A highly sensitive hemicyanine-based fluorescent chemodosimeter for mercury ions in aqueous solution and living cells

Abstract

A novel hemicyanine-based fluorescence turn-on chemodosimeter for Hg²⁺ by mercury triggered hydrolysis of the vinyl ether group has been reported. The probe has the unique advantages of easy-preparation, good water solubility, excellent selectivity, high sensitivity, and fast response time (∼60 s) towards Hg²⁺ in aqueous solution. Furthermore, the probe is demonstrated to be qualified to detect Hg²⁺ in living cells.

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Mercury is one of the most ubiquitous and poisonous heavy metals.¹ Mercury ions are not biodegradable, and hence can be concentrated through the food chain in the tissues of fish and marine mammals. Excess mercury accumulation may induce heavy damage to the central nervous system, various cognitive and motor disorders, and Minamata disease.² Therefore, the determination of mercury in biological and environmental samples is crucial both to the monitoring of environmental pollution and to the diagnosis of clinical disorders. Whereas standard techniques, such as atomic absorption-emission spectrometry,³ inductively coupled plasma mass spectrometry,⁴ and anodic stripping voltammetry,⁵ often require expensive and sophisticated instrumentation, and/or sample preparation, and are therefore not suitable for real-time and in situ analysis.

Optical sensors involving fluoroionophores are becoming popular because of their ease of application in solution as well as their high sensitivity to and selectivity for trace analytes with spatial and temporal resolution.⁶ In the past several years, considerable efforts have been made to develop fluorescent chemosensors for Hg²⁺ based on the coordination of Hg²⁺ to heteroatom-based ligands, Hg²⁺ catalyzed desulfurization, and Hg²⁺ promoted hydrolysis of the vinyl ether group and β-alkynyl ether group.⁷ However, most of them still have limitations such as interference from other coexisting metal ions, poor water-solubility, and long response time.⁸ Therefore, for practical applications, it is still strongly desirable to develop fluorescent sensors with good water solubility, high sensitivity, and quick response for real-time detection of Hg²⁺.

Merocyanine dye is a well-known chromophore exhibiting strong intramolecular charge transfer (ICT) due to “push–pull” substituent pairs which results in a large spectral shift. To date, a number of probes have been reported based on the hemicyanine moiety for different analytes through the modulation of ICT efficiency (Fig. 1).⁹ Inspired by these works, we report here a novel and simple hemicyanine-based fluorescence turn-on chemodosimeters MS2, a vinyl ether derivative of hemicyanine, for the detection of Hg²⁺. The design of probe MS2 is based on the mercury ion-promoted hydrolysis reaction of vinyl ethers. It is well known that the oxymercuration of vinyl ether will generate the corresponding hemiacetal intermediate, which undergoes fragmentation to release free hydroxyl group.^7j,n,10 We envisioned that the protection of the hydroxyl group of hemicyanine with a vinyl group would block the ICT process. However, the deprotection of the electron-withdrawing vinyl enol ether group of MS2 by Hg²⁺ promoted hydrolysis reaction would releases a hydroxy donor which will increase the “push–pull” character of the hemicyanine dye and recover its ICT property (Scheme 1).

As shown in Scheme S1 (ESI†), MS2 can be readily prepared in three convenient steps under facile conditions with high yield starting with commercially available 4-hydroxybenzaldehyde. The product (MS2) was well characterized by ¹H, ¹³C NMR, and HR-MS (ESI†).

Fig. 1 Hemicyanine-based probes.

Scheme 1 Mercury triggered hydrolysis of MS2.

We firstly assessed the UV-vis spectroscopic properties of MS2 in PBS buffer solution (10 mM, pH = 7.4, containing 1% CH₃CN). MS2 (20.0 μM) displayed a moderate UV-vis absorption around 400 nm. Upon incremental addition of Hg²⁺ (0–2.0 equiv.), the peak at 400 nm slightly decreased, and a new band at 520 nm, which is characteristic of hemicyanine fluorophore,^9f appeared instantly with a clear isosbestic point at 412 nm (Fig. 2). Furthermore, a good linear relationship was observed between the changes in the absorbance at 520 nm with Hg²⁺ in the range of 0–30.0 μM (Fig. S1, ESI†).

Fig. 2 Absorption spectra of MS2 (20.0 μM) in PBS buffer solution (10 mM, pH = 7.4, containing 1% CH₃CN) in the presence of different concentrations of Hg²⁺ (0–2.0 equiv.).

The emission spectra of MS2 and its fluorescence titration with Hg²⁺ were recorded in PBS buffer (10 mM, pH = 7.4, containing 1% CH₃CN). As expected, MS2 alone is almost non-fluorescent (λ_ex = 510 nm, Φ = 0.004, Table S1, ESI†). However, upon progressive addition of Hg²⁺, the emission band at 551 nm rapidly increased (Fig. 3), which was attributed to the cleavage of vinyl enol group by mercury ion promoted hydrolysis reaction and the formation of free ICT active hemicyanine fluorophore (Scheme 1). Moreover, the fluorescence titration curve revealed that the fluorescence intensity at 551 nm increased linearly with increasing concentration of Hg²⁺ (R² = 0.99) (Fig. 3 and S2, ESI†) and further smoothly increased until a maximum was reached up to 25.0 μM Hg²⁺ (λ_ex = 510 nm, Φ = 0.018, Table S1, ESI†).

Fig. 3 Fluorescence spectra of MS2 (10.0 μM) in PBS buffer solution (pH = 7.4, containing 1% CH₃CN) in the presence of different concentrations of Hg²⁺ (0–30.0 μM) (λ_ex = 510 nm). Inset: fluorescence intensity changes as a function of Hg²⁺ concentration.

Efforts were then made to check the detecting mechanism as envisioned that the Hg²⁺ induced hydrolytic cleavage of vinyl enol ether group of MS2 to the free hemicyanine. To this end, ¹H NMR titration experiment was conducted. As shown in Fig. 4, the three olefinic protons at δ 6.74 (dd, J = 13.5, 5.6 Hz, 1H), 4.81 (d, J = 13.5 Hz, 1H), and 4.55 (d, J = 5.6 Hz, 1H) attributed to the vinyl enol ether group of MS2, disappeared after the addition of Hg²⁺. Meanwhile, chemical shifts of three protons at δ 9.24 (t, J = 5.2 Hz, 1H), and 2.78 (d, J = 5.2 Hz, 2H) attributed to the (2-oxoethyl)mercury chloride were discovered, indicating that the hydrolytic cleavage of vinyl enol ether group of MS2 occurred in the presence of Hg²⁺ (Scheme 1). Furthermore, the aromatic protons at δ 7.08 (d, J = 8.4 Hz, 2H) attributed to H-h were dramatically shifted upfield after the addition of Hg²⁺ due to the deprotection process strengthens the electron-donating ability from oxygen atom. Those results are in agreement with the optical responses. We also carried out the HPLC-MS measurements for the MS2–Hg²⁺ solution (Fig. S10, ESI†). All those results agree well with the proposed Hg²⁺ induce deprotection of MS2.

Fig. 4 ¹H NMR spectra of MS2 in the absence and presence of Hg²⁺ (1.0 equiv. in D₂O).

Subsequently, the time-dependence of MS2 fluorescence was evaluated in the presence of Hg²⁺ in PBS buffer (10 mM, pH = 7.4, containing 1% CH₃CN). The results showed that the fluorescence of tested solutions remarkably increased to their maximum value within 1 minute (Fig. 5), which indicated that MS2 features the fastest response chemodosimeters to date for Hg²⁺.

Fig. 5 Time-dependent fluorescence intensity changes of MS2 (10.0 μM) upon addition of Hg²⁺ (3.0 equiv.) in PBS buffer solution (pH = 7.4, containing 1% CH₃CN) (λ_ex = 510 nm). Inset: fluorescence intensity changes as a function of response time.

Further, the fluorescence titration of MS2 with various metal ions was conducted to examine the selectivity (Fig. 6, and S3, ESI†). Much to our delight, the examined alkali, alkaline-earth metal ions, transition metal ions, and even Pd²⁺ ions showed nominal changes to the fluorescence spectra of MS2. It should be mentioned that MS2 still responded to Hg²⁺ sensitively even in the presence of other relevant competing ions (Fig. 6, and S4, ESI†). Therefore, these results suggest that MS2 displays high selectivity toward Hg²⁺ in aqueous solution.

Fig. 6 Fluorescence responses of MS2 to various metal ions (including Hg²⁺, Ag⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Li⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, Zn²⁺, and Pd²⁺). Black bars represent the addition of 3.0 equiv. of the appropriate metal ion to a 10.0 μM solution of MS2 (in PBS buffer solution, pH = 7.4, containing 1% CH₃CN). Red bars represent the addition of 3.0 equiv. of Hg²⁺ to the solutions containing MS2 (10.0 μM) and the appropriated metals (3.0 equiv.) (λ_ex = 510 nm).

pH impacts on the fluorescence of MS2 and the MS2–Hg²⁺ system were also investigated. As depicted in Fig. S11, ESI,†MS2 alone was inert to pH in the range of 5.5–9.8. But in the presence of Hg²⁺, the fluorescence response of MS2 decreased as pH of test solutions decreased, which was attributed to the formation of enol-form of free hemicyanine fluorophore by mercury-induced hydrolytic cleavage of probe (Scheme S2, ESI†).^9f Moreover, it reacted more difficultly with Hg²⁺ when increased the pH of test solutions due to the reaction rate of mercury ion-promoted hydrolysis of vinyl enol ether becomes slow at high pH value.^7j,10b However, satisfactory Hg²⁺-sensing abilities were exhibited in the range of pH from 7.0 to 9.0, indicating that MS2 could be used in living cells without interference from pH effects.

Due to the favorable properties of MS2in vitro, the potential utility of MS2 in living cells was studied. HeLa cells were incubated with 5.0 μM of MS2 for 30 min at 37 °C and exhibited only weak fluorescence (Fig. 7b). The cells were then treated with HgCl₂ (10.0 μM) for 30 min at 37 °C, which resulted in a dramatic increase of intracellular red fluorescence (Fig. 7d). These indicated that MS2 was cell membrane permeable and capable of image Hg²⁺ in living cells.

Fig. 7 Fluorescence image of HeLa cells incubated with MS2 (5.0 μM) for 0.5 h, and then washed quickly with PBS for imaging (b). The cells were then treated with HgCl₂ (10.0 μM) for 0.5 h which resulted in a dramatic increase in intracellular red fluorescence (d). (a) and (c) Bright-field images of live cells in (b) and (d).

In conclusion, we have rationally developed a novel and simple hemicyanine-based sensitive fluorescence turn-on chemodosimeter for Hg²⁺via mercury triggered hydrolysis reaction. The probe has the unique advantages of easy-preparation, good water solubility, excellent selectivity, high sensitivity, and fast response towards Hg²⁺ in aqueous solution. Furthermore, fluorescence imaging of Hg²⁺ in living cells indicates that this probe is favorable for biological applications.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (20902082), the Graduate Innovative Research Program of Zhejiang Sci-Tech University (YCX14002), and the Program for Innovative Research Team of Zhejiang Sci-Tech University (13060052-Y).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, characterization of the compounds, and additional spectroscopic data. See DOI: 10.1039/c5ra13802g

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