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A rhodamine-based chemosensor with diphenylselenium for highly selective fluorescence turn-on detection of Hg2+ in vitro and in vivo

A rhodamine-B based chemosensor with diphenylselenium, RhoSe, has been synthesized, and its detection behavior towards various metal ions is studied via UV/Vis and fluorescence spectroscopy. RhoSe shows a selective response to Hg2+ in CH3OH/H2O (v/v = 9 : 1) solutions over other metal ions. After addition of Hg2+, the solution of RhoSe displays an obvious color change from colorless to pink and a significant, 48-fold fluorescence enhancement. The color change and fluorescence enhancement are attributed to the ring-opening of the spirolactam in the rhodamine fluorophore, which is induced by Hg2+ binding. The binding ratio of RhoSe–Hg2+ was determined by a Job plot as a 1 : 1 ratio, and the effective pH range for Hg2+ detection was 4.0–10. Importantly, the reversibility of the RhoSe–Hg2+ complex was observed through the addition of Na2S. For practical applications, the strip method was utilized to detect Hg2+ in water/methanol solution. In addition, confocal fluorescence microscopy experiments demonstrated that RhoSe is an effective fluorescent probe for Hg2+ detection in vitro and in vivo.


Introduction
There is a growing demand to develop selective and sensitive chemosensors for toxic metal ions in environmental and biological studies. 1-4 Mercury is known as a highly toxic heavy metal even at very low concentrations. Mercury can be found in various products, such as batteries, light bulbs, and thermometers. Three forms of mercury exist: metallic mercury, ionic mercury, and organic mercury. Metallic mercury can be converted into methylated forms, mainly methylmercury and dimethylmercury, by bacteria. 5,6 Mercury ions show high affinities with the thiol groups in proteins, and their accumulation in the body has harmful effects on the brain, central nervous system, and kidneys. Minamata disease, discovered in Minamata city, Japan, is a typical disease caused by severe mercury poisoning. 7 Due to the high toxicity of mercury, the highest permitted concentration of mercury in environmental and dietary samples has been established by the EPA in the USA as 2 ppb. 8 Many analytical methods for mercury determination in environmental and clinical samples have been developed, such as inductively coupled plasma atomic emission spectrometry (ICP-AES), 9 atomic absorption spectroscopy (AA), 10 electrochemical method 11 and inductively coupled plasma mass spectrometry (ICP-MS). 12 Fluorescence chemosensors provide a direct way for mercury detection due to their high sensitivity, selectivity, and their applications in environmental and biological analysis. [13][14][15][16][17][18][19][20][21][22][23][24][25] Among various available uorophores, rhodamine is remarkable due to its exceptional optical properties, such as a long absorption and emission wavelength, high extinction coefficient and quantum yield, and low detection limit. 4,26 Furthermore, rhodamine exists in two forms, a spirolactam (non-uorescent) form and a ring-opened amide (uorescent) form, providing an ideal model for the design of metal ion sensing with light "off-on" switching. Binding metal ions to rhodamine derivative induces ring-opening of rhodamine. This resulted in a red emission and a color change from colorless to pink because of a shi in the equilibrium state from a spirolactam to a ring-open amide. [27][28][29][30][31][32][33][34][35] In this work, a chemosensor RhoSe containing a rhodamine B hydrazide linked with diphenyl selenium was synthesized for Hg 2+ detection (Scheme 1). Due to its spirolactam form, RhoSe has no visible absorption and is colorless. The binding of Hg 2+ to the chemosensor RhoSe induced a switch from the spirolactam form to the ring-opened amide form, which was observed as a pink color. More importantly, RhoSe could potentially be applied to test strips, living cell imaging, and zebrash studies.

Synthesis of RhoSe
The chemosensor RhoSe was synthesized by the reaction of rhodamine B hydrazide and 2-(phenylselanyl) benzaldehyde to form a Schiff base (Scheme 1). RhoSe was characterized by 1 H NMR, 13 C NMR, and HRMS (ESI) (see the ESI †).
The selectivity towards metal ions measured by UV-visible and uorescence spectra The metal ion detection ability of chemosensor RhoSe was further evaluated. Fig. 1 shows the metal ion selectivity of RhoSe; only Hg 2+ causes a color change and produces a yellow uorescence. Other metal ions cause no change in the uorescence and color. Fig. 2 shows the UV/Vis and uorescence spectra of chemosensor RhoSe in the presence of several metal ions. Only Hg 2+ caused signicant absorption and emission peaks at 561 and 584 nm, respectively. Hg 2+ titration of chemosensor RhoSe was monitored by UV/Vis spectra (Fig. 3a).
During the sequential addition of Hg 2+ ions, a new absorption band at 561 nm was observed with an intense pink color. Free RhoSe does not exhibit any absorption peak in the visible region due to its spirocyclic form. When RhoSe binds with Hg 2+ , the binding induces the ring-opening of rhodamine, resulting in the pink color. During Hg 2+ titration, an emission peak at 584 nm was produced (Fig. 3b). Based on the uorescence titration prole, the detection limit of RhoSe for the Hg 2+ ion was calculated to be 12 nM (see Fig. S5 in the ESI †).
In order to quantify the effective uorogenic change, a quantum yield calculation was carried out. The quantum yield of RhoSe was found to be as low as F ¼ 0.006. Upon coordination with Hg 2+ , the quantum yield of RhoSe-Hg 2+ was F ¼ 0.29, which is a 48-fold enhancement. Next, the binding ratio of   RhoSe-Hg 2+ was evaluated by Job plot experiments. The uorescence intensity at 584 nm was plotted against the molar fraction of RhoSe. The highest uorescence intensity was reached at a molar fraction of 0.5, indicating that RhoSe binds to Hg 2+ with a 1 : 1 ratio (see Fig. S6 in the ESI †). ESI mass spectra also provided further support for a 1 : 1 metal complex, with m/z ¼ 1002.13 (see Fig. S7 in the ESI †). Furthermore, the binding constant was obtained as 7.44 Â 10 4 M À1 , which indicates the strong binding affinity of RhoSe for the Hg 2+ ion (see Fig. S8 in the ESI †).
To further evaluate the inuence of other metal ions, experiments with coexisting ions were carried out. RhoSe (10 mM) was examined with various metal ions in the presence of Hg 2+ . As shown in Fig. 4a, the uorescence intensity with coexisting ions is similar to that from Hg 2+ alone. This observation indicates that there was no interference from the other metal ions. Owing to the importance of pH, we also studied the emission behavior of RhoSe towards the Hg 2+ ion with respect to different pH environments. As shown in Fig. 4b, only a low emission intensity was observed for RhoSe at pH 4.0-10.0. When the pH is lower than 4, the emission intensity increases remarkably; this is due to the protonation-induced ring opening in RhoSe. With Hg 2+ , a high emission intensity at 584 nm was observed at pH 4.0-10. Furthermore, the emission intensity of RhoSe decreased slightly when the pH was higher than 10.0. This is due to the reduced stability of RhoSe-Hg 2+ at high pH values. These observations conrm that RhoSe can be used to detect Hg 2+ over a wide pH range. In order to examine the reversibility of RhoSe-Hg 2+ , Na 2 S was added to remove the Hg 2+ . In Fig. 5, the emission peak at 584 nm decreased aer the addition of Na 2 S. With further addition of Hg 2+ , the uorescence of RhoSe-Hg 2+ was regained. The collective data clearly indicates the reversible binding character of the chemosensor RhoSe to Hg 2+ .
We further performed Hg 2+ titrations monitored by 1 H NMR to determine the binding interaction between RhoSe and Hg 2+ . As a heavy metal ion, Hg 2+ affects the proton signals near the Hg 2+ binding site. 36 The 1 H NMR signal of the imine nitrogen proton (-CH]N) was shied upeld upon the addition of Hg 2+ (Fig. 6). Two protons signals, H b and H f , which are located adjacent to the selenium atom on the diphenyl-selenium moiety, were also shied upeld and became much broader aer the sequential addition of Hg 2+ ions. Furthermore, the IR spectra of RhoSe and RhoSe-Hg 2+ were recorded (Fig. 7). Without Hg 2+ , two peaks at 1715 cm À1 and 1684 cm À1 are the strength mode of the carbonyl oxygen (C]O). Aer addition of   Hg 2+ ions, the peak corresponding to the carbonyl oxygen (C] O) in rhodamine disappeared. In addition, a new stretching frequency at 1618 cm À1 (C]N) is corresponding to its ring opened amide conformation. The collective data indicates that Hg 2+ binds to RhoSe through the selenium atom at a diphenylselenium moiety, one nitrogen atom, and one oxygen atom. Some recently reported uorescence Hg 2+ -sensors based on rhodamine derivatives are shown in Table 1. Some rhodamine based Hg 2+ sensors show response to Hg 2+ and the other metal ions, such as Cu 2+ and Zn 2+ . The probe RhoSe only responses to Hg 2+ with a low detection limit 12 nM. Also, the probe RhoSe can be applied in test strip detection and bioimaging in cells and zebrash.
According to the collective results, the binding mechanism is shown in Scheme 2. When the probe RhoSe is in the spirolactam form, it has no visible absorption and is colorless. Upon the complexation of RhoSe with Hg 2+ , Hg 2+ binding induced a switch from the spirolactam form to the ring-opened amide form, which was observed as a pink color.
In order to elucidate the structures of RhoSe and RhoSe-Hg 2+ , density functional theory (DFT) calculations using the Gaussian 09 soware package were carried out. We applied the B3LYP/LANL2DZ energy optimization to determine the optimal structures of RhoSe and RhoSe-Hg 2+ (Fig. 8). The structure of RhoSe-Hg 2+ shows that Hg 2+ binds to the chemosensor RhoSe by one nitrogen atom, one oxygen atom, and one selenium atom at distances of 2.30, 2.20, and 2.70Å, respectively. The energy gaps between the HOMO and LUMO levels of RhoSe and RhoSe-Hg 2+ are DE ¼ 3.81 eV and 1.08 eV, respectively (see Fig. S9 in the ESI †). RhoSe-Hg 2+ has a smaller energy gap compared with that of RhoSe.

Application of RhoSe in test strips
To apply the chemosensor RhoSe to a practical use, test strips were prepared by immersing lter paper in RhoSe solution,   followed by drying in air. The selectivity of the test strips for various metal ions was rst evaluated (see Fig. S10 in the ESI †).
Only in the presence of Hg 2+ were a pink color and yellow emission observed; other metal ions did not cause any color or uorescence changes in the test strips. In addition, the test strips were immersed in different concentrations of Hg 2+ , and exhibited a clear color change from colorless to pink with increasing Hg 2+ concentration (Fig. 9). The detection limit of the test strips was found to be 1 Â 10 À6 M, allowing for sensitive detection of Hg 2+ ions.

Bioimaging of RhoSe
The bioimaging potential of RhoSe for Hg 2+ was further explored. Firstly, the cytotoxicity of RhoSe was evaluated by an MTT assay using HeLa cells. The cellular viability of HeLa cells treated with RhoSe was greater than 80%, which indicates a low cytotoxicity of RhoSe (<20 mM) (see Fig. S11 in the ESI †). Thereaer, RhoSe was applied to image Hg 2+ using confocal uorescence microscopy. Fig. 10a shows that no uorescence was observed from HeLa cells treated with RhoSe. With Hg 2+ , a bright red uorescence emission in the HeLa cells can be clearly observed (Fig. 10b). The overlay image revealed that the red uorescence was distributed throughout the intracellular area. This observation shows the good cell-membrane permeability of RhoSe.

Fluorescence imaging experiments of RhoSe in zebrash
Furthermore, RhoSe was used for Hg 2+ imaging in zebrash. First, 3 day-old sh were treated with the chemosensor RhoSe (20 mM) for 30 min at 28 C. No red uorescence was found (Fig. 11a). With Hg 2+ , a strong red uorescence in the 3 day-old zebrash was observed (Fig. 11b). Remarkably, the organs in the zebrash, such as the heart, liver, and brain, were clearly seen. 37,38 In addition, a lower uorescence intensity was observed in the brain (Fig. 12). This indicates a relatively lower level of mercury in the brain. The zebrash uorescent images demonstrate that the chemosensor RhoSe can be used to study the accumulation of mercury species in vivo.

Instruments
Hitachi F-7000 uorescence spectrophotometer was used for uorescence spectra measurements. 1 H and 13 C NMR spectra were obtained with a 300 MHz Bruker spectrometer. UV/Vis spectra were recorded on an Agilent 8453 UV/Vis spectrometer. Fluorescence microscope images were obtained from a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope.

Synthesis of RhoSe
Rhodamine B hydrazide 39 (500 mg, 1.0 mmol) and 2-(phenylselanyl) benzaldehyde 40 (314 mg, 1.1 mmol) were added to methanol (20 mL). The reaction mixture was reuxed for 24 h. The subsequent precipitate was ltrated and column chromatography (DCM: hexane ¼ 1 : 1) was used to purify the compound.      Job plot was used to decide the binding ratio of the complex RhoSe-Hg 2+ . The molar fraction of RhoSe was plotted against the uorescence intensity at 584 nm. The total concentration of RhoSe and Hg 2+ was 50 mM. The binding stoichiometry of the complex RhoSe-Hg 2+ was determined from the molar ratio with maximum uorescence intensity. The association constant (K a ) of the complex RhoSe-Hg 2+ was obtained by using the Benesi-Hilderbrand equation. 41 where DF is the intensity difference at 584 nm and DF sat is the maximum intensity difference at 584 nm. The plot (1/DF vs. 1/[Hg 2+ ]) was tted by using eqn (1) and the association constant K a was obtained from the intercept and slope of the line.

Cytotoxicity assay
The cytotoxicity of RhoSe was determined by the methyl thiazolyl tetrazolium (MTT) assay. HeLa cells were grown in Dulbecco's modied Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) at 37 C, 5% CO 2 . Then HeLa cells were grown into a 96-well cell-culture plate. The cells were added with several concentrations (5, 10, 15, 20, 25 mM) of RhoSe and then incubated at 37 C, 5% CO 2 for 24 h. Subsequently, each well was added with MTT (10 mL, 5 mg mL À1 ) and then incubated at 37 C, 5% CO 2 for 4 h. Yellow precipitates (formazan) formed in plates then were collected and dissolved in DMSO (200 mL) and Sorenson's glycine buffer (25 mL, 0.1 M glycine and 0.1 M NaCl). The absorbance at 570 nm of each well was recorded by Multiskan GO microplate reader. Cell viability was calculated by the following equation: Cell viability (%) ¼ (average absorbance of treatment group)/ (average absorbance of control group).

Fluorescence imaging of RhoSe
HeLa cells were treated with Hg(ClO 4 ) 2 (30 mM, dissolved in PBS) and incubated for 30 min at 37 C. Excess metal ions were washed away by PBS (3 Â 2 mL). The cells were added with culture media (2 mL) and then incubated with RhoSe (10 mM, dissolved in DMSO). Aer 30 min incubation at 37 C, the culture media was removed and cells were washed with PBS (3 Â 2 mL). Cell imaging was obtained by a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope (Germany) with the excited wavelength, 510 nm and the collected emission at 580-590 nm.

Hg 2+ imaging in zebrash
Animals were maintained in accordance with the guidelines of the National Institute of Health, Taiwan, and approved by the Institutional Animal Care and Use Committee (IACUC) of National Chiao Tung University. Zebrash was kept at best breeding conditions at 28 C. For mating, male and female zebrash were put in the same tank at 28 C in a 12 h light/12 h dark cycle. The spawning of eggs was triggered by light stimulation in the morning. Most eggs were fertilized immediately. The zebrash was growed in 5 mL of embryo medium supplemented with 1-phenyl-2-thiourea (PTU) in 6-well plates for 24 h at 28 C. Furthermore, 3 day-old zebrash embryos were anaesthetized with 50 mg L À1 tricaine and incubated with mercury(II) perchlorate hydrate (60 mM) in E3 media for 30 min at 28 C. PBS buffer was used to remove the remaining mercury ions. The zebrash was further incubated with RhoSe (20 mM) for 30 min at 28 C. Aer washing with PBS to remove the remaining probe, the image of zebrash was observed by a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope.

Computational methods
Quantum chemical calculations based on density functional theory (DFT) were carried out using a Gaussian 09 program. The optimal structures of RhoSe and RhoSe-Hg 2+ were obtained using the density functional theory (DFT) method with the hybrid-generalized gradient approximation (HGGA) functional B3LYP. For RhoSe-Hg 2+ , the LANL2DZ basis set was used for Hg 2+ , whereas the 6-31G basis set was used for RhoSe.

Conclusions
In conclusion, a rhodamine-based probe RhoSe with diphenylselenium has been designed for selective and sensitive Hg 2+ detection. RhoSe showed a rapid response to Hg 2+ alone among other metal ions, with colorimetric and uorescent turn-on responses. RhoSe can be used for Hg 2+ detection over a pH range of 4.0-10.0. In addition, the stoichiometry of the RhoSe-Hg 2+ complex was calculated as a 1 : 1 binding ratio using a Job plot, and was further conrmed by ESI-MS. RhoSe shows high sensitivity towards Hg 2+ with a detection limit of 12 nM. Importantly, the practical application of RhoSe for Hg 2+ detection was successfully demonstrated through test strips, live cell images, and live zebrash images. RhoSe has been developed as a useful tool for monitoring Hg 2+ distribution in biological samples.