Double-mode detection of HClO by naked eye and concurrent fluorescence increasing in absolute PBS

Abstract

A water soluble fluorescent probe WCN was successfully designed and synthesized based on acenaphthenequinone. It can be employed for double-mode detection of HClO by the naked eye and concurrent significant fluorescence increasing in absolute PBS. The detection limit was measured down to 77 pM. Especially important, WCN works well in both living cells and a living mouse.

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Study of reactive oxygen species (ROS) is attracting more and more attention due to their essential roles in mediating a wide variety of biological events such as aging and immunity.¹ Among them, hypochlorous acid (HClO), normally produced by myeloperoxidase (MPO)-catalyzed per-oxidation of chloride ions in phagolysosome,² plays a vital role in killing a wide range of pathogens in the innate immune system.³ Unfortunately, uncontrolled production of HClO derived from phagocytes is involved in some diseases such as cardiovascular diseases, rheumatoid arthritis, and cancer etc.⁴ Therefore, it is of great importance to develop methods for sensitive and selective detection of HClO/ClO⁻ for both disease diagnosis and exploration of its diverse pathophysiology.⁵

In recent years, a number of methods for detection of HClO/ClO⁻ have been developed.⁶ Among them, synthetic fluorescent probes are generally superior in terms of high sensitivity, low cost, real-time detection, and simple manipulation.⁷ Typically, the design strategies are based on specific reactions between recognition groups of the probes and HClO, which give highly fluorescent products. And the most commonly employed fluorophores are rhodamine,⁸ fluorescein,⁹ BODIPY,¹⁰ etc. Although many HClO/ClO⁻ fluorescent probes have been developed, most of them still have drawbacks such as single-mode detection only, poor water solubility, relatively complicated operating process, low sensitivity and selectivity, etc.¹¹ Therefore, further studies to develop a double, or even multi-mode, detection probe together with more convenient operating conditions for biological applications are still needed.

To fulfil this need, a water soluble fluorescent probe WCN (Scheme 1) was successfully designed and synthesized based on acenaphthenequinone via one-pot synthetic strategy, which was fully characterized by ¹H NMR, HRMS (Fig. S10–S12 in the ESI†). As expected, WCN exhibits a remarkable color change from blue to pink, together with a significant fluorescence increasing as soon as it encounters with HClO in absolute PBS, owing to oxidative cleavage of malononitrile in WCN.¹² This can be employed for double-mode detection of HClO by either naked eye or significant enhanced fluorescence.

Scheme 1 The proposed double-mode detection mechanism of HClO by WCN.

The water solubility of WCN was first confirmed in PBS (20 mM, pH = 7.4) (Fig. S1†). Next, the absorption properties of WCN (5 μM) were investigated in absolute PBS (Fig. S2†). Then, the fluorescence dynamics of WCN were recorded in PBS buffer (20 mM, pH 7.4) at room temperature. As shown in Fig. S3,† a remarkable fluorescence increase was observed at 560 nm within 5 min and no further significant change occurred, demonstrating that WCN can be used to monitor HClO in real time. Thus, all of the following measurements were performed under the same conditions. To verify the sensitivity of WCN to HClO, it was treated with various concentrations of hypochlorite. Upon the addition of HClO, an immediate fluorescence increase was observed at 560 nm, and about a 100-fold increase was found in fluorescence intensity (Fig. 1a) with the addition of 60 μM HClO. The fluorescence intensity at 560 nm was linearly related to the concentration of HClO added over a range of 0–60 μM (Fig. 1b), together with a lower detection limit (77 pM, LOD = 3σ/S), indicating that WCN was quite suitable for accurate detection of HClO.

Fig. 1 Fluorescence titration studies of WCN upon addition of HClO. (a) Fluorescence spectra of WCN (5 μM) upon addition of HClO (0–70 μM) in PBS buffer (20 mM, pH 7.4) at room temperature. (b) The linear relationship between the fluorescent intensity and HClO concentration (0–60 μM). All data were collected 5 min after the addition of HClO. λ_ex = 530 nm, λ_em = 560 nm. Error bars indicate the mean value of three experiments.

Meanwhile, WCN exhibited a remarkable color change from blue to pink (Fig. 2) along with the addition of HClO. Especially important, good linear relationships were also reached between the absorbance of WCN at the wavelength of 595 nm, 645 nm and the concentration of HClO as shown in Fig. S4,† demonstrating that WCN can be a good candidate for double-mode detection of HClO by either the naked eye or simultaneously enhanced fluorescence in absolute PBS.

Fig. 2 (a) The color changes of WCN (5 μM) upon addition of various concentrations of HClO (from left to right: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μM). (b) UV-vis spectra of WCN (10 μM) upon the addition of increasing concentrations of sodium hypochlorite (0–90 μM) in PBS buffer (20 mM, pH 7.4).

Next, we examined the selectivity of WCN towards HClO over other oxidants and anions under simulated physiological conditions. WCN was incubated with HClO and the other relevant ROS and RNS including H₂O₂, ¹O₂, ˙OH, O₂˙⁻, TBHP (tert-butyl hydroperoxide), TBO˙ (tert-butoxy radical) and ˙NO (nitric oxide), respectively.¹³ As shown in Fig. S5,† almost no changes in fluorescence intensity were observed after the addition of excess ROS and RNS, demonstrating WCN has high selectivity towards HClO. Furthermore, the studies of some cations and anions such as Li⁺, K⁺, Cu²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Al³⁺, F⁻, Cl⁻, Br⁻, I⁻, ClO₄⁻, NO₂⁻, S₂O₃²⁻ and SCN⁻ (100 μM for each) were also carried out (Fig. S6†). As expected, none of these cations and anions exhibited any interference. The excellent selectivity for HClO over other analytes shows that WCN has potential applications for HClO detection in complex biological environments. All these can be attributed to the HClO induced specific oxidative cleavage of malononitrile in WCN as shown in Scheme 1, which was confirmed by ESI-MS (Fig. S11 and S12†).

Furthermore, the effect of pH was also checked on the fluorescence of WCN. As shown in Fig. S7,† WCN exhibited a stable performance with HClO over pH values ranging from 1 to 10, suggesting that WCN is quite suitable for biological applications.

With these results in hand, WCN was applied to image HClO in HeLa cells using a confocal laser microscope with widely employed 488 nm laser excitation. HeLa cells were incubated with WCN (5 μM) for 30 min at 37 °C in DMEM and washed three times with PBS to remove the excess WCN. Upon excitation at 488 nm, there was negligible intracellular green fluorescence at 540–620 nm (Fig. 3a–c). When NaClO (40 μM) was added and then incubated for another 30 min, intense fluorescence emerged in the green channel showing that WCN can detect HClO rapidly in living cells (Fig. 3d–f). MTT assay showed that 5 μM WCN has no obvious cytotoxicity to the HeLa cells (Fig. S9†).

Fig. 3 Fluorescence, bright-field, and merged images of WCN-loaded HeLa cells in the absence (a–c) and presence (d–f) of HClO (50 μM).

We next assessed the ability of our probe to visualize HClO in a living mouse. The nude mouse was first subcutaneously injected with WCN (20 μM, 50 μL, PBS buffer), and 5 min later, 10 equiv. HClO (in PBS buffer) was injected into the same region, and strong fluorescence was observed after 30 min (Fig. 4b). In contrast, a control nude mouse that was injected with saline only followed by the probe showed no significant fluorescence (Fig. 4a). Thus, WCN is applicable for not only in vitro but also in vivo imaging of HClO.

Fig. 4 Fluorescence images of living nude mice. Subcutaneous injection of the solution of WCN and then a solution of HClO (a) and PBS (b) were injected separately.

In summary, a water soluble fluorescent probe WCN was designed and synthesized based on acenaphthenequinone via one-pot synthetic strategy. WCN can not only detect HClO in absolute PBS by naked eye and enhanced fluorescence simultaneously, but also works well for imaging living cells and a living mouse. For more practical applications, the probe can be made into a portable tool like test paper, etc. Thus, this probe is expected to provide a valuable reference for double and multi-mode investigation of HClO in the future.

All of the experiments were performed in compliance with the relevant laws and institutional guidelines, and were approved by Northwest A&F University.

Acknowledgements

This work was financially supported by the Scientific Research Foundation of Northwest A&F University (Z111021103 and Z111021107), the National Natural Science Foundation of China (No. 21472016, 21272030 and 21476185), State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (No. 2013005).

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Footnotes

† Electronic supplementary information (ESI) available: Experimental procedures and spectroscopic data for isolated compounds. See DOI: 10.1039/c6ra03959f

‡ These authors contributed equally.

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