A reversible biocompatible “turn-on” fluorescent probe for the detection of mercury(II)

Author’s Accepted Manuscript A reversible biocompatible “turn-on” fluorescent probe for the detection of mercury (II) Tao Zhang, Baoyan Wu, Zhengzhi Zou, Yunxia Wu, Judun Zheng, Wai-Kwok Wong, Ka-Leung Wong www.elsevier.com/locate/jlumin

PII: DOI: Reference:

S0022-2313(15)30211-8 http://dx.doi.org/10.1016/j.jlumin.2015.10.037 LUMIN13660

To appear in: Journal of Luminescence Received date: 7 July 2015 Revised date: 26 September 2015 Accepted date: 17 October 2015 Cite this article as: Tao Zhang, Baoyan Wu, Zhengzhi Zou, Yunxia Wu, Judun Zheng, Wai-Kwok Wong and Ka-Leung Wong, A reversible biocompatible “turn-on” fluorescent probe for the detection of mercury (II), Journal of Luminescence, http://dx.doi.org/10.1016/j.jlumin.2015.10.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A reversible biocompatible “turn-on” fluorescent probe for the detection of mercury (II) Tao Zhang,a, * Baoyan Wu,a Zhengzhi Zou,a Yunxia Wu,a Judun Zheng,a Wai-Kwok Wong, b, ** Ka-Leung Wongb, **

a

MOE key Laboratory of Laser Life Science & Institute of Laser Life Science,

College of Biophotonics, South China Normal University, Guangzhou 510631, P.R. China b

Department of Chemistry, Hong Kong Baptist University, Waterloo Road, Hong

Kong, P.R. China * Corresponding Author. Tel: +8620-8521-6052; Fax: +8620-8521-6052 **

Corresponding

Author.

E-mail

address:

[email protected]

(T.

Zhang);

[email protected] (W.-K. Wong); [email protected] (K.-L. Wong)

Abstract A new rhodamine-based fluorescent probe based on the known ring-opening mechanism of rhodamine derivative, was successfully synthesized and demonstrated to detect the Hg2+ with high selectivity and sensitivity in aqueous solution and living cells. In the presence of Hg2+, the probe exhibit remarkably amplified absorption with a binding constant determined as 8.76 ± 0.13, followed by a distinct change in color and 560-fold enhancement of fluorescence with a reversible response and little interference with other biological relevant metal ions. Moreover, the confocal 1

microscopic switch-on imaging in MCF-7 cells of the probe towards Hg2+, together with its non-cytotoxicity and efficient cellular uptake, confirmed that the probe can be used as a biocompatible probe for monitoring of Hg2+ in living cells. Key words: rhodamine ; fluorescent probe; mercury; reversible; cellular uptake

2

1. Introduction The development of probes for sensing and recognition with high selectivity and sensitivity of heavy metal ions, particular mercury ions, have attracted considerable attention over the past few decades.[1-3] As one of the most toxic heavy metals, global mercury contamination has long been a great concern. The mercury and its derivatives can be converted into methyl mercury by bacteria in the environment and subsequently bioaccumulates through the food chain to produce indelible pollution.[4-6] Furthermore, as the high affinity of mercury to thiol groups in proteins and enzymes, these accumulated mercury-substances may lead to the dysfunction of cells and consequently causing health problems. Therefore, concerted attempts have been made to develop fluorescent probes for monitoring the level of Hg2+ in vitro and in vivo.[7-9] Rhodamine-based off–on functionality with a metal-induced ring opening process, among the created chemical sensing systems on metal ions, is one of the most spectacular modalities due to its photo-stability, high extinction coefficient and high fluorescence quantum yield.[10] Based on this, with an awareness of the biohazard of mercury-substances

mounting in

the

past

decade,

many rhodamine-based

chemosensors are reported for the detection of Hg2+ ions.[11-17] However, the choice of these kind of bio-probes available is small and some key factors of these kind probes, such as their cytotoxicities and cellular uptake efficiency, are less investigated.[18] The need for the development of new biocompatible probe for Hg2+ is still as real and pressing.

3

Recently, based on the high affinity of mercury to thiol groups, some sensors in the framework of rhodamine-thiol patterns have been reported.[19-21] And we also successfully demonstrated a visible to NIR probe to detect the Hg2+ with high sensitivity and selectivity in aqueous solution.[22] Herein, we will demonstrate the synthesis and characterization in detail of a new biocompatible probe Rh-SS with a mercury-binding thiol group for monitoring Hg2+ in aqueous solution and living cells, including its cytotoxicity and cellular uptake efficiency. 2. Experimental 2.1 Materials and characterization All chemicals used were of reagent-grade and were purchased from Sigma–Aldrich and used without further purification. Analytical-grade solvents were dried by standard procedures, distilled and deaerated before use. The freshly distilled solvents were bubbled with nitrogen gas for at least 10 min to remove any residual oxygen. NMR spectra were recorded on a Bruker Ultrashield 400 Plus NMR spectrometer. 1H and

13

C NMR chemical shifts were referenced to tetramethylsilane

(δ = 0.00 ppm). High-resolution mass spectra were recorded with a Bruker Autoflex matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker). 2.2 Spectroscopic measurements Electronic absorption spectra in the UV/Vis region were recorded on a Hewlett Packard 8453 UV/Vis spectrophotometer. Luminescence spectra were recorded on an Edinburgh Instrument FLS920. The data was recorded in a solution of CH3CN/PBS 4

buffer (5:1, v/v, pH = 7.4). 2.3 Binding Constants The binding constants for 1:1 complexation Kb were determined by nonlinear least quares analysis with the Equation (1).[23, 24] ⁄

{

[(

)

]

}

(1)

Y was the recorded intensity (absorption or emission), Y0 was the start value without the addition of Hg2+, Yl was the limiting value (absorption or emission), CHg was Hg2+ concentration, and cL was the sensor concentration. 2.4 Cell culture Human breast carcinoma MCF-7 cell line was maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco). The medium was supplemented with 10% FBS (Gibco) and antibiotics (penicillin 50 U/mL; streptomycin 50 μg/mL). The cells were incubated at 37 ℃ in humidified incubator with 5% CO2 when used.Confocal microscope. 2.5 Confocal microscope MCF-7 cells (2 × 104 in 35-mm culture dishes) were incubated with 10 μM of Rh-SS separately in culture medium containing 0.25% DMSO for 1 h in dark and then treated with 1 μM, 5μM, 10 μM of Hg(ClO4)2 for 30 min, respectively. The cells were then washed with PBS and then loaded with fresh culture medium. The imaging of Rh-SS with Hg2+ in MCF-7 cells was examined using a ZEISS LSM 510 META confocal microscope. A 40× objective was used for image capturing. Images were processed and analyzed using the LSM software. 5

2.6 MTT cell viability assay MCF-7 cells (2 × 104 per well in wells of 96-well plate) were incubated with a gradient concentration of Rh-SS in culture medium containing 0.25% DMSO for 24 h in dark. And then 20 μl of MTT solution (5mg/mL in PBS) was added to each well and incubated further for 4h. The cells were then washed with phosphate-buffered saline (PBS). After that, the formazan was extracted and dissolved with DMSO. The absorbance of the subsequent solutions was measured at 540 and 690 nm using a 96-well plate reader (ELx800 Absorbance Microplate Reader). Quadruplicates were performed and the data obtained were analyzed using the GraphPad Prism 5 software. 2.7 Cellular uptake MCF-7 cells (2 × 105 per well in 6-well plate) were incubated with 10 μM of Rh-SS in culture medium containing 0.25 % DMSO for 0.5 h, 1 h, 2 h in dark, respectively. The cells were then washed with PBS, loaded with culture medium containing 0.1 mM Hg(ClO4)2 and incubated further for 30 min. Then the cells were washed with PBS and trypsinized. The cell-suspended solutions were centrifuged and then washed several times with PBS for subsequent flow cytometric analysis. The flow cytometric experiments were performed on a FACS Calibur (Becton Dickinson) flow cytometer using 488 nm laser for excitation and the fluorescence signal produced was collected using the FL-3 channels equipped with long-pass filters. 2.8 Synthesis of SS Under a nitrogen atmosphere, a mixture of pyrrolidine-1-dithiocarboxylic acid ammonium salt (1.64 g, 0.01 mol), 2-bromoacetophenone (1.99 g, 0.01 mol) and 6

acetone (30 mL) was stirred for 1.5 h at room temperature. After that, solvent was removed under reduced pressure. The residue was redissolved in CH2Cl2 (30 mL) and subsequently washed with brine (5 mL × 3). Then the extract was dried over anhydrous Na2SO4 and solvent was removed. The crude product was purified by column chromatography on silica gel eluting with CH2Cl2, affording SS as white solids. Yield: 71%; 1H NMR (400M, CDCl3): δ 1.96 (m, 2H), 2.08 (m, 2H), 3.74 (t, J = 7.2 Hz, 2H), 3.91 (t, J = 7.2 Hz, 2H), 4.92 (s, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.81 (t, J = 6.4 Hz, 1H), 8.08 (m, 2H); 13C NMR (101M, CDCl3): δ 24.39, 26.18, 44.51, 50.82, 55.50, 128.59, 128.71, 136.08, 191.00, 193.44; FAB-MS: m/z = 265. 2.9 Synthesis of Rh-SS A mixture of SS (265 mg, 1.0 mmol), hydrazine hydrate (200 μL), a drop of acetic acid and THF/ ethanol (20 mL, v/v, 1:1) was heated to reflux for 3 h under a nitrogen atmosphere. After cooled to room temperature, the solvent was removed under reduced pressure. The residue was redissolved in CH2Cl2 (20 mL) and washed with brine (5 mL × 3). After dried by anhydrous Na2SO4 and removal of the solvent, the crude SS-NH2 was obtained and used directly for next step without further purification. At the same time, a mixture of rhodamine B (480 mg, 1.0 mmol), POCl 3 (1 mL) and CH2Cl2 (10 mL) was heated to reflux for 3 h. After cooled to room temperature, the solvent was removed under reduced pressure. The obtained crude rhodamine B acid chloride was redissolved in anhydrous CH2Cl2 10 mL) and then added to a mixture of SS-NH2, triethylamine (2 mL) and CH2Cl2 (10 mL). The reaction mixture was stirred for 12 h at room temperature. After that, the reaction 7

solution was washed with brine (5 mL × 3), dried over anhydrous Na2SO4 and then condensed. The crude product was purified by column chromatography on silica gel eluting with CH2Cl2/CH3OH (40/1, v/v), affording Rh-SS as pale yellow solids. Then pale yellow X-ray quality crystals were collected by vaporing diffusion of the methanol solution of Rh-SS at room temperature. Yield: 68%; 1H NMR (400M, CDCl3): δ 1.14 (t, J = 8.0 Hz, 12H), 1.89 (m, 4H), 3.31 (m, 8H), 3.50 (t, J = 8.0 Hz, 2H), 3.88 (t, J = 8.0 Hz, 2H), 4.74 (s, 2H), 6.29 (dd, J = 4.0 Hz, J = 8.0 Hz, 2H), 6.38 (d, J = 4.0 Hz, 2H), 6.57 (d, J = 8.0 Hz, 2H), 7.21 (m, 3H), 7.29 (m, 1H), 7.47 (m, 2H), 7.68 (d, J = 4.0 Hz, 1H), 7.70 (d, J = 4.0 Hz, 1H), 7.96 (m, 1H);

13

C NMR

(101M, CDCl3): δ 12.63, 24.21, 25.99, 35.86, 44.35, 50.35, 55.55, 67.55, 97.93, 106.78, 107.94, 123.39, 124.12, 127.94, 128.04, 128.34, 128.56, 130.24, 130.61, 132.63, 135.18, 148.62, 151.38, 153.71, 161.23, 166.79, 192.48; MALDI-TOF MS: calcd. for [M+] 703.9583, found: 704.3036. 2.10 Crystal Structure Determination Single crystal of Rh-SS of suitable dimension was mounted onto a thin glass fiber. The intensity data was collected at 296 K on a Bruker SMART CCD diffractometer (Mo-K radiation, = 0.71073 Å) in μΦ and scan modes. Structure was solved by direct methods followed by difference Fourier syntheses, and then refined by full-matrix least-square technique against F2 using SHELXTL.[25] All other non-hydrogen atoms were refined with anisotropic thermal parameters. Absorption corrections were applied using SADABS.[26] All hydrogen atoms were placed in calculated positions and refined isotropically using a riding model. Crystallographic data and refinement 8

parameters for Rh-SS are recorded (Table S1). Relevant atomic distances (Table S2) and bond angles (Table S3) are also collected. The crystallographic data of Rh-SS has been deposited at the Cambridge Crystallographic Data Center, CCDC - 1426642. 3. Results and discussion 3.1 Synthesis and characterization Synthetic procedures for the probe Rh-SS are shown in Scheme 1. The intermediate SS-NH2 and rhodamine B chloride was readily prepared from the starting compound SS and rhodamine B, respectively. The aimed product was subsequently prepared by treating the intermediate SS-NH2 with rhodamine B chloride in the presence of organic base NEt3 in CH2Cl2 at room temperature in high yield and can be obtained in gram scale. The product was then purified by column chromatography on silica gel and recrystallized in methanol to afford the pure final product Rh-SS. The product Rh-SS was characterized by 1H and

13

C NMR spectroscopy and

MALDI-TOF mass spectrometry (Figure S1-3). Rh-SS was also characterized by X-ray diffraction analysis. As shown in the otrep diagram of Rh-SS (Figure 1), the probe exists in a form of spirocyclic structure. And, moreover, the atoms O4-N8-N10-S2-S9 seem to form together a mercury coordination trap for the mercury detection. 3.2 Absorption and emission 9

The UV/Vis absorption Hg2+-titration spectra of Rh-SS was recorded as depicted in Figure 2a (CH3CN/PBS buffer, pH = 7.4, v/v, 5:1), displaying no obvious absorption at λ > 450 nm in the absence of Hg2+, which suggests that Rh-SS exists in the form of spirocyclic structure in the solution. Upon addition of 10.0 equiv Hg2+ ion (Hg(ClO4)2 as Hg2+ source) to the buffer solution of Rh-SS, a 70-fold enhancement of the absorption centered at 567 nm was observed, accompanying with the solution color converted from colorless to magenta (Figure 2a inset ). This can be interpreted that Rh-SS in spirocyclic structure turned to ring-opened structural style after binding with Hg2+, which is in accordance with rhodamine-based metal ions sensing systems. The binding stoichiometry was confirmed as 1:1 by a Job’s plot (Figure 2b) and MALDI-TOF mass spectrometry (Figure S5). The binding constant Kb (log Kb = 8.76 ± 0.13) for Hg2+ with Rh-SS in the buffer solutions was estimated by means of absorption titration at 567 nm based on the theoretical nonlinear fitting of the titration curve. Furthermore, the binding mode of Rh-SS with Hg2+ was elucidated by 1H NMR titration (Figure S6). After addition of 0.2 equiv. of Hg2+ solution in methanol-d4 into a solution of Rh-SS in CDCl3, the triplet peak of Ha proton and quartet peak of Hc of the two (CH3CH2)2N- groups of the probe display a downfield shift of 0.01 ppm and 0.03 ppm, respectively. In addition, along with the coordination of Hg2+ with Rh-SS, the singlet peak of Hf of the -S-CH2-C(Ph)=N- linker exhibits a slight upfield shift and the intensity decreased dramatically. And the aryl proton Hh , Hg and Hl in the rhodamine B matrix show significant downfield shift, from 6.20 ~ 6.70 ppm to 7.0 ~ 7.90 ppm, due to the Hg2+-induced ring-opening process of the 10

rhodamine B spirocycle. Meanwhile, upon addition of 10.0 eq Hg2+ and excitation at 540 nm, an intense emission band appeared at 590 nm with an enhancement of 560 fold (Figure 3). The switch-on luminescent response observed at 590 nm together with the response in the absorption spectral band centered at 567 nm triggered by Hg2+ suggest opening of the ring in Rh-SS due to the metal ion coordination. Association constant value log Kb, calculated from the nonlinear fitting of the emission titration (Figure 3 inset), for binding of Rh-SS with Hg2+ was found to be 8.85 ± 0.06, which is in good agreement with the results from the absorbance titration. This further indicates that the visible responsive signal at 590 nm was attributed to the ring-opened rhodamine sensing unit. Based on this, the detection limit was determined as 0.1 μM.[27] 3.3 Reversibility Based on the Hg2+-titration spectra of MALDI-TOF mass, 1H NMR, absorption and emission , the possible binding mode between Rh-SS towards Hg2+ is proposed in Scheme 2 with the binding stoichiometry as 1:1.[28] In the absence of Hg2+ ions, Rh-SS prefers to exist in form of spirocyclic state and only weak fluorescence signal could be observed. While upon the addition of Hg2+ ions, the S and N atoms of Rh-SS molecular are involved in the binding with Hg2+ ions to form a stable chelating rings required for the opening of the spirocyclic ring of Rh-SS to establish the delocalized xanthene moiety, displaying absorption and fluorescence enhancement. Moreover, the 11

addition of an aqueous solution of 10.0 equiv Na2S to the solutions of Rh-SS-Hg2+ almost restored the initial spectroscopic feature of the free probe (Figure 4), which confirmed further the interaction process of the Rh-SS with Hg2+ ion. For a chemical probe to be widely employed in the detection of specific analyses, this reversibility is a profitable feature. 3.4 Selectivity The binding selectivity of the probe Rh-SS was then investigated based on emission spectroscopies in the buffer solution towards various metal ions, such as Al3+, Cr3+, Cd2+, Cu2+, Fe2+, Pb2+, Co2+, Ni2+, Ag+, Zn2+, Hg2+. As shown in Figure 5a, with the addition of each metal listed, it was only Hg2+ that gave a large off-on response (560-fold amplification) in the visible region. The competition experiments with high concentrations of the above-mentioned metal ions confirm further that the probe Rh-SS has high selectivity towards Hg2+ ion over the other competitive metal ions (Figure 5b). 3.5 PH effect The effects of PH on the fluorescence response of Rh-SS towards Hg2+ were then investigated at different PH values in the absence and presence of Hg2+. As shown in Figure 6, in the PH range from 4.5 to 8.5, no obvious characteristic fluorescence of rhodamine was observed for Rh-SS in the absence of Hg2+. Upon addition of Hg2+, a 12

stably fluorescent amplified-response was recorded in the same PH condition. The results show that Rh-SS may be able to sense Hg2+ under approximate physiological conditions with very low background fluorescence, although the sensitivity decrease slightly in alkaline condition (PH > 8.0). [15, 29] 3.6 Cell imaging To evaluate the potential application of Rh-SS as bio-probe for the detection of mercury in living cells, its behavior in human breast carcinoma MCF-7 cells were investigated using confocal microscopic. As shown in Figure 7, a bright cell imaging was captured after the Rh-SS-loaded MCF-7 cells was treated with 10 μM of Hg(ClO4)2 for 30 min. Meanwhile, the brightness of cell imaging exhibit a character of Hg2+ concentration dependence, which was in accordance with the founding in the aqueous solution. 3.7 Cytotoxicity and cellular uptake As a promising bio-probe, the cytotoxicity and cellular uptake efficiency of the probe are also critical determinants for its long- and real-time and sensitively monitoring of the analysts. However, there are few reports on the investigation of these features of the rhodamine-based sensor towards metal ions. So we then studied the cytotoxicity and cellular uptake efficiency of Rh-SS in MCF-7 cells using MTT reduction assay (Figure 8a). We found that Rh-SS exhibit low cytotoxicity after treated with various concentrations of Rh-SS at dark for 24h. Then we investigated 13

the cellular uptake efficiency using flow cytometric method. As shown in Figure 8b, after the MCF-7 cell incubated with Rh-SS for 30 minutes and then treated with 0.1 mM Hg2+, significant (3-fold) enhancement of fluorescence intensity was observed in Rh-SS loaded cells compared to those controls. Moreover, after 2 h of further incubation and then loaded with 0.1 mM Hg2+, a 16-fold increase in fluorescence intensity from Rh-SS loaded cells relative to those untreated cells. These indicated that Rh-SS can be uptaken very efficiently by the living MCF-7 cells. 4. Conclusion In summary, we have demonstrated that a new rhodamine-based probe Rh-SS exhibits Hg2+-triggered amplified absorption and turn-on responsive fluorescence via a 1:1 binding mode in aqueous solution with a detection limit of 0.1 μM determined. A feature of reversible fluorescent response of Rh-SS towards Hg2+ has been founded in the presence of Na2S. And its selectivity toward Hg2+ over other metal ions has also confirmed very high. Furthermore, Rh-SS is successfully applied for monitoring of Hg2+ in living MCF-7 cells with low cytotoxicity and efficient cellular uptake, which will supply more alternative and evidence for rhodamine-based bio-probe for detection of metal ions in living cells. Acknowledgements This research is supported by the National Natural Science Foundation of China (21405052, 81371646) and the Natural Science Foundation of Guangdong Province, China (2014A030310255). 14

Appendix A. Supplementarymaterial Supplementary data associated with this article can be found in the online version at doi:

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Figure Captions Figure 1. Crystal structure of Rh-SS Figure 2. (a) Changes in absorption spectra of Rh-SS (10 μM) in CH3CN/PBS buffer (5:1, v/v, pH = 7.4) solution upon addition of Hg2+ (0 ~ 10.0 eq); inset: color change of Rh-SS. (b) Job’s plot between Rh-SS and Hg2+. Figure 3. Emission spectra excited at 540 nm Rh-SS (10 μM) in the CH3CN/PBS buffer (5:1, v/v, pH = 7.4) solution upon addition of Hg2+ (0 → 10 eq); Inset: Plot of emission at 590 nm as a function of the concentration of Hg2+ with non-linear theoretical fit. Figure 4. Emission spectra of Rh-SS (10 μM, CH3CN/PBS buffer 5:1, v/v, pH = 7.4) in the presence of 3.0 equiv. of Hg2+ and 10.0 equiv. of Na2S at the excitation of 540 nm. Figure 5. (a) Emission changes of Rh-SS (10 μM) excited at 540 nm with various metal ions monitored at 590 nm in CH3CN/PBS buffer (5:1, v/v, PH = 7.4) solution with various 10.0 eq metal ions; (b) Emission response of Rh-SS (10 μM) excited at 540 nm to various metal ions (10.0 eq) monitored at 590 nm without and with Hg2+ (10.0 eq) in CH3CN/PBS buffer (5:1, v/v, pH = 7.4) solution. Figure 6. Fluorescence changes of Rh-SS (10 μM)) excited at 540 nm in the absence and presence of Hg2+ (10.0 eq) monitored at 590 nm in CH3CN/PBS buffer (5:1, v/v) with different PH values. Figure 7. Confocal microscopic images of MCF-7 cells treated with 10 μM of Rh-SS for 1h in dark and then treated with (a) Hg2+ (1 μM), (b) Hg2+ (5 μM), (c) Hg2+ (10 μM) for 30 min, respectively, under excitation at 543 nm. Figure 8. (a) Dark cytotoxicity of Rh-SS towards MCF-7 cells (The cells were incubated with a gradient concentration of Rh-SS for 24 h in dark); (b) Flow cytometric analysis of the cellular 19

uptake by monitoring the emission of Rh-SS in the MCF-7 cells: the cells were incubated with 10 μM of Rh-SS for 0 h (control), 0.5 h, 1 h and 3 h in dark, and then loaded with 0.1 mM of Hg(ClO4)2 for 30min. Scheme 1. Synthetic route to Rh-SS. Scheme 2. Proposed reversible 1:1 binding mode between Rh–SS and Hg2+.

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