mass spectrum and ¹H NMR analysis (Fig. S8 and S9†).

Scheme 2 The proposed reaction mechanism for the oxidative desulfurization of PIPT induced by HOCl via the formation of sulfinic acid intermediate, followed by hydrolysis to generate an ylide product and sulphurous acid as side product.

For potential bioimaging and biosensing application of the probe in living cells, the cytotoxicity should be evaluated with MTT viability assay because the fluorescence cell images only represent a number of cells limited in the vision. The MTT viability assay was performed on A549 cells incubated with the probe at various concentrations from 0 to 10.0 μM for 12 hours and 24 hours, respectively (Fig. 3). The MTT results clearly show that the cell viability is higher than 90% after incubated with 10.0 μM of PIPT for 24 hours, suggesting the low cytotoxicity of the probe PIPT to A549 cells.

Fig. 3 Cell viability of A549 cells incubated with PIPT at varying concentrations for 12 hours and 24 hours, respectively.

The fluorescence imaging was performed in the human lung adenocarcinoma A549 cells with a Zeiss LSM710 confocal microscopy (Fig. 4). Clearly, after incubation with PIPT (7.5 μM) for 10 hours, the cells show bright green fluorescence, which indicate that the probe is greatly cell-membrane permeable (Fig. 4b). Subsequent treatment with HOCl cause ratiometric response on the fluorescence of the stained cells and blue fluorescence is observed (Fig. 4c). Even incubation for a prolonged period of 24 hours (Fig. 4f) and 48 hours (Fig. 4g), the fluorescence of the cells nearly keeps constant and the cells remain in good shape, suggesting the low cytotoxicity of the probe. The results indicate that the probe PIPT is cell-permeable, low-toxic, and capable for cell imaging and detection of HOCl in vivo. A simple and portable PIPT-based fluorescent indicating paper has also been fabricated for the visual detection of aerosol HOCl as another application (see part 13 in the ESI, Fig. S19†).

Fig. 4 Visualization of HOCl in living cells using PIPT. Top panel: bright field image (a) and fluorescence image (b) of A549 cells incubated with 7.5 μM of PIPT for 10 hours; fluorescence image (c) and bright field image (d) of A549 cells incubated with PIPT and further treated with 100 μM of HOCl for 2 hours. Bottom panel (control): bright field image (e) and fluorescence image (f) A549 cells incubated with only 7.5 μM of PIPT for 24 hours; bright field image (g) and fluorescence image (h) A549 cells incubated with only 7.5 μM of PIPT for 48 hours.

In conclusion, we have successfully developed a facile approach for the synthesis of a fluorescent imidazole compound PIPT for the detection of HOCl. The probe is rapidly reacted with HOCl and produces remarkable ratiometric fluorescence response in a dose-response manner to the amount of HOCl. Distinct fluorescence color change from green to blue is observed after the reaction. This method is highly selective and sensitive toward HOCl over other ROS. Moreover, the probe has been successfully employed for the fluorescence imaging of HOCl in A549 cells. As the probe features good cell-membrane permeability, low cytotoxicity and a detection limit at micromolar level for HOCl, it is expected to serve as an effective probe for HOCl in biological studies.

We are grateful for the financial support from the National Basic Research Program of China (2011CB933700), the National Natural Science Foundation of China (nos 21228702, 21302187, 21205120).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra09611h

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