English

• Fluorescent probes can be used as an efficient tool for the detection and imaging of biological trace species, pesticide residues and heavy metal cations^[1]. Among various heavy metal cations, Hg²⁺ is extremely toxic, and it becomes methylmercury which accumulates in the body through the food chain, leading to a series of mental illnesses and the well-known Minamata disease^[2-4]. Consequently, selective and sensitive fluorescent probes for Hg²⁺ detection are highly desirable.

  Generally, most of papers published about Hg²⁺ probes have investigated the high thiophilicity of Hg^{2+ [5-8]}. Furthermore, rhodamine^{[6-7, 9, 12]}, coumarin^[10], and naphthalimide^[7] have been frequently employed as signal fluorophores for the probe design. Some of them have been utilized for fluorescence imaging of Hg²⁺ in living cells (Table S1, Supporting information) ^[5-14]. It is worth noting that lysosomes digest unwanted substances and cell fragments and contain a variety of hydrolases, so they are considered"enzyme banks"or"cell cleaners"in cells^[15]. In fact, Hg²⁺ has been found to accumulate in lysosomes, causing abnormal lysosomal pH fluctuation, which is closely associated with lysosome dysfunctions^[16]. In this regard, lysosomes are one of the important organelle targets for Hg²⁺ toxicity^[17]. However, the probes that can be used in lysosomes for Hg²⁺ detection are barely reported^{[5-6, 11]}. It is well known that the pH of lysosomes in normal cells ranges from 4.5 to 6.0, while that of cancer cells is even lower^[18]. Nevertheless, some of the reported probes, especially those based on rhodamine backbone, could not perform normally under such extremely acidic conditions^[12]. In this regard, it is still a great challenge to the development of lysosome-targeting probes toward Hg²⁺.

  Recently, our group reported a pyrrole-containingbis-hydrazone (1a, Scheme 1) capable of detecting Hg²⁺ in real water samples^[14]. Herein, a lysosome - targeting group of 4-(2-aminoethyl)morpholine was introduced to 1a to fabricate a simple and effective fluorescent turn-on probe 1 for Hg²⁺ detection in lysosomes of HeLa cells. It is worth noting that the primary starting materi-al, 5 - formyl - 2, 4 - dimethyl - 1H - pyrrole - 3 - carboxylic acid, is a commercial intermediate of Sunitinib (an inhibitor used in cancer therapies) favoring the real -world application of the as-synthesized probe.

  Scheme 1

  

  Scheme 1.  Synthesis route of probe 1

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  1.   Experimental

  1.1   Materials and instrumentation

  Solvents and starting materials for syntheses were purchased commercially and used as received. Elemental analyses were carried out on an Elemental Vario ELanalyzer. ¹H NMR spectra were recorded on a Bruker AV400 NMR spectrometer in DMSO - d₆ solution. The UV spectra were recorded on a Purkinje General TU-1800 spectrophotometer. Fluorescence spectra were determined on a Varian CARY Eclipse spectrophotometer, in the measurements of emission and excitation spectra the pass width was 5 nm. Time-resolved photoluminescence spectra were determined on an Edinburgh FLS980 spectrophotometer. ESI-MS spectra were obtained on a Bruker Daltonics Esquire 6000 mass spectrometer. The cytotoxic effect exerted by 1 on cultured HeLa cells was ascertained by a standard MTT assay according to the literature method^[19]. Fluorescent images were taken on Zeiss Leica inverted epifluorescence/reflectance laser scanning confocal microscope.

  1.2   Synthesis of 1

  5-Formyl-2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-morpholin-4-yl-ethyl)-amide (2) was prepared from 5-formyl-2, 4-dimethyl-1H-pyrrole-3-carboxylic acid according to literature method^[20]. Hydrazinium hydroxide (100 mg, 85%, 1.7 mmol) and 2 (279 mg, 1mmol) were added to an EtOH solution (10 mL). The mixture was refluxed for 3 h with two drops of acetic acid. After cooling to room temperature, the separated solid was filtered, washed with EtOH, and then dried in air. Yield: 58%. Anal. Calcd. for C₂₈H₄₂N₈O₄(%): C, 60.63; H, 7.63; N, 20.20. Found(%): C, 60.48; H, 7.78;N, 20.33. ¹H NMR (400 MHz, DMSO - d₆): δ 11.35 (s, 1H, NH), 8.40 (s, 1H, CH), 7.24 (s, 1H, NH), 3.57 (t, 4H, 2CH₂), 3.30 (2H, merged by peak of H₂O), 2.41 -2.44 (m, 8H, 4CH₂), 2.35 (s, 3H, CH₃), 2.25 (s, 3H, CH₃). ¹³C NMR was not recorded due to the poor solubility of 1. ESI-MS: m/z=555.321 8 for [M+H] ⁺ (Calcd.555.330 0), 278.179 8 for [M+2H]²⁺ (Calcd. 278.1750), 185.787 0 for [M+3H]³⁺ (Calcd. 185.786 6).

  1.3   General UV-Vis and fluorescence spectra measurements

  The spectral analyses were accomplished in EtOH/H₂O (7∶3, V/V) solution at room temperature. The concentration of probe 1 for UV-Vis and fluorescence measurement was 10 µmol·L^-1. Solutions of metal ions were prepared with nitrate or chloride salts in H₂O. UV-Vis and fluorescence spectrophotometric titration were conducted directly in a 2 mL cuvette by successive addition of corresponding chemical reagent using a microliter syringe. Upon addition of every aliquot, the solution was well mixed then the spectrumwas measured.

  2.   Results and discussion

  2.1   Synthesis and characterization

  Probe 1 was produced by the condensation of 2 and hydrazinium hydroxide in a moderate yield, and was further characterized by elemental analyses, ¹H NMR, and ESI-MS methods. It should be noted that the peak of one CH₂ group was covered by the H₂O peak in the ¹H NMR spectrum of 1 (Fig. S1), which is assumed according to the spectrum of 2 shown in Fig.S2.

  2.2   UV-Vis spectroscopic studies for Hg²⁺ sensoractivity

  The absorption spectrum of probe 1 (10 µmol·L^-1) in EtOH/H₂O (7∶3, V/V) solution displayed one band centered at 380 nm (Fig. 1a), attributed to the n - π^*transition of imine units^[14]. When probe 1 bound to Hg²⁺ (3 equiv.), the band had an obvious redshift to 413 nm, a hyperchromatic effect, along with the generation of a shoulder at 440 nm, assigned to the ligand to metal charge transfer (LMCT) ^[13]. This finding indicates that the N atom of the imine bond is involved in the coordination with Hg²⁺. The change of the color of the liquid in the cuvette from colorless to yellow leads to the conclusion that the response of probe 1 to Hg²⁺ is more pronounced and more selective than the other metal ions (3 equiv.) tested (Fig. 1a, Inset), including Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺, which enables the potential application of detection of Hg²⁺ in water samples by naked eyes. The absorbance ratio A₄₄₀/A₃₈₀ linearly increased by increasing the Hg²⁺ concentration from 4.5 to 16.5 µmol·L^-1 (Fig. 1b, Inset). The two isosbestic points at 308 and 403 nm reveal the existence of only one intermediate complex (Fig. 1b). The Job′s plot obtained by varying the concentration ratio of Hg²⁺ and 1 gained the 1∶1 combined stoichiometric ratio of 1 and Hg²⁺ (Fig.S3), with an association constant (K_a) of 3.48×10⁴ L·mol^-1 according to the Benesi - Hildebrand expression (Fig. S4) ^[21]. The limit of detection (LOD) of 1 to Hg²⁺ was 59.6 nmol·L^-1 based on 3σ/k (σ: standard deviation, k: slope)^[20], which was lower than the LOD of 1a.

  Figure 1

  

  Figure 1.  (a) UV-Vis spectra of 10 µmol·L^-1 probe 1 in EtOH/H₂O (7∶3, V/V) solution with 3 equiv. of metal ions: Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺ ions and blank; (b) UV-Vis spectra of 10 µmol·L^-1 probe 1 upon the addition of Hg²⁺ (0-5 equiv.) in EtOH/H₂O (7∶3, V/V) solution

  Inset in a: the color change of 1 solution in the presence of Hg²⁺; Inset in b: the absorbance ratio of A₄₄₀/A₃₈₀ as a function of Hg²⁺ concentration (4.5-16.5 µmol·L^-1)

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  2.3   Fluorescence spectroscopic studies for Hg²⁺ sensor activity

  Fluorescence emission spectral changes of 1, caused by the presence of several metal ions in the EtOH/H₂O (7: 3, V/V) solution, are demonstrated in Fig. 2. When excited at 410 nm, the initial solution of 1 displayed a weak emission at 475 nm (quantum yield Φ =0.02, determined with quinine sulfate, and Φ_c=0.546 in 0.05 mol·L^-1 H₂SO₄)^[22]. Significant changes in the emission spectrum were observed in the presence of Hg²⁺ (3 equiv.), except in the case of other metal cations (3 equiv.). When 1 was combined with Hg²⁺ (3equiv.), the fluorescence intensity was significantly enhanced as the color changed from colorless to cyan (Fig. 2a, Inset) under a 365 nm UV lamp (Φ =0.21).Furthermore, it can be seen from Fig.S5 that the average decay constant (τ) value of 1 obtained by the singleexponential decay fitting method was 1.56 ns, which was far less than that of 1-Hg²⁺ (6.12 ns), indicating thecoordination between 1 and Hg²⁺.

  Figure 2

  

  Figure 2.  (a) Fluorescence emission spectra of 10 µmol·L^-1 probe 1 in EtOH/H₂O (7∶3, V/V) solution with 3 equiv. of metal ions: Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺ ions and blank. The inset shows. (b) Fluorescence emission spectra of 10 µmol·L^-1 probe 1 upon the addition of Hg²⁺ (0-5 equiv.) in EtOH/H₂O (7∶3, V/V) solution

  Inset in a: the color change of 1 solution in the presence of Hg²⁺ under 365 nm UV lamp; Inset in b: the fluorescence intensity at 475 nm as a function of Hg²⁺ concentration (6.0-18.0 µ mol·L^-1); λ_ex=410 nm

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  Similarly, the linear response concentration range of Hg²⁺ was 6.0-18.0 µmol·L^-1 according to the fluorescent titration results (Fig. 2b, Inset). The K_a and LOD were 2.61×10⁴ L·mol^-1 (Fig. S6) and 45.8 nmol·L^-1, respectively, which are in the same order of magnitude when compared with the data from UV spectra. All these facts show that the response of probe 1 to Hg²⁺ is characterized by efficient selectivity and sensitivity in colorimetric and fluorescent detection, so it can be used to research Hg²⁺ quantitatively in water or biological samples.

  2.4   Investigation of environmental factors and dynamics analysis

  The specificity of Hg²⁺ to probe 1 was determined by testing the interference of other metal cations (Fig.S7 and S8). No distinct changes in the UV and fluorescence spectra of 1+Hg²⁺ were observed in the presence of other metal cations, in addition to trivalent cations (Al³⁺, Cr³⁺, and Fe³⁺) in which their strong Lewis acidity results in imine - bond cleavage^[23], thereby indicating that the ability of probe 1 to detect Hg²⁺ is less affected by other metal cations and has high selectivity.

  It was concluded that 1 can detect Hg²⁺ in a pH range of 4.0 to 10.0 by studying the fluorescence intensity changes of probe 1 and probe 1+Hg²⁺ at different pH values (Fig.S9). Due to this unique property, probe 1 can be used for practical studies of Hg²⁺ in lysosomes. Compared with the probe 1a described in our previous work^[14], the introduction of 4 - (2 - aminoethyl) morpholine not only improves the selectivity of the probe (interference from Cu²⁺ in the case of 1a) but also enhances the tolerance to acidic environments.

  A fast time response is an important characteristic of chemosensors. The changes in the fluorescence of 1 (10 mol·L^-1) in EtOH/H₂O (7∶3, V/V) solution with 3 equiv. of Hg²⁺ was detected in less than 2 min, which is a faster analysis time for the Hg²⁺ detection than that of 1a (ca. 3 min) (Fig.S10).

  The reversibility of the probe toward Hg²⁺ was investigated by adding EDTA to the 1+Hg²⁺ system. The results showed that the emission of 1+Hg²⁺ wastotally quenched upon the addition of EDTA (3 equiv.), and it was recovered by further addition of Hg²⁺. The on-off phenomenon was reversible even after four cycles (Fig.S11), indicating that the probe can be easily regenerated for repeated use.

  2.5   Reaction mechanism

  To analyze the coordination mechanism of 1 with Hg²⁺, ESI-MS measurements were performed in EtOH solution (Fig. 3). The spectrum of 1+Hg²⁺ displayed a peak at m/z 754.275 1 corresponding to the protonated cation of [Hg(1-2H)] (1 - Hg²⁺, C₂₈H₄₀N₈O₄Hg, Calcd.753.29). Furthermore, in the ¹H NMR spectrum of 1+Hg²⁺ (Fig. 4), the signal of the CH=N groups of 1 at δ 8.40 shifted to the lower field and split into two peaks at δ 8.50 and 8.60, indicating the coordination of one imine N atom and the change in the environment of the other imine N atom. The amide NH signal of 1 was greatly broadened at δ 7.24 and significantly shifted to δ 7.89 in the presence of Hg²⁺, it may be because of the effect of the two pyrrole N atoms to protons and coordination effect. Therefore, a donor set of two pyrrole and one imine N atoms involving the binding of Hg²⁺ is suggested, as illustrated in Scheme 2.

  Figure 3

  

  Figure 3.  ESI-MS spectral changes of 1 with the addition of Hg²⁺ in EtOH solution

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  Figure 4

  

  Figure 4.  ¹H NMR spectral changes of 1 with the addition of Hg²⁺ in DMSO-d₆ solution

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  Scheme 2

  

  Scheme 2.  Proposed reaction mechanism of 1 with Hg²⁺ ions

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  To gain more insights into the sensing mechanism, 1 and 1-Hg²⁺ were examined by density functional theory (DFT) calculation^[24]. The results show that both HOMO and LUMO are dispersed on pyrrole and imine moieties in probe 1 (Fig. 5), while the C=N isomerization leads to weak fluorescence. In contrast, the C=N isomerization is not possible in the 1 - Hg²⁺ complex. Meanwhile, the HOMO of 1-Hg²⁺ is localized on the pyrrole and imine moieties, while the LUMO is almost centered on the Hg²⁺ ion, clearly indicating that the emission of 1-Hg²⁺ is mainly from the LMCT. The corresponding energy difference (ΔE) of 1 - Hg²⁺ (3.40eV) is lower than that of 1 (3.58 eV), reflecting the longer maximum absorbance wavelength of 1-Hg²⁺ compared to that of 1. The Hg—N_pyrrole bond lengths in complex 1-Hg²⁺ were 0.236 0 and 0.237 5 nm, respectively, which are shorter than that of Hg—N_imine (0.253 2 nm). Nevertheless, these bond lengths are comparable with those in the calculated 1a-Hg²⁺ complex, thus suggesting stable emission of complex 1-Hg²⁺ in an aqueous solution^[14].

  Figure 5

  

  Figure 5.  Optimized structures and HOMO/LUMO of 1 and 1-Hg²⁺ by DFT calculation

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  2.6   Bioimaging applications

  The MTT assay was used to detect the effect of probe 1 on cell viability as shown in Fig.S12, the cell viability was still 82.6% even while the probe concentration reached 40 µmol·L^-1, which indicates that probe 1 had low cytotoxicity. The biological suitability of probe 1 was assessed by detecting its ability to examine Hg²⁺ in HeLa cells. HeLa cells without the treatment of probe 1 exhibited no fluorescence signal(Fig. 6a), while after incubation with 1 (10 µmol·L^-1) at 37 ℃ for 30 min, a faint blue fluorescence was observed in the cells (Fig. 6d), which should be attributed to the emission of probe 1. Next, the brighter cellular blue fluorescence was shown after the cells were treated with 20 µmol·L^-1 Hg²⁺ (Fig. 6g). This finding indicates the capability of 1 in imaging Hg²⁺ in living cells.

  Figure 6

  

  Figure 6.  Confocal fluorescence images of Hela cells: confocal fluorescence (a), brightfield (b), and overlay (c) images of HeLa cells incubated for 30 min at 37 ℃; confocal fluorescence (d), brightfield (e), and overlay (f) images of HeLa cells incubated with 10 µmol·L^-1 of 1 for 30 min at 37 ℃; confocal fluorescence (g), brightfield (h), and overlay (i) images of HeLa cells incubated with 10 µmol·L^-1 of 1 for 30 min at 37 ℃ and then incubated with 20 µmol·L^-1 Hg²⁺ for another 30 min at 37 ℃

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  To determine the capability of 1 to detect Hg²⁺ in the lysosomes of HeLa cells, colocalization images were performed with LysoTracker Red (Fig. 7). Whenthe image was merged, it could be observed that the blue fluorescence channel of 1+Hg²⁺ has a coincidence rate of 0.91 with the red channel of the lysosome tracker. The above results indicate that the probe can detect Hg²⁺ in the lysosomes of living cells.

  Figure 7

  

  Figure 7.  Bright field and fluorescence images of HeLa cells stained with 10 µmol·L^-1 of probe 1+Hg²⁺ (20 µmol·L^-1) and LysoTracker Red: (a) from the blue channel, (b) from the red channel (lysosomes staining), (c) an overlay blue and red channels, (d) bright field image, (e) an overlay of bright field, blue, and red channels; (f) Intensity profile of the linear region of interest across the HeLa cell co-stained with LysoTracker Red and the blue channel of 1+Hg²⁺

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  3.   Conclusions

  In summary, a pyrrole-based bis-hydrazone appended with 4-(2-aminoethyl)morpholine moiety has been prepared, which acts as a fluorescent off-on sensor toward Hg²⁺. The mechanism was completely investigated by UV - Vis, fluorescence spectroscopy, time -resolved fluorescence spectroscopy, ESI-MS, ¹H NMR, and DFT calculations. probe 1 can be used as an Hg²⁺ sensor. In addition, the costained experiment reveals that the probe was able to image Hg²⁺ in lysosomes.

  Supporting information is available at http://www.wjhxxb.cn

  
  
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• 

  Scheme 1  Synthesis route of probe 1

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  Figure 1  (a) UV-Vis spectra of 10 µmol·L^-1 probe 1 in EtOH/H₂O (7∶3, V/V) solution with 3 equiv. of metal ions: Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺ ions and blank; (b) UV-Vis spectra of 10 µmol·L^-1 probe 1 upon the addition of Hg²⁺ (0-5 equiv.) in EtOH/H₂O (7∶3, V/V) solution

  Inset in a: the color change of 1 solution in the presence of Hg²⁺; Inset in b: the absorbance ratio of A₄₄₀/A₃₈₀ as a function of Hg²⁺ concentration (4.5-16.5 µmol·L^-1)

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  Figure 2  (a) Fluorescence emission spectra of 10 µmol·L^-1 probe 1 in EtOH/H₂O (7∶3, V/V) solution with 3 equiv. of metal ions: Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺ ions and blank. The inset shows. (b) Fluorescence emission spectra of 10 µmol·L^-1 probe 1 upon the addition of Hg²⁺ (0-5 equiv.) in EtOH/H₂O (7∶3, V/V) solution

  Inset in a: the color change of 1 solution in the presence of Hg²⁺ under 365 nm UV lamp; Inset in b: the fluorescence intensity at 475 nm as a function of Hg²⁺ concentration (6.0-18.0 µ mol·L^-1); λ_ex=410 nm

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  Figure 3  ESI-MS spectral changes of 1 with the addition of Hg²⁺ in EtOH solution

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  Figure 4  ¹H NMR spectral changes of 1 with the addition of Hg²⁺ in DMSO-d₆ solution

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  Scheme 2  Proposed reaction mechanism of 1 with Hg²⁺ ions

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  Figure 5  Optimized structures and HOMO/LUMO of 1 and 1-Hg²⁺ by DFT calculation

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  Figure 6  Confocal fluorescence images of Hela cells: confocal fluorescence (a), brightfield (b), and overlay (c) images of HeLa cells incubated for 30 min at 37 ℃; confocal fluorescence (d), brightfield (e), and overlay (f) images of HeLa cells incubated with 10 µmol·L^-1 of 1 for 30 min at 37 ℃; confocal fluorescence (g), brightfield (h), and overlay (i) images of HeLa cells incubated with 10 µmol·L^-1 of 1 for 30 min at 37 ℃ and then incubated with 20 µmol·L^-1 Hg²⁺ for another 30 min at 37 ℃

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  Figure 7  Bright field and fluorescence images of HeLa cells stained with 10 µmol·L^-1 of probe 1+Hg²⁺ (20 µmol·L^-1) and LysoTracker Red: (a) from the blue channel, (b) from the red channel (lysosomes staining), (c) an overlay blue and red channels, (d) bright field image, (e) an overlay of bright field, blue, and red channels; (f) Intensity profile of the linear region of interest across the HeLa cell co-stained with LysoTracker Red and the blue channel of 1+Hg²⁺

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