Colorimetric detection of Fe3+ and Fe2+ and sequential fluorescent detection of Al3+ and pyrophosphate by an imidazole-based chemosensor in a near-perfect aqueous solution

Accepted Manuscript 3+ 2+ 3+ Colorimetric detection of Fe and Fe and sequential fluorescent detection of Al and pyrophosphate by an imidazole-based chemosensor in a near-perfect aqueous solution Tae Geun Jo, Kwon Hee Bok, Jiyeon Han, Mi Hee Lim, Cheal Kim PII:

S0143-7208(16)30926-3

DOI:

10.1016/j.dyepig.2016.11.052

Reference:

DYPI 5621

To appear in:

Dyes and Pigments

Received Date: 10 October 2016 Revised Date:

26 November 2016

Accepted Date: 26 November 2016

3+ Please cite this article as: Jo TG, Bok KH, Han J, Lim MH, Kim C, Colorimetric detection of Fe 2+ 3+ and Fe and sequential fluorescent detection of Al and pyrophosphate by an imidazolebased chemosensor in a near-perfect aqueous solution, Dyes and Pigments (2016), doi: 10.1016/ j.dyepig.2016.11.052. 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.

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Graphical Abstract

ACCEPTED MANUSCRIPT Colorimetric detection of Fe3+ and Fe2+ and sequential fluorescent detection of Al3+ and pyrophosphate by an imidazole-based chemosensor in a near-perfect aqueous

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solution

a

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Tae Geun Jo,a Kwon Hee Bok,a Jiyeon Han,b Mi Hee Lim,b Cheal Kima*

Department of Fine Chemistry and Department of Interdisciplinary Bio IT Materials,

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Seoul National University of Science and Technology, Seoul 139-743, Republic of Korea. Fax: +82-2-973-9149; Tel: +82-2-970-6693; E-mail: [email protected] b

Department of Chemistry, Ulsan National Institute of Science and Technology

Abstract

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(UNIST), Ulsan 44919, Republic of Korea.

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A novel chemosensor was designed and synthesized for various analytes: Fe3+, Fe2+, Al3+ and pyrophosphate. The sensor showed a selective color change from yellow to

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orange toward both Fe3+ and Fe2+ in a near-perfect aqueous solution, which could be reusable simply through treatment with ethylenediaminetetraacetic acid. The detection limits (0.27 µM and 0.32 µM) for Fe3+ and Fe2+ were much lower than the environmental protection agency guideline (5.37 µM) in drinking water. The sensor could be used to quantify Fe3+ in real water samples. Moreover, this sensor acted as a ‘turn-on’ and ‘turnoff’ type fluorescent sensor toward Al3+ and pyrophosphate. The sensing mechanism of 1

ACCEPTED MANUSCRIPT the sensor for Al3+ could be explained by chelation-enhanced fluorescence effect, which was supported by theoretical calculations. Through a metal-complex displacement method, the sensor-Al3+ complex selectively responded to pyrophosphate over various

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anions especially including phosphate-based anions. Interestingly, the sensor could be used to sequentially detect both Al3+ and pyrophosphate in the living cells.

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Keywords: Multiple analytes, Colorimetric, Fluorescent, Theoretical calculations, Cell

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imaging.

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ACCEPTED MANUSCRIPT 1. Introduction The development of chemical sensors for biologically and environmentally important metal ions and anions has received considerable attention because of their significant

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roles in industry, medicine, human health and the environment [1-6]. The chemical sensors have analytical merits, such as high selectivity, eidetic recognition, rapid response and real time monitoring [7-11]. Among heavy metals, iron is one of the

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indispensable metal ions and plays an important function in a wide range of organic and biological processes such as oxygen-carrying, cellular metabolism, enzymatic reaction

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and various bio-syntheses [12,13]. However, the deficiency or overload of iron in humans cause various diseases such as anemia, liver damages and hemochromatosis [14-16]. For these reasons, detecting iron ions has steadily attracted a great deal of attention in various areas [17,18].

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Aluminium, the third most prevalent metallic element in the earth, is extensively used in various fields, including food packaging, pharmaceuticals, water purification, and the

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manufacturing industry [19,20]. Because of its wide use, aluminium ion can be easily accumulated in human body. The accumulation of the ion can lead to many hazardous

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diseases such as Parkinson’s disease and Alzheimer’s disease [21-23]. Hence, the development of sensors for aluminium is highly desirable in environmental and biological systems [24].

Pyrophosphate (P2O74-, PPi), the product of adenosine triphosphate (ATP) hydrolysis under cellular conditions, has been received attention due to its important roles in many crucial reactions, such as energy transduction, metabolic processes and DNA/RNA 3

ACCEPTED MANUSCRIPT polymerization [25,26]. Also, the detection and quantification of PPi are of importance in cancer and various disease research areas. Therefore, there have been tremendous efforts to develop the sensors for PPi [27,28]. Recently, a metal-complex displacement method

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was recognized as one of the successful strategies in the design of detector for PPi, with a specific interaction between metal ions and PPi [29]. Up to now, most of the metalcomplex displacement-type probes for PPi used Zn2+, Fe3+ and Cu2+ as a metal source

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[25-27,29-32], while only few examples were reported for Al3+ used as a metal source [33-37].

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Imidazole derivatives have been utilized as a good sensor to detect the metal ions because they have excellent fluorogenic and chromogenic properties [38-41]. Also, the julolidine moiety is well known as a good fluorophore and chromophore [42-44]. In this regard, we designed and synthesized a new chemosensor 1 based on the imidazole and

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julolidine moieties, which was expected to detect various analytes through the change of unique photophysical properties.

Herein, we report on the development of a multiple-target colorimetric and fluorescent

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chemosensor 1, which could detect Fe2+ and Fe3+ by color change from yellow to orange

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and Al3+ by fluorescence enhancement in a near-perfect aqueous environment. Moreover, the resulting 1-Al3+ complex could be used for detection of PPi by the displacement reaction. Based on Job plots, UV-vis titrations, ESI-mass spectrometry analyses, 1H NMR titrations and theoretical calculations, their binding structures and sensing mechanisms were proposed and explained.

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ACCEPTED MANUSCRIPT 2. Experimental section 2.1. General information All solvents and reagents (analytical grade and spectroscopic grade) were purchased 13

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from Sigma-Aldrich and used without further purification. Both 1H NMR and

C NMR

were recorded on a Varian 400 MHz and 100 MHz spectrometer, respectively. The chemical shifts (δ) were recorded in ppm. Absorption spectra were recorded at room

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temperature using a Perkin Elmer model Lambda 2S UV/Vis spectrophotometer.

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Electrospray ionization mass spectra (ESI-mass) were collected on a Thermo Finnigan (San Jose, CA, USA) LCQTM Advantage MAX quadrupole ion trap instrument. Fluorescence measurements were performed on a Perkin Elmer model LS45 fluorescence spectrophotometer. Elemental analysis for carbon, nitrogen, and hydrogen was carried out by using a Flash EA 1112 elemental analyzer (thermo) in Organic Chemistry Research

2.2 Synthesis of 1

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Center of Sogang University, Korea.

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A synthesis of sensor 1 was performed by stirring a mixture of 5-amino-1H-imidazole-

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4-carboxamide (0.13 g, 1 mmol) and 8-hydroxyjulolidine-9-carboxaldehyde (0.22 g, 1 mmol) in ethanol (15 mL) for 5 h at room temperature until a red precipitate appeared. The resulting precipitate was filtered and washed with methanol and diethyl ether. The yield: 0.19 g (60.0 %) and the melting point: 230-235 ºC. IR (KBr): ν (cm-1) = 3136 (m), 2944 (m), 2853 (m), 2360 (s), 1613(s), 1535(s), 1439(s), 1302 (s), 1183(s). 1H NMR (DMSO-d6, 400 MHz): δ 13.66 (s, 1H), 12.61 (s, 1H), 8.69 (s, 1H), 7.93 (m, 3H), 7.25 (s, 1H), 3.42 (m, 4H), 2.63 (m, 4H), 1.86 (m, 4H). 13C NMR (DMSO-d6, 100 MHz): δ 161.8, 5

ACCEPTED MANUSCRIPT 158.4, 152.0, 151.9, 142.3, 136.1, 134.89, 117.1, 111.6, 106.8, 105.8, 50.9, 50.0, 26.8, 21.0, 20.6, 20.0 ppm. ESI-mass: m/z calcd for C17H19N5O2+H+ ([M+H+]), 326.16; found, 326.10. Anal. Calc. for C17H19N5O2 (325.37): C 62.75; H, 5.89; N, 21.52 %; found: C,

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62.42; H, 5.96; N, 21.88 %. 2.3 Colorimetric sensing for iron

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UV-vis titration measurements

A stock solution (3 mM) of sensor 1 in DMSO was diluted in bis-tris buffer solution (10

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mM, pH 7.0) to make the final concentration of 20 µM (3 mL). Fe(ClO4)2 (or Fe(NO3)3) (0.01 mmol) was dissolved in bis-tris buffer solution (500 µL). Then, 0-4.8 µL of the Fe2+ (or 0-3.6 µL of the Fe3+) solution (20 mM) were transferred to the solution prepared above. After stirring the solutions for a few seconds, UV-vis spectra were recorded at

Job plot measurements

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room temperature.

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The stock solutions of sensor 1 (3 mM) in DMSO and Fe(ClO4)2 (or Fe(NO3)3) (20 mM) in bis-tris buffer solution were prepared, respectively. The sensor 1 solution (400 µL) was

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diluted to 29.6 mL of bis-tris buffer solution (10 mM, pH 7.0) to make the final concentration of 40 µM. 60 µL of Fe(ClO4)2 (or Fe(NO3)3) solution (20 mM) was diluted to 29.94 mL of bis-tris buffer solution (10 mM, pH 7.0). 2.7, 2.4, 2.1, 1.8, 1.5, 1.2, 0.9, 0.6 and 0.3 mL of the sensor 1 solution were taken and transferred to vials. 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4 and 2.7 mL of the Fe2+ (or Fe3+) solution were added to each sensor 1 solution to make a total volume of 3 mL, seperately. After stirring the solutions for a 6

ACCEPTED MANUSCRIPT few seconds, UV-vis spectra were recorded at room temperature. Competition tests

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MNO3 (M = Na, K, 0.01 mmol) or M(NO3)2 (M = Zn, Cd, Cu, Mg, Co, Ni, Ca, Mn, Pb, 0.01 mmol) or M(NO3)3 (M = Al, Ga, In, Fe, Cr, 0.01 mmol) or M(ClO4)2 (M = Fe, 0.01 mmol) was separately dissolved in bis-tris buffer solution (500 µL). 4.2 µL of each metal solution (20 mM) was diluted to bis-tris buffer solution. 4.2 µL of the Fe2+ (or 3.0 µL of

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the Fe3+) solution (20 mM) was added to the solutions prepared above. Then, 20 µL of the

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sensor 1 solution (3 mM) was added to the mixed solutions to make a total volume of 3 mL. After stirring the solutions for a few seconds, UV-vis spectra were recorded at room temperature. pH experiments

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A series of buffers with pH values ranging from 2 to 12 was prepared by mixing sodium hydroxide solution and hydrochloric acid in bis-tris buffer. After the solution with a desired pH was achieved, a stock solution of 1 (3 mM) was diluted with buffers to make

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the final concentration of 20 µM (3 mL). Then, 4.2 µL of the Fe2+ (or 3.0 µL of the Fe3+)

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solution (20 mM) was transferred to each sensor 1 solution prepared above. After stirring the solutions for a few seconds, UV-vis spectra were recorded at room temperature. Determination of Fe3+ in water samples UV-vis spectral measurements of water samples (drinking, tap and artificial polluted water) containing Fe3+ were performed by adding 20 µL of 3 mM stock solution of 1 and 0.60 mL of 50 mM bis-tris buffer stock solution to 2.38 mL sample solutions. After 7

ACCEPTED MANUSCRIPT stirring the solutions for a few seconds, UV-vis spectra were recorded at room temperature.

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2.4. Fluorescent sensing for Al3+ and PPi Fluorescence titration measurements

For Al3+, a stock solution (3 mM) of sensor 1 in DMSO was diluted in bis-tris buffer

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solution (10 mM, pH 7.0) to make the final concentration of 20 µM (3 mL). Al(NO3)3 (0.1 mmol) was dissolved in bis-tris buffer solution (1 mL). Then, 0-144 µL of the Al3+

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solution (100 mM) were transferred to the solution of 1 (20 µM, 3 mL) prepared above. After stirring the solutions for a few seconds, fluorescence spectra were recorded at room temperature.

For PPi, a stock solution (100 mM, 104 µL) of Al3+ in bis-tris buffer solution was

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diluted in the solution of 1 (20 µM, 3 mL). Na4P2O7 (PPi, 0.1 mmol) was dissolved in bistris buffer solution (1 mL). Then, 0-12.6 µL of the PPi stock solution (100 mM) were transferred to the solution of 1-Al3+ complex prepared above. After stirring the solutions

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for a few seconds, fluorescence spectra were recorded at room temperature.

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UV-vis titration measurements

For Al3+, a stock solution (3 mM) of sensor 1 in DMSO was diluted in bis-tris buffer solution (10 mM, pH 7.0) to make the final concentration of 20 µM (3 mL). Al(NO3)3 (0.1 mmol) was dissolved in bis-tris buffer solution (1 mL). Then, 0-144 µL of the Al3+ solution (100 mM) were transferred to the 1 solution (20 µM, 3 mL) prepared above. After stirring the solutions for a few seconds, UV-vis spectra were recorded at room 8

ACCEPTED MANUSCRIPT temperature. For PPi, a stock solution (100 mM, 104 µL) of Al3+ in bis-tris buffer solution was diluted in the solution of 1 (20 µM, 3 mL). PPi (0.1 mmol) was dissolved in bis-tris buffer

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solution (1 mL). Then, 0-25.2 µL of the PPi solution (100 mM) were transferred to the solution of 1-Al3+ complex prepared above. After stirring the solutions for a few seconds, UV-vis spectra were recorded at room temperature.

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Job plot measurements

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For Al3+, 400 µL of the sensor 1 solution (3 mM) was diluted to 29.6 mL of bis-tris buffer solution to make the concentration of 40 µM. 12 µL of Al(NO3)3 solution (100 mM) was diluted to 29.988 mL of bis-tris buffer solution. 2.7, 2.4, 2.1, 1.8, 1.5, 1.2, 0.9, 0.6 and 0.3 mL of the sensor 1 solution were taken and transferred to vials. 0.3, 0.6, 0.9, 1.2,

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1.5, 1.8, 2.1, 2.4 and 2.7 mL of the Al3+ solution were added to each sensor 1 solution seperately. Each vial had a total volume of 3 mL. After stirring the solutions for a few seconds, fluorescence spectra were recorded at room temperature.

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For PPi, 1 (0.02 mmol) in DMSO (1 mL) and Al(NO3)3 (0.02 mmol) in bist-ris buffer

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were prepared, respectively. The two solutions were mixed to make 1-Al3+ complex. 27, 24, 21, 18, 15, 12, 9, 6 and 3 µL of the 1-Al3+ complex solution (3 mM) were taken and transferred to vials. Each vial was diluted with bis-tris buffer solution to make a total volume of 2.985 mL. PPi (0.01 mmol) was dissolved in bis-tris buffer (1 mL). 3, 6, 9, 12, 15, 18, 21, 24 and 27 µL of the PPi solution were added to each diluted 1-Al3+ solution. Each vial had a total volume of 3 mL. After stirring the solutions for a few, UV-vis spectra were taken at room temperature. 9

ACCEPTED MANUSCRIPT Competition tests For Al3+, MNO3 (M = Na, K, 0.1 mmol) or M(NO3)2 (M = Zn, Cd, Cu, Mg, Co, Ni, Ca, Mn, Pb, 0.1 mmol) or M(NO3)3 (M = Al, Ga, In, Fe, Cr, 0.05 mmol) or M(ClO4)2 (M = Fe,

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0.1 mmol) was separately dissolved in bis-tris buffer solution (1 mL). 104 µL of each metal solution (100 mM) was diluted to 2.772 mL of bis-tris buffer solution, separately. 104 µL of the Al3+ solution (100 mM) was taken and added to the solutions prepared

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above. Then, 20 µL (3 mM) of the sensor 1 was taken and added to the mixed solutions. After stirring the solutions for a few seconds, fluorescence spectra were recorded at room

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temperature.

For PPi, a stock solution (100 mM, 104 µL) of Al3+ in bis-tris buffer solution was diluted in the solution of 1 (20 µM, 3 mL). The tetraethylammonium salts (0.1 mmol) of F-, CN-, Cl-, Br- and I-, the tetrabutylammonium salts (0.1 mmol) of AcO-, H2PO4-, BzO-,

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N3- and SCN- and the sodium salts (0.1 mmol) of SH-, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and PPi were separately dissolved in bis-tris buffer (1 mL). 21.6 µL of each anion solution (100 mM) was dilluted

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to 2.978 mL of 1-Al3+ complex solution, separately. Then, 21.6 µL of the PPi solution

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(100 mM) was taken and added to the solutions prepared above. After stirring the solutions for a few seconds, fluorescence spectra were recorded at room temperature. pH effect

For Al3+, a series of buffers with pH values ranging from 2 to 12 was prepared by mixing sodium hydroxide solution and hydrochloric acid in bis-tris buffer. After the solution with a desired pH was achieved, the stock solution (3 mM) of 1 was diluted in 10

ACCEPTED MANUSCRIPT buffers to make the final concentration of 20 µM (3 mL). Then, 104 µL of the Al3+ stock solution (100 mM) was transferred to each sensor 1 solution prepared above. After stirring the solutions for a few seconds, fluorescence spectra were recorded at room

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temperature. For PPi, a series of buffers with pH values ranging from 2 to 12 was prepared by mixing sodium hydroxide solution and hydrochloric acid in bis-tris buffer. After the solution with

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a desired pH was achieved, the stock solutions of Al3+ (100 mM, 104 µL) and 1 (3 mM, 20 µL) were diluted and mixed in the buffer solution. Then, 21.6 µL of the PPi stock

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solution (100 mM) was transferred to each 1-Al3+ solution prepared above. After stirring the solutions for a few seconds, fluorescence spectra were recorded at room temperature. Imaging experiments in living cells

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HeLa cells (ATCC, Manassas, USA) were maintained in media containing Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA), 100 U/mL penicillin (GIBCO), and 100 mg/mL streptomycin (GIBCO). The

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cells grew in a humidified atmosphere with 5 % CO2 at 37 °C. Cells were seeded onto 6 well plate (SPL Life Sciences Co., Ltd., South Korea) at a density of 150,000 cells per 1

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mL and then incubated at 37 °C for 12 h. For fluorescence imaging experiments, cells were first treated with 1 (dissolved in DMSO; 1 % v/v final DMSO concentration; 20 µM; at room temperature) for 10 min. After incubation with various concentrations of Al(NO3)3 (dissolved in water; 1 % v/v) for 10 min, cells were washed twice with 2 mL of 10 mM bis-tris buffer (pH 7.4, 150 mM NaCl). In case of fluorescence quenching experiments, cells were first treated with 1 (dissolved in DMSO; 1 % v/v final DMSO 11

ACCEPTED MANUSCRIPT concentration; 20 µM; at room temperature). After 5 min, Al(NO3)3 (dissolved in water; 200 µM; 1% v/v) was incubated with cells for 10 min. Various concentrations of PPi (dissolved in bis-tris buffer; 1% v/v) were introduced to cells for 5 min and the cells were

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washed with 3 mL of bis-tris buffer three times. Imaging was performed with an EVOS FL fluorescence microscope (Life technologies) using a GFP light cube [excitation 470 (± 11) nm; emission 510 (± 21) nm]. H NMR titrations

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1

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For 1H NMR titrations of sensor 1 with Al3+, three NMR tubes of sensor 1 (3.3 mg, 0.01 mmol) dissolved in DMSO-d6 were prepared and then three different concentrations (0, 0.005 and 0.01 mmol) of Al(NO3)3 dissolved in DMSO-d6 were added to each sensor 1 solution. After stirring the solutions for a few seconds, 1H NMR spectra were recorded at

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room temperature. Theoretical calculations

All DFT/TDDFT calculations based on the hybrid exchange correlation functional

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B3LYP [45,46] with 6-31G** basis set [47,48] were carried out using Gaussian 03 program [49]. In vibrational frequency calculations, there was no imaginary frequency for

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the optimized geometries of 1 and 1-Al3+, suggesting that these geometries represented local minima. For all calculations, the solvent effect of water was considered by using the Cossi and Barone’s CPCM (conductor-like polarizable continuum model) [50,51]. To investigate the electronic properties of singlet excited states, time-dependent DFT (TDDFT) was performed in the ground state geometries of 1 and 1-Al3+. The twenty lowest singlet states were calculated and analyzed. The GaussSum 2.1 [52] was used to 12

ACCEPTED MANUSCRIPT calculate the contributions of molecular orbitals in electronic transitions.

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3. Results and discussion The chemosensor 1 was synthesized by the condensation reaction of 8hydroxyjulolidine-9-carboxaldehyde

with

5-amino-1H-imidazole-4-carboxamide

mass spectrometry and elemental analysis.

C NMR, ESI-

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3.1 Colorimetric sensing for Fe2+ and Fe3+

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ethanol at room temperature (Scheme 1), and characterized by 1H and

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The UV-vis spectral changes of 1 were investigated with the addition of various metal ions such as Al3+, Ga3+, In3+, Zn2+, Cd2+, Cu2+, Fe2+, Fe3+, Mg2+, Cr3+, Co2+, Ni2+, Na+, K+, Ca2+, Mn2+ and Pb2+ in bis-tris buffer solution (10 mM, pH 7.0). As shown in Fig. 1, there

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were no significant spectral and color changes in the presence of most metal ions, whereas Fe2+ and Fe3+ ions caused both distinct spectral changes and pronounced colour

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changes from yellow to orange. These results suggested that 1 could be used as a colorimetric chemosensor for Fe2+ and Fe3+ ions via direct visualization in a near-perfect

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aqueous media. First of all, the UV-vis titration experiments were conducted to investigate the binding property of 1 with Fe3+ ions (Fig. 2). Upon the addition of Fe3+, the absorbance band at 433 nm decreased gradually, while the absorbance at 325 nm and 520 nm increased with two clear isosbestic points. The points indicated that the binding between 1 and Fe3+ ions afforded only one species. The UV-vis variation of 1-Fe2+ complex was also nearly identical to that of 1-Fe3+ (Fig. S1). The peaks at 520 nm with high molar extinction coefficients, 3.3 x 103 M-1cm-1 (ε520nm) for Fe3+and 2.5 x 103 M13

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cm-1 (ε520nm) for Fe2+, are too large to be Fe-based d-d transitions. Thus, these new peaks

might be attributed to a metal-to-ligand charge-transfer (MLCT) [53-57], resulting in the color change of the solutions.

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The binding stoichiometries between 1 and Fe2+/3+ were determined by the Job plot analyses [58], which showed 2:1 complexations of 1 and Fe2+/3+ (Fig. S2). The stoichiometries of 1 and Fe2+/3+ were further confirmed by ESI-mass data analyses (Figs.

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3 and S3). The positive mass spectrum of 1 for Fe3+ indicated that the first major peak at m/z = 704.20 was assignable to 2·1-2H++Fe3+ [calcd, m/z: 704.23] (Fig. 3). The positive-

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ion mass spectrum of 1 with Fe2+ was also nearly identical to that of Fe3+ (Fig. S3). These results led us to propose that Fe2+ of the complex formed from the reaction of Fe2+ with 1 might be rapidly oxidized to Fe3+ by air [54,55].

To verify our proposal, we examined the spectral changes of the solution of 1 with Fe2+

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under the degassed conditions (Fig. S4). When Fe2+ reacted with 1 under the anaerobic condition, there was no spectral and color change. Under the aerobic condition, however, we observed the significant spectral and color changes of the solution, which were

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identical to those of the Fe3+-2·1 complex. These results indicated that the Fe2+-2·1

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complex formed under the degassed conditions was oxidized to the Fe3+-2·1 complex in air. In addition, the time-dependent changes for the reaction of sensor 1 with Fe2+/3+ was evaluated (Fig. S5). The formation time (120 s) of Fe3+-2·1 obtained from the reaction of 1 with Fe2+ was 3 times slower than that (40 s) obtained from the reaction of 1 with Fe3+. This observation further proved that Fe2+ of the Fe2+-2·1 complex formed from the reaction of Fe2+ with 1 might be rapidly oxidized to Fe3+ by air. Based on the Job plot, ESI-Mass spectrometry analysis, and the degassing experiment, we proposed the 14

ACCEPTED MANUSCRIPT structure of Fe3+-2·1 complex as shown in Scheme 2. On the basis of the Li’s equations [59], the association constants (K) of 1 with Fe2+ and Fe3+ were calculated as 1.4 x 104 and 2.8 x 104, respectively (Fig. S6). The detection

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limits of sensor 1 for Fe2+ and Fe3+ were calculated to be 0.32 µM and 0.27 µM on the basis of 3σ/K (Fig. S7) [60]. These values are much lower than the guideline (5.37 µM) set by the Environmental Protection Agency (EPA) for iron in drinking water [61]. In

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order to check the selectivity of 1 towards Fe3+ ions over the various metal ions, competitive studies were carried out (Fig. 4). In presence of the competing metal ions,

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there was no significant interference in the detection of Fe3+. Only Cu2+ influenced somewhat the interaction of 1 with Fe3+. Fe2+ also showed a similar tendency to Fe3+ (Fig. S8). These results indicated that sensor 1 could be efficiently used for the selective detection of Fe2+/3+. For practical applications, the effect of pH on the color and

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absorbance changes of 1 to Fe2+ and Fe3+ was studied in various pH range (2-12) (Fig. S9). The Fe2+/3+-2·1 complexes exhibited the stable absorption and color changes between pH 3 and pH 11, which warrant that 1 could detect Fe2+ and Fe3+ via naked eye in a wide

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range of pH (3-11).

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For the practical application of the sensor 1 toward Fe3+, the real sample analysis was performed for quantitative measurement of Fe3+. As shown in Fig. S10, a good calibration curve was obtained for the determination of Fe3+. Then, 1 was applied for the determination of Fe3+ in both tap and drinking water samples. As shown in Table 1, a suitable recovery and Relative Standard Deviation (R.S.D.) values were obtained. These results indicated both the suitability and applicability of the sensor for the detection of Fe3+ in the real samples. The reusability of sensor is an imperative ability to develop the 15

ACCEPTED MANUSCRIPT practical chemosensor. Hence, the reversible ability of 1 was examined by adding ethylenediaminetetraacetic acid (EDTA) to the solution of Fe3+-2·1 complex (Fig. S11). The complex solution color was recovered from orange to yellow (the original color of 1).

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Upon the sequentially alternative addition of Fe3+ and EDTA, the absorbance and color changes of the solution were almost reversible even after several cycles. These results

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showed that sensor 1 could be used as a recyclable sensor for Fe3+.

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3.2 Fluorescence and absorption studies of 1 toward Al3+

The fluorescence selectivity of sensor 1 toward various metal ions was also conducted in bis-tris buffer solution (10 mM, pH 7.0). Sensor 1 exhibited no fluorescence intensity upon excitation at 455 nm (Fig. 5). When 180 equiv of various metal ions (Al3+, Ga3+,

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In3+, Zn2+, Cd2+, Cu2+, Fe2+, Fe3+, Mg2+, Cr3+, Co2+, Ni2+, Na+, K+, Ca2+, Mn2 and Pb2+) were added to the solution of 1, only Al3+ induced a significant fluorescence enhancement at 489 nm. This result indicated that sensor 1 could act as a “turn-on” type fluorescence

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chemosensor for Al3+ in a near-perfect aqueous solution. Importantly, 1 could selectively detect Al3+ from Ga3+ and In3+, while many chemosensors reported for Al3+ have

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difficulty in discriminating Al3+ from Ga3+ and In3+ due to their similar chemical behavior [62-64].

To understand the spectroscopic properties of 1 towards Al3+, we conducted a fluorescence titration. Upon the addition of Al3+ into 1, the fluorescence intensity at 489 nm steadily increased until the amount of Al3+ reached at 180 equiv (Fig. S12). Then, UV-vis titration experiment of 1 with Al3+ was also conducted. The addition of Al3+ to a 16

ACCEPTED MANUSCRIPT solution of 1 showed that the absorption band at 433 nm decreased and a new band at 464 nm increased gradually (Fig. S13). Two isosbestic points were observed at 336 nm and 443 nm, indicating that only one product was generated from 1 upon binding to Al3+. The

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Job plot [58] was carried out to determine the binding mode between 1 and Al3+ (Fig. S14). The fluorescence intensity at 489 nm reached the maximum at the molar ratio of 0.5, indicating a 1:1 binding mode. Moreover, the binding mode between 1 and Al3+ was

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further confirmed by ESI-mass spectrometry analysis (Fig. S15). The positive mass spectrum showed that two peaks at m/z = 470.7 and at m/z = 490.7 were assignable to 1-

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2H++Al3++NO3-+Na++2H2O [calcd, m/z: 471.12] and 1-H++Al3++NO3-+DMSO [calcd, m/z: 491.13], respectively. Based on the fluorescence titration measurement, the association constant (K) of the 1-Al3+ complex was determined as 2.3 × 102 M-1 through Benesi-Hildebrand equation [65] (Fig. S16), which was within the range of those (102~108) previously reported for Al3+ binding chemosensors [66-69]. The detection limit

be 9.24 µM (Fig. S17).

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(3σ/k) [60] of sensor 1 as a fluorescence sensor for the analysis of Al3+ ions was found to

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In order to check the possible interference from other metal ions in the detection of Al3+, we examined the fluorescence intensity change of 1 to Al3+ in the presence of other

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competitive metal ions (Fig. S18). In the presence of Cu2+, Fe2+ and Fe3+, the fluorescence emission intensity was inhibited, and some interference was observed in presence of Ga3+. However, most other competitive metal ions did not interfere with the detection of Al3+. To investigate the practicality of 1 as sensor, the detecting ability was tested in various pH (2-12). (Fig. S19). In the absence of Al3+, sensor 1 exhibited no fluorescence intensity over the pH rage of 2-12. Upon the addition of Al3+, an intense and 17

ACCEPTED MANUSCRIPT stable fluorescence of 1-Al3+ was observed in the pH range of 4.0-11.0 which warrants its application under physiological conditions. To evaluate the feasibility of 1 for monitoring Al3+ in biological systems, we conducted the cell imaging experiments. Cells were

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preincubated with 1 for 10 min prior to addition of various concentrations of Al3+ (0-200 µM) (Fig. 6). The background fluorescence in the cells was not observed in absence of Al3+. By contrast, the fluorescence intensity in the cells gradually increased as the Al3+ concentration increased from 0 to 200 µM. These observations showed that sensor 1

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could be an appropriate and biocompatible detector to successfully sense Al3+ in

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biological systems.

To get the insight into the binding mechanism of 1 with Al3+, 1H NMR titration was conducted (Fig. 7). Upon the addition of Al3+, the proton H9 disappeared and the integral of the proton H1 decreased by half. The rest protons H2, H3, H4, and H8 showed slightly

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down-field shifts. These results suggested that the N atom of the amide moiety and the O atom of the hydroxyl group of 1 might coordinate to Al3+ ion. When more than 1 equiv of Al3+ were added, there was no longer a spectral change, which indicates the 1:1 binding

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mode of 1 to Al3+.

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To further elucidate the fluorescent sensing mechanism of 1 to Al3+, geometric optimizations and theoretical calculations were performed for 1 and 1-Al3+ complex by utilizing the B3LYP/6-31g** methods with CPCM/water. The energy-minimized structure (1C, 2N, 3C, 4N = -25.049o) of 1 showed a twisted shape (Fig. 8). After combined with Al3+, the structure o f 1-Al3+ complex was flattened (1C, 2N, 3C, 4N = 0.343 o), which showed that 1 coordinated to Al3+ via two N atoms in the Schiff-base and the amide moiety and the O atom in the hydroxyl group. The singlet excited states of 1 18

ACCEPTED MANUSCRIPT and 1-Al3+ complex were investigated using the TD-DFT (time dependent-density functional theory) methods. The first lowest excited state of both 1 and 1-Al3+ was determined for HOMO → LUMO transition (404.91 nm for 1 (Fig. S20) and 429.78 nm

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for 1-Al3+ (Fig. S21)), which indicated an intramolecular charge transfer (ICT) transition. The calculations were consistent with the experimental absorption wavelengths of 1 and 1-Al3+. On the other hand, there was no obvious change in the electronic transitions between 1 and 1-Al3+ complex except the hypochromic shift (404.91 to 429.78 nm) upon

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chelating of 1 with Al3+ (Fig. S22). Based on these calculations, the fluorescent sensing

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mechanism could be explained by chelation-enhanced fluorescence (CHEF) effect [44,70,71]. The chelation of 1 with Al3+ caused the rigid structure and the restriction of the free rotation around the C=N bond, which might inhibit the non-radiative process. Based on a Job plot, ESI-mass spectrometry analysis, 1H NMR titration, and theoretical

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calculations, we propose the binding structure of 1-Al3+ complex in Scheme 3.

3.3 Fluorescence and absorption studies of 1-Al3+ complex toward PPi

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The fluorescence variation of 1-Al3+ complex was examined upon the addition of

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various anions, such as PPi, AMP, ADP, ATP, CN-, AcO-, F-, Cl-, Br-, I-, BzO-, N3-, SCN-, H2PO4-, HS-, NO3-, SO42- and PO43- in a near-perfect aqueous solution. Upon the addition of 36 equiv of various anions to 1-Al3+ solution, only PPi induced a fluorescence quenching (Fig. 9). These results suggested that 1-Al3+ complex could be a selective chemosensor for PPi over other various anions, especially including phosphate-based anions.

19

ACCEPTED MANUSCRIPT The fluorescence titration of 1-Al3+ with PPi was investigated to understand fluorescence spectral variation (Fig. S24). Upon the addition of PPi into 1-Al3+ complex, the fluorescence intensity at 489 nm steadily decreased until the amount of PPi reached

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36 equiv. The UV-vis titration experiments were also conducted (Fig. S25). Upon the addition of PPi to the solution of 1-Al3+, the absorbance at 466 nm considerably decreased, and a new band at 433 nm appeared and reached a maximum at 36 equiv of PPi. Two isosbestic points were observed at 440 nm and 496 nm, indicating that only one

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species was formed by the reaction of 1-Al3+ with PPi. The final UV-vis spectrum of 1-

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Al3+ with PPi was almost identical to that of 1 itself. This result led us to propose that 1 was released from the 1-Al3+ complex by the chelation of PPi with Al3+ (Scheme 3). The binding mode of PPi and 1-Al3+ was determined by Job plot method, which revealed a 1:1 stoichiometric ratio (Fig. S26) [58]. Further, the demetallation of 1-Al3+ complex by PPi was confirmed by an ESI-mass spectrometry analysis (Fig. S27). When PPi was

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added into 1-Al3+ solution, the positive ion mass spectrum showed that a peak at m/z 256.87 was assigned to P2O73-(PPi)+Al3++Na++MeOH [calcd. 256.92], indicating the

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demetallation of 1-Al3+ complex by PPi. Based on UV-vis titrations, Job plot and ESImass spectrometry analysis, we proposed the sensing mechanism of the 1-Al3+ complex

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toward PPi (Scheme 3). The association constant was calculated to be 3.70 × 103 M-1 from a Benesi-Hildebrand plot (Fig. S27) [65]. The detection limit (3σ/k) for PPi was found to be 20.5 µM (Fig. S28) [60]. To examine the practical applicability of 1-Al3+ complex as a PPi selective sensor, competitive experiments were carried out in the presence of PPi (36 equiv) with competing anions (36 equiv) (Fig. S29). There was no interference for the detection of 20

ACCEPTED MANUSCRIPT PPi by 1-Al3+, while ADP and ATP showed a slight inhibition. These results indicated that the detection of PPi by 1-Al3+ was not disturbed from various anions even including phosphate-based anions. In order to investigate the pH dependence of 1-Al3+ toward PPi,

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the pH effect was conducted in a wide range of pH. The optimal range for the fluorescent sensing of PPi by 1-Al3+ was turned out to be between pH 4 and pH 11 (Fig. S30).

Based on the response of 1-Al3+ complex toward PPi at the physiological pH range in

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aqueous solution, we further investigated whether the complex could detect the PPi in live cells (Fig. 10). When the cells were preincubated with 1 and Al3+, green fluorescence

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could be observed. After PPi (0-200 µM) was gradually added to the preincubated cells, the fluorescence in the cells reduced and finally disappeared. These results indicated that 1-Al3+ complex has a huge potential as a suitable detector for monitoring PPi in living cells. To the best of our knowledge, this is the first example to sequentially detect both

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4. Conclusion

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Al3+ and PPi in living cells [33-37].

We have developed a simple, selective and efficient sensor 1, which can detect Fe2+/3+

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via direct visualization and Al3+ and PPi by fluorescence change in a near-perfect aqueous media. The senor 1 could selectively and preferentially bind with Fe2+/3+ among other competitive metal ions, which can detect Fe2+/3+ ions at low concentration ca. 0.28 µM, which is lower than the EPA guidelines for drinking water of 5.37 µM. Based on the various experiments, we have demonstrated that Fe2+ of the 1-Fe2+ complex was rapidly oxidized to Fe3+ in air. In addition, 1 could successfully detect Fe3+ in real water samples 21

ACCEPTED MANUSCRIPT and be also reusable simply by treatment with EDTA. Moreover, 1 could act as an ‘offon-off’ type fluorescence sensor for sequential detection of Al3+ and PPi in a near-perfect aqueous solution. The sensor 1 showed high selectivity toward Al3+ over other

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competitive metal ions including Ga3+ and In3+. The fluorescent sensing mechanism of 1 with Al3+ could be explained by the effect of CHEF with theoretical calculations. Furthermore, 1-Al3+ complex could selectively detect PPi in the presence of other various anions, especially including phosphate-based anions. The spectroscopic experiments

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demonstrated that 1-Al3+ complex detected PPi through a metal-complex displacement

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method. Importantly, this is the first example that sensor 1 could sequentially detect both Al3+ and PPi in living cells, to the best of our knowledge. Therefore, we believe that the sensor 1 might contribute to the development of the multiple target chemosensors in

Acknowledgements

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aqueous and biological environments.

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Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-

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2014R1A2A1A11051794,

NRF-2015R1A2A2A09001301

and

NRF-

2014S1A2A2028270) are gratefully acknowledged.

Supplementary Information Supplementary data (experimental procedures and additional experimental data) 22

ACCEPTED MANUSCRIPT associated with this article can be found. This material is available free of charge via the

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Internet at http:// ~.

[1]

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imaging in human blood cells. New J Chem 2015;39:8582-8587.

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Scheme 1. Synthetic procedure of 1.

33

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Scheme 2. Proposed structure of Fe3+-2·1 complex.

35

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Scheme 3. Proposed sequential sensing process of Al3+ and PPi by 1.

36

ACCEPTED MANUSCRIPT Table 1. Determination of Fe3+ in water samplesa

Fe(III) added

Fe(III) found

(µmol/L)

(µmol/L)

0.00

0.00

-

3.00

2.94

98.0

2.2

0.00

0.00

-

-

3.00

3.26

108.7

6.4

Sample

Recovery (%)

Artificial 0.00 b

polluted water

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0.00

-

-

6.01

100.1

1.5

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6.00

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Drink water

(%)

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Tap water

Condition: [1] = 20 µmol/L in bis-tris buffer (10 mM, pH 7.0)

b

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a

R.S.D (n=3)

Prepared by deionized water, 6.0 µmol/L Zn2+, Cd2+, Pb2+, Hg2+ and 9.0 µmol/L Na+, K+,

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Ca2+, Mg2+.

Figure captions 37

ACCEPTED MANUSCRIPT Fig. 1 (a) UV-vis absorption and (b) color changes of 1 (20 µM) upon addition of 1.4 equiv of different metal ions in buffer (10 mM bis-tris, pH 7.0) Fig. 2 UV-vis absorption change of 1 (20 µM) with Fe3+ ions (0-1.2 equiv) in bis-tris

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buffer (10 mM, pH 7.0).

Fig. 3 Positive-ion ESI-mass spectrum of 1 (100 µM) upon addition of 1 equiv of Fe3+

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Fig. 4 (a) UV-vis absorption (at 520 nm) and (b) color changes of 1 (20 µM) upon

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addition of Fe3+ (1.0 equiv) in the absence and presence of other metal ions (1.0 equiv). Fig. 5 Fluorescence spectra of 1 (20 µM) upon addition of 180 equiv of different metal ions in buffer (10 mM bis-tris, pH 7.0)

Fig. 6 Fluorescent responses of 1 to Al3+ in HeLa cells. Cells were preincubated with 1

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for 10 min prior to addition of various concentrations of Al3+. Conditions: [1] = 20 µM; [Al3+] = 0, 100 and 200 µM; 37 °C; 5% CO2. The scale bar is 50 µm.

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Fig. 7 1H NMR titrations of 1 with the addition of Al3+ (0, 0.5 and 1.0 equiv). Fig. 8 The energy-minimized structures of (a) 1 and (b) 1-Al3+.

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Fig. 9 Fluorescence spectra of 1-Al3+ upon addition of 36 equiv of different anions in buffer (10 mM bis-tris, pH 7.0) Fig. 10 Fluorescent imaging of HeLa cells incubated with 1 and Al3+ followed by addition of PPi. Fluorescence of the cells treated by 1 and Al3+ was quenched by PPi introduction. Cells incubated with 1 (for 5 min) followed by addition of Al3+ for 10 min were treated with various concentrations of PPi. Conditions: [1] = 20 µM; [Al3+] = 200 µM; 38

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[PPi] = 0, 100 and 200 µM; 37 °C; 5% CO2. The scale bar is 50 µm.

39

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0.8

0.6 0.5

3+

3+

3+

2+

1, 1+Al , Ga , In , Zn , 2+ 2+ 2+ 3+ Cd , Cu , Mg , Cr , 2+ 2+ + + 2+ Co , Ni , Na , K , Ca , 2+ 2+ Mn , Pb

RI PT

Absorbance

0.7

2+

3+

1+Fe , 1+Fe

0.4 0.3

0.1 0.0 400

500

M AN U

300

SC

0.2

Wavelength (nm)

AC C

EP

TE D

(a)

(b)

Fig. 1

40

600

AC C

EP

Fig. 2

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

41

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 3

42

AC C

EP

(a)

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

(b)

Fig. 4 43

1.5x10

4

1.0x10

4

5.0x10

3

RI PT

4

3+

1+Al

SC

2.0x10

M AN U

Fluorescence Intensity

ACCEPTED MANUSCRIPT

3+

3+

2+

1, 1+Ga , In , Zn , 2+ 2+ 2+ 3+ Cd , Cu , Fe , Fe , 2+ 3+ 2+ 2+ Mg , Cr , Co , Ni , + + 2+ 2+ 2+ Na , K , Ca , Mn , Pb

0.0

500

550

TE D

450

Wavelength (nm)

AC C

EP

Fig. 5

44

600

AC C

EP

Fig. 6

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

45

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 7

46

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

(a)

(b)

Fig. 8

47

1+Al Complex, 3+ 1+Al Complex + AMP, ADP, ATP, - CN , AcO , F , Br , Cl , I , BzO , N3 ,

4

SCN , H2PO4 , HS , NO3 , SO4 , PO4

-

-

4

1.0x10

3

5.0x10

1

500

550

AC C

EP

TE D

Wavelength (nm)

Fig. 9

2-

1+Al3++PPi

0.0

450

-

3-

SC

-

1.5x10

RI PT

3+

4

2.0x10

M AN U

Fluorescence Intensity

ACCEPTED MANUSCRIPT

48

600

AC C

EP

Fig. 10

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

49

ACCEPTED MANUSCRIPT Highlights

► New sensor 1 was developed for colorimetric detection of Fe2+ and Fe3+ and fluorescent

RI PT

detection of Al3+ and PPi. ► Sensor 1 could selectively sense Fe2+/3+ by color change from yellow to orange.

SC

► Sensor 1 could be used to quantify Fe3+ in real water samples.

AC C

EP

TE D

M AN U

► 1 showed the sequential fluorescent imaging toward Al3+ and PPi in living cells.