A novel facilely prepared rhodamine-based Hg2+ fluorescent probe with three thiourea receptors

Accepted Manuscript A novel facilely prepared rhodamine-based Hg thiourea receptors

2+

fluorescent probe with three

Miaomiao Hong, Shengzhou Lu, Feng Lv, Dongmei Xu PII:

S0143-7208(15)00510-0

DOI:

10.1016/j.dyepig.2015.12.023

Reference:

DYPI 5046

To appear in:

Dyes and Pigments

Received Date: 19 October 2015 Revised Date:

25 December 2015

Accepted Date: 26 December 2015

Please cite this article as: Hong M, Lu S, Lv F, Xu D, A novel facilely prepared rhodamine-based 2+ Hg fluorescent probe with three thiourea receptors, Dyes and Pigments (2016), doi: 10.1016/ j.dyepig.2015.12.023. 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 proof before it is published in its final 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.

ACCEPTED MANUSCRIPT

A novel facilely prepared rhodamine-based Hg2+ fluorescent probe with three thiourea receptors

a

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Miaomiao Hong a,b, Shengzhou Lu c, Feng Lv a,b, Dongmei Xu a,b∗

College of Chemistry, Chemical Engineering and Materials Science, Soochow University,

Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow

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b

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Suzhou, Jiangsu 215123, China

University, Suzhou, Jiangsu 215123, China c

College of Textile and Clothing Engineering, Soochow University, Suzhou, Jiangsu 215123,

Abstract

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China

A novel rhodamine-based turn-on fluorescent probe with three thiourea receptors was

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designed, synthesized and fully characterized. In acetonitrile/4-(2-hydroxyethyl)-

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1-piperazineethanesulfonic acid buffer (9/1, v/v, pH=7.21), the probe showed high selectivity and sensitivity to Hg2+ with a 20-fold fluorescence enhancement at 583 nm. The increase in fluorescence intensity was linearly proportional to the concentration of Hg2+ in the range of 25–200 µM with a detection limit of 3.04×10-7 M. The probe could work in a nearly neutral pH span of 6.41–8.33 and exhibited excellent ∗

Correspondence author. Tel.: +86-512-65882027, Fax: +86-512-65880089. E-mail address: [email protected]. Full postal address: College of Chemistry, Chemical Engineering and Materials Science, Soochow University. No.199 Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu, People’s Republic of China. 1

ACCEPTED MANUSCRIPT interference immunity. Real sample assay showed the probe had good practicability. The results from Job's plot, reversibility experiment, mass and infrared spectra analysis suggested a 1:3 probe/Hg2+ complex with an association constant of

Keywords: Rhodamine; Thiourea; Fluorescent probe; Hg2+

1. Introduction

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9.88×1011 M-3 and a new interaction way between the probe and Hg2+.

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Mercury, one of the common toxic elements in the environment, contaminates the

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atmosphere, soil and water ubiquitously. When absorbed in human body, mercury will cause damage to the brain, central nervous system, endocrine system and so on, leading to motional and cognitive disorders [1,2]. Thus, it is crucial to detect the presence of mercury. Due to high sensitivity, simplicity, non-destructive, as well as

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onsite and instantaneous response, fluorescent probe has been particularly attractive in the detection of noble, heavy or transition metal ions [3-7]. Rhodamine derivatives are regarded as a popular framework for fluorescent probe

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because of their long absorption and emission wavelengths, large extinction

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coefficients, high fluorescence quantum yields, and typical analyte-mediated spectroscopic changes resulting from lactonization-delactonization [3,8-10]. Despite the reported existing studies on rhodamine-based fluorescent probes for Hg2+ [1,2,11-16], there are still much room for improvement in order to attain probes with simple preparation method, good selectivity, high sensitivity, fast response speed, aqueous working media and ecological working pH range [17]. Herein, we designed a rhodamine-based Hg2+ fluorescent probe as advancement in

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ACCEPTED MANUSCRIPT this field. The probe is easily prepared, highly selective and sensitive, and quickly responsive to Hg2+ at room temperature in CH3CN/HEPES buffer (9/1, v/v, pH=7.21). A rare 1:3 probe/Hg2+ complex [18] formed and a new sensing mechanism was

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explored.

2. Experimental 2.1. Reagents and chemicals

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The reagents and chemicals comprise rhodamine B (RB) (AR, The Third Reagent

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Factory, Shanghai), triethylenetetramine (TETA) (CR, Sinopharm Chemical Reagent Co., Ltd.), phenyl isothiocyanate (PITC) (≥98%, Aladdin Industries, Inc.), ethanol (EtOH), acetonitrile (CH3CN), dichloromethane (CH2Cl2) (AR, Jiangsu Powerful Features Chemical Co., Ltd.,), NaCl, KCl, MgCl2, CaCl2, FeCl3·6H2O, FeCl2·7H2O,

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CuSO4·5H2O, Zn(NO3)2·6H2O, CrCl3·6H2O, Pb(NO3)2, Ni(NO3)2·6H2O, MnSO4·H2O, CoCl2·6H2O, CdCl2·2.5H2O and HgCl2 (Sinopharm Chemical Reagent Co., Ltd.). HEPES buffer (0.02 mol/L, pH=7.21) was prepared in deionized water (H2O). The

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solvents used in synthesis were of analytical grade, other solvents were spectroscopic

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grade. All reagents were commercially available and were put into use without further purification.

2.2. Apparatus

MALDI-TOF mass spectrum was recorded on an ultrafleXtreme Mass

spectrometer (Bruker, America) using trans-2-[3-(4-tert-butylphenyl)-2-methyl-2propenyl] malononitrile (DCTB) as matrix. LC-mass was recorded on a Brukermicro TOF-QIII LC/MS (Bruker Daltonics Co., Germany). 1H NMR and

3

13

C NMR spectra

ACCEPTED MANUSCRIPT were carried out on an UNITY INOVA400 and an UNITY INOVA 300 high-resolution superconducting NMR spectrometer (Varian, America) in CDCl3 respectively. Infrared (IR) spectra were recorded on a MagNa-IR550 Fourier

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transform infrared spectrometer (Nicolet, America) using KBr pellet. Elemental analysis (EA) was done on an EA1110 CHNO-S elemental analyzer (Carlo-Elmer, Italy). Fluorescence spectra measurements were obtained by a Fluoromax-4

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spectrofluorometer (HORIBA Jobin Yvon, France). Absorption spectra were obtained

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by a U-3900 spectrophotometer (Perkin-Elmer, America). pH value was tested on a METTLER TOLEDO FE20 pH meter (Mettler Toledo Co., Ltd. Shanghai, China). Melting point was determined on an X-6 Microscopic melting point tester (Beijing Taike Instrument Co., Ltd., China). The spectra and pH values were measured at

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25 °C 2.3. Synthesis of the probe RTTU

was

synthesized

from

the

reaction

of

rhodamine

B

(RB),

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triethylenetetramine (TETA) and phenyl isothiocyanate (PITC), as illustrated in

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Scheme 1. The structure of RTTU was fully characterized by 1H NMR,

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C NMR,

MALDI-TOF-MS, IR, and elemental analysis.

Scheme 1. Synthetic route of RTTU.

The intermediate RTTA was synthesized following the references [11,19,20]. RB (0.1 g, 0.209 mmol) was dissolved in EtOH (10 mL) and then TETA (1 mL, 5.2 mmol) was added dropwise. The solution was heated to reflux for 24 h until it turned from 4

ACCEPTED MANUSCRIPT red to colorless. The solvent was removed under reduced pressure. Water was added to the residue and the mixture was extracted with CH2Cl2. The organic phase was separated and washed three times with water, followed by the evaporation of the solvent. The resultant mixture was then dried in vacuo to afford RTTA as a yellow

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solid (0.103 g, 86.6%). Next, to a stirring solution of RTTA (0.1 g, 0.163 mmol) in CH3CN (10 mL), PITC (200 µL, 1.67 mmol) was added dropwise and the resulted mixture was reacted for 5 h under room temperature until white solid was precipitated.

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After standing for 12 h, the solid was filtrated, washed three times with CH3CN to get RTTU as a white solid. Yield: 0.089 g (52.0%), m.p. 181.8 oC. 1H NMR (400 MHz,

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CDCl3) (Fig. S1, Supporting information (ESI)), δ /ppm: 1.16 (t, 12H, J=6.4 Hz), 3.33 (q, 8H, J=6.8 Hz), 3.43 (t, 2H, J=4.4 Hz), 3.51 (s, 3H), 3.87 (t, 8H, J=3.6 Hz), 4.05 (t, 2H, J=4.4 Hz), 6.31 (d, 2H, J=6.8 Hz), 6.41-6.43 (m, 4H), 7.13 (d, 6H, J=7.2 Hz), 7.20-7.24 (m, 6H), 7.30-7.39 (m, 3H), 7.48 (q, 3H, J=6.8 Hz), 7.87 (d, 1H, J=7.2 Hz),

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9.50 (s, 1H). 13C NMR (400 MHz, CDCl3) (Fig. S2, ESI), δ /ppm: 12.65, 29.47, 38.20, 44.43, 49.52, 65.70, 97.94, 104.15, 108.40, 122.84, 124.09, 125.65, 126.08, 126.45, 127.53, 128.22, 128.53, 130.07, 133.10, 135.86, 139.68, 149.08, 153.40, 181.41,

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181.80. MALDI-TOF-MS (Fig. S3, ESI): [M+H]+=976.420, [M+Na]+=998.415. FTIR

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(cm-1) (Fig. S4, ESI): v(NH) 3206.49; v(CH3, CH2) 2969.26, 2923.94, 2854.50; v(C=O) 1665.44; v(ArH) 3037.73, 1615.30, 1515.01, 1494.76. Elemental analysis: Calculated for C55H61N9O2S3 (%): C, 67.66; N, 12.91; H, 6.30. Found (%): C, 67.34; N, 12.81; H, 6.24.

2.4. Testing and calculating Methods 2.4.1. Sample preparation RTTU was dissolved in CH3CN to form a 0.1 mM stock solution. Metal salts were dissolved in H2O to get 10 mM stock solutions. When the sensing behavior of RTTU 5

ACCEPTED MANUSCRIPT towards metal ions was studied, 5 mL of the RTTU stock solution was mixed with one of the metal salt stock solutions (250 µL) in a 10 mL volumetric flask and diluted with HEPES buffer solution (0.02 mol/L, pH=7.21) and CH3CN to volume. The

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concentration of RTTU was 50 µM. The solvent was CH3CN/HEPES buffer (9/1, v/v, pH=7.21). The pH was adjusted by 0.02 mol/L HEPES buffer with different pH values.

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2.4.2. Fluorescent quantum yield [21]

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The fluorescent quantum yield was estimated from the absorption and fluorescence spectra of RTTU according to Eq. (1), where the subscript s and r stand for the sample and the reference (rhodamine B, φr=0.97 in ethanol), respectively. φ is the quantum yield, A represents the absorbance at the excitation wavelength, S refers

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to the integrated emission band areas and nD is the solvent refractive index. The absorbance of the solutions was kept under 0.05 in order to make the testing results reliable.

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φs = φr

2 Ss A r n Ds 2 S r A s n Dr

(1)

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2.4.3. Detection limit [21]

The detection limit (δ) was calculated based on Eq. (2), where S is the standard

deviation of blank measurement and K is the slope of the fit line in fluorescence titration. The emission fluorescence intensity of RTTU in CH3CN/HEPES buffer (9/1, v/v, pH=7.21) without any metal ions was measured 5 times. δ=3S/K

(2)

2.4.4. Association constant [18] 6

ACCEPTED MANUSCRIPT The association constant for RTTU/Hg2+ was obtained from nonlinear curve fitting of the fluorescence titration data with Benesi-Hildebrand Eq. (3), where F is the fluorescence intensity at the respective wavelength, Fmin and Fmax denote the values at

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the start and end point of the titration, n is the binding stoichiometry for RTTU and Hg2+, [Hg2+] is the Hg2+ concentration, and K is the association constant. ( F − Fmin ) = n log[Hg 2+ ]+ log K ( Fmax − F )

(3)

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log

2.4.5. Real sample assay [22]

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In the real sample assay, 0.5 mL pond water (after filtration) or tap water from Dushu Lake Campus of Soochow University was added in 10 mL volumetric flask, then 5 mL stock solution of RTTU and a certain amount of Hg2+ was introduced. The mixture was diluted to volume with CH3CN and HEPES buffer and the fluorescence

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spectra were recorded. The concentration of Hg2+ in real water samples was obtained from the linear relationship between the maximal fluorescence intensity of the sample

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and the concentration of Hg2+.

3. Results and discussion

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3.1. Design and synthesis of RTTU We designed three S atoms in thiourea as the binding sites for the thiophilic Hg2+.

The reaction between the −NH2 and −NH groups in TETA and the −NCS group in PITC was employed to import three thiourea units into RTTU in one-pot under very mild conditions. Moreover, RTTU could be achieved in good yield through washing the precipitate from the reaction mixture with CH3CN instead of tedious extraction, drying, and column chromatography. Both the quasi Click reaction and the facile 7

ACCEPTED MANUSCRIPT postprocessing method made the synthesis process highly efficient and energy-saving.

3.2. Selectivity and sensitivity of RTTU to Hg2+ Na+, K+, Mg2+, Ca2+, Fe3+, Fe2+, Cu2+, Zn2+, Cr3+, Pb2+, Ni2+, Mn2+, Co2+, Cd2+ and

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Hg2+ were added to the CH3CN/HEPES (9/1, v/v, pH=7.21) buffer solutions of RTTU respectively to investigate the response of RTTU to metal ions. Most of the metal

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cations showed no significant influence on the UV-vis absorption spectra of the RTTU solution, while only Hg2+ produced a very weak peak around 565 nm and turned the

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solution from colorless to pink, as shown in Fig. 1. On the other hand, a significant fluorescence enhancement (about 20 folds at 583 nm) accompanied by a brilliant orange-red color emerged soon after Hg2+ instead of other metal cations was added into the RTTU solution, as shown in Fig. 2. These results imply the structure

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conversion of RTTU from its initial spirolactam form to its ring opened form. Hence, RTTU can be used as a naked-eye and fluorometric probe for Hg2+ in CH3CN/HEPES

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buffer (9/1, v/v, pH=7.21).

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Fig. 1. UV-vis absorption spectra of RTTU in the absence and presence of various cations. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for metal ions.

Fig. 2. Fluorescence spectra of RTTU in the absence and presence of various cations. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for metal ions. λex: 520 nm, slit width: 10 nm.

3.3. Fluorescence titration with Hg2+ The influence of Hg2+ concentration on the fluorescence of RTTU was studied and

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ACCEPTED MANUSCRIPT the result was shown in Fig. 3. The intensity of RTTU was strengthened upon the addition of Hg2+ until a concentration of 300 µM, and then approximately leveled with addition of excess Hg2+. The increase of fluorescence intensity at 583 nm was linearly

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proportional to the Hg2+ concentration in the range 25–200 µM, with a correlation coefficient (R) of 0.9906. The detection limit evaluated from this fluorescence

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titration was 3.04×10-7 mol/L.

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3.4. Effect of coexisting ions

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Fig. 3. Fluorescence spectra of RTTU (50 µM) with various concentrations of Hg2+. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), λex: 520 nm, slit width: 10 nm. From bottom to top, the concentration of Hg2+: 0, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400 µM. Inset: The relationship between the maximal fluorescence intensity and the concentration of Hg2+.

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Fig. 4. Effects of coexisting ions on the fluorescence maxima of RTTU/Hg2+. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for Na+, Mg2+, Ca2+, Fe3+, Cu2+, Zn2+, Cr3+, Fe2+, Cd2+, and Hg2+, 150 µM for K+, Pb2+, Ni2+, Mn2+, Co2+. λex: 520 nm, slit width: 10 nm.

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In order to know the interference from other common cations in the determination of Hg2+ by RTTU, Na+, K+, Mg2+, Ca2+, Fe3+, Fe2+, Cu2+, Zn2+, Cr3+, Pb2+, Ni2+, Mn2+, Co2+ and Cd2+ were introduced into the solution of RTTU/Hg2+ individually and the fluorescence spectra were recorded. As showed in Fig. 4, those ions showed no pronounced interference in the fluorescence of the RTTU/Hg2+ solution. Therefore, the result suggests that RTTU possesses excellent interference immunity for monitoring Hg2+, demonstrating great practical value.

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ACCEPTED MANUSCRIPT 3.5. Effect of time Reaction time is an important factor for probe, so the time response of RTTU to Hg2+ was investigated. As shown in Fig. 5, the fluorescence intensity increased to the

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maximum within about 30 min and then leveled off, while the fluorescence intensity of the blank solution was almost stable at a very low level. The result indicates the

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reliability and efficiency of RTTU for the detection of Hg2+.

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Fig. 5. Time response of RTTU (blank triangle) and RTTU/Hg2+ (solid triangle) solutions. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for Hg2+, λex: 520 nm, slit width: 10 nm.

3.6. Effect of pH

For practical applications, the effects of pH on the fluorescence intensity of RTTU

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were studied. It can be seen from Fig. 6 that the fluorescence intensity of RTTU at 583 nm with or without Hg2+ gradually decreased when pH value increased from 6.41

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to 8.33. However, the fluorescence intensity of RTTU with Hg2+ was much higher than that of RTTU solely. Thus, RTTU can detect Hg2+ in a pH span of 6.41–8.33

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which covers most of the physiological pH ranges.

Fig. 6. Effect of pH on the fluorescence maxima of RTTU (50 µM) in the absence (blank circle) and presence (solid circle) of Hg2+ (250 µM) in CH3CN/HEPES buffer (9/1, v/v) pH: 6.41, 6.52, 6.65, 6.94, 7.05, 7.21, 7.29, 7.61, 8.01, 8.33. λex: 520 nm, slit width: 10 nm.

3.7. Sensing mechanism To investigate the sensing mechanism of RTTU for Hg2+, Job’s plot experiment

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ACCEPTED MANUSCRIPT was conducted. By keeping the concentration of RTTU and Hg2+ constant at 90 µM and changing the mole fraction of Hg2+ from 0 to 1, the fluorescence enhancement of the solutions was tested. As shown in Fig. 7, the fluorescence intensity at 583 nm had

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a maximal increment at Hg2+ molar fraction of 0.75, indicating a 1:3 stoichiometric ratio of RTTU to Hg2+. The estimated association constant was 9.88×1011 M-3.

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Fig. 7. Job’s plot for Hg2+ versus RTTU. Total concentration ([RTTU]+[Hg2+]): 90 µM, F0 and F: fluorescence maxima before and after addition of Hg2+ at 583 nm, respectively. λex: 520 nm, slit width: 10 nm.

Furthermore, when excess EDTA was added to RTTU/Hg2+ in CH3CN/HEPES buffer (9/1, v/v, pH=7.21), the fluorescence of the solution could not be quenched,

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indicating the chemically irreversible coordination of RTTU with Hg2+ (Fig. 8).

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Fig. 8. Reversibility of the fluorescent detection of Hg2+ with RTTU. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for Hg2+. λex: 520 nm, slit width: 10 nm.

To further understand the mechanism of RTTU with Hg2+, LC-MS analysis of the

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RTTU/Hg2+ in CH3CN/HEPES buffer (9/1, v/v, pH=7.21) was carried out. As shown in Fig. 9, the 1:3 RTTU/Hg2+ complex was confirmed by the molecular ion peak of [RTTU+3Hg2++2H2O+K+] at m/z 1652.3459. In addition, the peaks at m/z 819.4337, 807.4206, 797.5231, 773.4339, 739.5234, and 661.4739 could be ascribed to [RTTU3+H++K+], [RTTU1+], [RTTU3++H2O], [RTTU2+2Na+], [RTTU4+K+], and [RTTU5+K+], RTTU1-C6H5,

respectively,

where

RTTU1=RTTU+3Hg2+-3HgS-C6H5, RTTU2=

RTTU3=RTTU1-CNH2, 11

RTTU4=RTTU3-C6H5,

and

ACCEPTED MANUSCRIPT RTTU5=RTTU4-C6H5.

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Fig. 9. LC-MS of RTTU/Hg2+ in CH3CN/HEPES buffer (9/1, v/v, pH=7.21).

The Job’s plot and reversibility experiments, and the LC-MS analysis results suggest a new interaction way between RTTU and Hg2+ which is different from the

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hydrolysis [21,23] and the cyclization reactions [24,25] reported in the references. We

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deduce the mechanism of RTTU with Hg2+ as Scheme 2. When Hg2+ was added to the RTTU solution, it was bound to the S atom in the thiourea receptor, leading to the formation of the RTTU/Hg2+ complex as well as the conversion of the RTTU structure from the spirolactam to the ring-opened amide. Then HgS and fragments such as

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phenyl and –CNH2 are removed to produce a series of new compounds.

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Scheme 2. Proposed sensing mechanism of RTTU for Hg2+.

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To confirm the binding mode of RTTU with Hg2+, 1H NMR test was done, but the spectrum of RTTU/Hg2+ in CD3CN/D2O (9/1, v/v) was unreadable (Fig. S5). In addition, the IR spectra of RTTU before and after addition of Hg2+ were collected (Fig. 10). The disappearance of the peak at 1665.45 cm-1 matched the variation of the carbonyl (C=O) in spirolactam to the carbon-oxygen single bond (C−O). The great changes of the peaks between 1616 and 1495 cm-1 (corresponding to the phenyl and the C=N groups) as well as the peaks from 1375 to 1150 cm-1 (related to the C−O group)

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ACCEPTED MANUSCRIPT supported the speculated sensing mechanism.

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Fig. 10. IR spectra of RTTU before (a) and after (b) addition of Hg2+.

3.8. Application

The amount of Hg2+ in tap water and pool water were determined by the

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fluorescence assay method we proposed above under the same condition, and the

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results were showed in Table 1. The concentration of Hg2+ detected was close to that of the added. The recovery was between 93.5% and 101% and the relative standard deviation (RSD) of three measurements was less than 4.57%. Thus, the presented

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method could be used to determine Hg2+ in real environmental samples.

Table 1. Determination of Hg2+ in pond water and tap water (n=3) a.

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4. Conclusions

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In summary, we designed and synthesized a novel rhodamine-based fluorescent probe for Hg2+ with three thiourea receptors (RTTU). RTTU has many good properties, such as simple and efficient synthetic method, aqueous working medium, high sensitivity and selectivity, wide linear Hg2+ concentration range, low detection limit, strong interference immunity, favorable working pH span, and good validity for signaling Hg2+ in real samples. Job's plot and reversibility experiments as well as LC-MS and IR analysis results infer a 1:3 stoichiometry and a new interaction way

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ACCEPTED MANUSCRIPT between the rhodamine-based fluorescent probe and Hg2+.

Acknowledgment This work was supported by the National Natural Science Foundation of China

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(21074085), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, and the Graduate Student Innovation Training Project

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of Jiangsu Province (KYLX_1241).

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Supplementary data

Supplementary data associated with this article can be found in the online version. References

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ACCEPTED MANUSCRIPT [19] Mao J, Wang LN, Dou W, Tang XL, Yan Y, Liu WS. Tuning the selectivity of two chemosensors to Fe(III) and Cr(III). Org Lett 2007;9:4567-70. [20]

Bag

B,

Pal

A.

Rhodamine-based

probes

for

metal

ion-induced

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chromo-fluorogenic dual signaling and their selectivity towards Hg(II) ion. Org Biomol Chem 2011;9:4467-80.

[21] Liu AF, Yang L, Zhang ZY, Zhang ZL, Xu DM. A novel rhodamine-based

SC

colorimetric and fluorescent sensor for the dual-channel detection of Cu2+ and

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Fe3+ in aqueous solutions. Dyes Pigm 2013;99:472-9.

[22] Zhang ZY, Lu SZ, Sha CM, Xu DM. A single thiourea-appended 1,8-naphthalimide chemosensor for three heavy metal ions: Fe3+, Pb2+, and Hg2+. Sens Actuators B 2015;208:258-66.

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[23] Shi W, Ma HM. Rhodamine B thiolactone: a simple chemosensor for Hg2+ in aqueous media. Chem Commun 2008;16:1856-8. [24] Wu JS, Hwang IC, Kim KS, Kim JS. Rhodamine-based Hg2+-selective in

aqueous

EP

chemodosimeter

solution:

fluorescent

off-on.

Org

Lett

AC C

2007;9:907-10.

[25] Angupillai S, Hwang JY, Lee JY, Rao BA, Son YA. Efficient rhodamine-thiosemicarbazide-based

colorimetric/fluorescent

‘turn-on’

chemodosimeters for the detection of Hg2+ in aqueous samples. Sens Actuators B 2015;214:101-10.

17

ACCEPTED MANUSCRIPT

Tables Table 1. Determination of Hg2+ in pond water and tap water (n=3) a.

/µM

/µM

50

50.5

75

70.1

50

47.5

75

76.0

Recovery

RSD

Tap water

/%

/%

101

3.81

93.5

4.57

SC

RTTU

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Sample

Pond water

a

Hg2+ found

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Sensor

Hg2+ added

95.0

3.52

101

3.64

: Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 20 µM for RTTU, RSD：Relative

AC C

EP

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standard deviation.

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-3H gS

-C

6H 5

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SC

Scheme 1. Synthetic route of RTTU.

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Schemes

AC C

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Scheme 2. Proposed sensing mechanism of RTTU for Hg2+.

ACCEPTED MANUSCRIPT

Figures

3 RTTU RTTU 2+ +Hg

2+

Hg

2+

3+

2+

Cu ,Fe ,Pb , 2+ 2+ 3+ 2+ Zn ,Ni ,Cr ,Co , 2+ + 2+ + 2+ Mn ,Na ,Ca ,K ,Cd , 2+ 2+ Fe ,Mg ,None

1

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Absorbance

2

300

450

600

SC

0

750

M AN U

Wavelength /nm

Fig. 1. UV-vis absorption spectra of RTTU in the absence and presence of various cations. Solvent:

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CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for metal ions.

EP

FL Intensity

5

6.0x10

AC C

2+

RTTU RTTU 2+ +Hg

Hg

6

1.2x10

+

+

2+

2+

Na , K , Mg , Ca , 3+ 2+ 2+ 3+ Fe , Cu , Zn , Cr , 2+ 2+ 2+ 2+ Pb , Ni , Fe , Mn , 2+ 2+ Co , Cd , None

0.0 550

600 650 700 Wavelength /nm

750

Fig. 2. Fluorescence spectra of RTTU in the absence and presence of various cations. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for metal ions. λex: 520 nm, slit width: 10 nm.

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6

1.2x10

6 FL Intensity

5

6.0x10

0.0 0

5

6.0x10

10

20 2+

30 -5

40

RI PT

FL Intensity

1.2x10

[Hg ]/10 mol/L

0.0 600 650 700 Wavelength /nm

750

SC

550

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Fig. 3. Fluorescence spectra of RTTU (50 µM) with various concentrations of Hg2+. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), λex: 520 nm, slit width: 10 nm. From bottom to top, the concentration of Hg2+: 0, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400 µM. Inset: The

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relationship between the maximal fluorescence intensity and the concentration of Hg2+.

6

EP

FL Intensity

1.2x10

5

2+

+

Hg 2 O n l + y + Hg 2Na + Hg 2 + + + + k Hg 2+M + g 2+ Hg +2 C 2 + a + Hg 2 + F + e 2 + + Hg 2 C u 2 + + Hg 2+Zn 2 + + Hg 2 + C + r 3+ Hg +2Pb 2 + + Hg 2 +N + i 2+ Hg 2 +F + e 3 + Hg 2+M + n 2+ + Hg 2 C + o 2+ +C d2

0.0 Hg

AC C

6.0x10

Fig. 4. Effects of coexisting ions on the fluorescence maxima of RTTU/Hg2+. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for Na+, Mg2+, Ca2+, Fe3+, Cu2+, Zn2+, Cr3+, Fe2+, Cd2+, and Hg2+, 150 µM for K+, Pb2+, Ni2+, Mn2+, Co2+. λex: 520 nm, slit width: 10 nm.

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6

1.2x10

2+

5

6.0x10

RTTU

0.0 20

40 60 Time/min

80

100

SC

0

RI PT

FL Intensity

RTTU/Hg

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Fig. 5. Time response of RTTU (blank triangle) and RTTU/Hg2+ (solid triangle) solutions. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for Hg2+, λex: 520 nm, slit width: 10 nm.

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6

1.2x10

AC C

6.0x10

RTTU/Hg

2+

6

EP

FL Intensity

1.8x10

5

RTTU

0.0 6.5

7.0

7.5 pH

8.0

8.5

Fig. 6. Effect of pH on the fluorescence maxima of RTTU (50 µM) in the absence (blank circle) and presence (solid circle) of Hg2+ (250 µM) in CH3CN/HEPES buffer (9/1, v/v) pH: 6.41, 6.52, 6.65, 6.94, 7.05, 7.21, 7.29, 7.61, 8.01, 8.33. λex: 520 nm, slit width: 10 nm.

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6

1.8x10

6

F-F0

1.2x10

0.0 0.2 0.4 0.6 0.8 2+ 2+ [Hg ]/([Hg ]+[RTTU])

1.0

SC

0.0

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5

6.0x10

Fig. 7. Job’s plot for Hg2+ versus RTTU. Total concentration ([RTTU]+[Hg2+]): 90 µM, F0 and F:

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fluorescence maxima before and after addition of Hg2+ at 583 nm, respectively. λex: 520 nm, slit

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width: 10 nm.

6

2+

RTTU+Hg +EDTA 2+

RTTU+Hg

5

6.0x10

RTTU Only

EP

FL Intensity

1.2x10

AC C

0.0

550

600 650 700 Wavelength /nm

750

Fig. 8. Reversibility of the fluorescent detection of Hg2+ with RTTU. Solvent: CH3CN/HEPES buffer (9/1, v/v, pH=7.21), c: 50 µM for RTTU, 250 µM for Hg2+. λex: 520 nm, slit width: 10 nm.

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SC

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ACCEPTED MANUSCRIPT

75

a

AC C

EP

Transmittance %

90

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Fig. 9. LC-MS of RTTU/Hg2+ in CH3CN/HEPES buffer (9/1, v/v, pH=7.21).

b

90 80

4000

3000 2000 -1 Wavenumber/cm

1000

Fig. 10. IR spectra of RTTU before (a) and after (b) addition of Hg2+.

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Highlights (1) A facilely prepared new rhodamine-thiourea conjugate is reported. (2) The dye is a fluorescent probe for Hg2+ with good overall performance.

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(3) The probe formed a rare 1:3 complex with Hg2+.

AC C

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(4) A new interaction way between Hg2+ and the probe is discussed.