A fluorescent and colorimetric Schiff base chemosensor for the detection of Zn2+ and Cu2+: Application in live cell imaging and colorimetric test kit

Accepted Manuscript A fluorescent and colorimetric Schiff base chemosensor for the detection of Zn2+ and Cu2+: Application in live cell imaging and colorimetric test kit

Min Seon Kim, Tae Geun Jo, Minuk Yang, Jiyeon Han, Mi Hee Lim, Cheal Kim PII: DOI: Reference:

S1386-1425(18)31055-2 https://doi.org/10.1016/j.saa.2018.11.058 SAA 16622

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

8 August 2018 13 November 2018 22 November 2018

Please cite this article as: Min Seon Kim, Tae Geun Jo, Minuk Yang, Jiyeon Han, Mi Hee Lim, Cheal Kim , A fluorescent and colorimetric Schiff base chemosensor for the detection of Zn2+ and Cu2+: Application in live cell imaging and colorimetric test kit. Saa (2018), https://doi.org/10.1016/j.saa.2018.11.058

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A fluorescent and colorimetric Schiff base chemosensor for the detection of Zn2+ and Cu2+: application in live cell imaging and colorimetric test kit

Department of Fine Chem., Seoul National Univ. of Sci. and Tech. (SNUT), Seoul 01811,

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Min Seon Kim,a Tae Geun Jo,a Minuk Yang,a Jiyeon Han,b Mi Hee Lim,b* Cheal Kima*

Department of Chem., KAIST, Daejeon 34141, Korea. E-mail: [email protected]

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Korea. Tel: +82-2-970-6690; Fax: +82-2-973-9147; E-mail: [email protected]

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novel

Schiff

base

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Abstract

chemosensor

HMID,

((E)-1-((2-hydroxy-3-

methoxybenzylidene)amino)imidazolidine-2,4-dione), have been designed and synthesized.

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Sensor HMID showed a selectivity to Zn2+ through fluorescence enhancement in aqueous

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solution. Its detection limit was analyzed as 11.9 μM. Importantly, compound HMID could be applied to image Zn2+ in live cells. Detection mechanism of Zn2+ by HMID was suggested to be an effect of chelation-enhanced fluorescence (CHEF) by DFT calculations. Moreover, HMID could detect Cu2+ with a change of color from colorless to pink. The selective detection

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mechanism of Cu2+ by HMID was demonstrated to be the promotion of intramolecular charge transfer band by DFT calculations. Additionally, HMID could be employed as a naked-eye

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colorimetric kit for Cu2+. Therefore, HMID has the ability as a 'single sensor for dual targets'.

Keywords: chemosensor, colorimetric, fluorescent, zinc ion, cell imaging, copper ion

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1. Introduction Detection of metal ions is of special significance in biomedical and analytical areas since they display pivotal roles in diverse biological systems [1–4]. Generally, proper level of metal ions could maintain the biological processes in good ways [5]. Among the metal ions, zinc ion exists plentifully in living organism due to its various coordination properties [6]. While most

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Zn2+ ions tightly bind to metalloproteins in a cell, free Zn2+ ions are also detected. In brain, 520 % of the overall Zn2+ accumulated in presynaptic cells, and much intracellular free Zn2+ is

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observed in the hippocampus [7]. Zn2+ controls brain excitability and acts as a modulatory core in synaptic plasticity [8]. Also, chelatable Zn2+ ion displays a pivotal role in synaptic plasticity

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and enzyme regulation in human body [9,10]. Meanwhile, excess of zinc would cause unstable metabolism, resulting in various cerebropathia disorders, high blood cholesterol, and slow

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growth in children [11]. Hence, the design of chemosensors for a sensitive and efficient detection of Zn2+ is a very crucial subject in neurobiology and an active research area [12,13].

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Copper, an essential transition metal ion in biological systems, also performs important roles in various fundamental processes [14]. For example, it operates as an essential cofactor related to activities of a broad range of metalloenzymes like tyrosinase and superoxide dismutase [15].

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However, at higher concentration, copper motivates neurodegenerative troubles like Menkes,

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Wilson and Alzheimer disease [16]. Meanwhile, copper can affect a serious environmental pollution, owing to its broad use in industrial and agricultural fields [17]. Thus, the

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development of novel analytical tools for an efficient detection of Cu2+ is very important [18]. Several analytical tools have been employed for the analysis of copper and zinc ions in the past decades like ICP-AES, AAS, and instrumental neutron activation analysis [19-22].

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However, most of the tools need expensive equipment, multi-step sample preparation and time consuming procedures [23]. Therefore, the fluorescence and colorimetric chemosensors are especially popular due to their great sensitivities, rapid response time, simple operation, and significantly low costs. These advantages prompted researchers to develop new chemosensors as an all-around tool in chemistry, cell imaging, and biochemistry [24–29]. Thus, various chemosensors have been developed using acetamidoquinoline [30], iminocoumarin [31] and tetrazole [32] to detect Zn2+ ion and aldazine [33], squaraine [34] and rhodamine [35] to detect Cu2+ ion. 2

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The salicylidene Schiff bases could be simply synthesized by condensing the salicylaldehyde moiety and amine-containing chromophore derivatives [36], and was expected to tightly bind to the metal ions, resulting in various optical properties toward the sort of metal ion [37-39]. Therefore, we considered a novel type of salicylidene Schiff base, which includes 3-methoxysalicylaldehyde with an electron donating group and a new amine-containing

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chromophore aminohydantoin. Herein, we demonstrate a novel Schiff base sensor HMID for Zn2+ and Cu2+, which was

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produced in a single step by condensation reaction of 1-aminohydantoin hydrochloride and 3methoxysalicylaldehyde (Scheme 1). The compound HMID could detect Zn2+ by a significant

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fluorescence enhancement and Cu2+ by the change of color from colorless to pink.

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2. Experimental section 2.1 Materials and equipment

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From Sigma-Aldrich, chemicals (analytical and spectroscopic grade) were purchased commercially. A Varian spectrometer was conducted to obtain

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C and 1H NMR spectra. By

using Perkin Elmer 25 UV-Vis and LS45 fluorescence spectrometers, absorption and emission

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spectra were measured. A Thermo MAX instrument was applied to collect ESI-MS spectra. A

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MICRO CUBE elemental analyzer was used for elemental analysis of nitrogen, hydrogen, and carbon. FT-IR spectra were collected on an Agilent Cary 670 FTIR spectrometer.

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2.2 Synthesis of compound HMID

1-Aminohydantoin hydrochloride (80 mg, 5x10-1 mmol) and 3-methoxysalicylaldehyde

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(120 mg, 0.7 mmol) were dissolved in ethanol (12.0 mL). The mixed solution was reacted for 1 d at 20 oC. White powder produced was filtered and washed sequentially with chilly ethanol and ether (90 mg, 74 %). 1H NMR (DMSO-d6): δ 11.31 (s, 1H), 10.24 (s, 1H), 7.98 (s, 1H), 7.17 (d, J = 7.2 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.86 (t, J = 8.0 Hz, 1H), 4.39 (s, 2H), 3.81 (s, 3H); 13C NMR (DMSO-d6): 169.43, 153.81, 148.36, 146.79, 142.84, 120.35, 119.74, 119.60, 113.83, 56.26, 48.92. ESI-MS m/z [HMID+Na]+: calcd, 272.06; found, 272.00. Anal. Calc. for C11H11N3O4: C, 53.01; N, 16.86; H, 4.45. Found: C, 52.84; N, 16.54; H, 4.81 %.

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2.3 Fluorescent titration Sensor HMID (2.5x10-1 mg, 1x10-3 mmol) was dissolved in 1x10-1 mL of DMSO and its final concentration of 30 μM was obtained by adding 9 μL of the stock solution (1x10-2 M) into 2.991 mL bis-tris buffer (1x10-2 M, pH 7.0). 2.25-45 μL of the Zn(NO3)2 (2x10-2 M) dissolved in bis-tris buffer were added to compound HMID (30 μM, 3.0 mL). Fluorescence spectra of

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the solutions well mixed were taken.

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2.4 UV-vis titrations

For Zn2+ ion, DMSO (100 L) was employed to dissolve sensor HMID (2.5x10-1 mg, 1x10mmol), and its final concentration of 30 μM was obtained by adding 9 μL of the stock solution

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(1x10-2 M) into 2.991 mL buffer. 4.5-49.5 μL of the Zn(NO3)2 (2x10-2 M) dissolved in bis-tris

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buffer were transferred to the HMID (3x10-2 mM). The solutions were well mixed and UV-vis spectra taken.

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For Cu2+ ion, DMSO (100 μL) was employed to dissolve sensor HMID (2.5x10-1 mg, 1x10mmol), and its final concentration of 10 μM was obtained by adding 3 μL of the stock solution

(1x10-2 M) into 2.997 mL acetonitrile. 0.3-3 μL of the Cu(NO3)2 (2x10-2 M) dissolved in bis-

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tris buffer was transferred to the diluted HMID (1x10-2 M) prepared above. The solutions were

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well mixed and UV-vis spectra taken. 2.5 Job plots

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For Zn2+ ion, a stock solution of compound HMID (1x10-3 M) was made in 1.0 mL of DMSO. Zn2+ solution (1x10-3 M) was obtained with its nitrate salt in buffer (1.0 mL). 0.27-

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0.03 mL of the HMID was transferred to several quartzes. Each quartz was diluted with buffer to give 2.70 mL. 0.03-0.27 mL of the Zn2+ solution was added to diluted HMID. Each quartz was filled with buffer to make 3 mL. The solutions were well mixed and fluorescence spectra taken. For Cu2+ ion, a stock solution of compound HMID (1x10-3 M) was made in 1.0 mL of DMSO. Cu2+ solution (1x10-3 M) was obtained with its nitrate salt in MeCN (1.0 mL). 0.270.03 mL of the HMID was transferred to several quartzes. Each quartz was slowly diluted with 4

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MeCN to give 2.70 mL. 0.03-0.27 mL of the Cu2+ solution was added to diluted HMID. Each quartz was filled with buffer to make 3 mL. The solutions were well mixed and UV-vis spectra taken. 2.6 Competition experiments For Zn2+ ion, DMSO (100 L) was employed to dissolve compound HMID (2.5x10-1 mg,

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1x10-3 mmol). Stock solutions of KNO3, NaNO3, M(NO3)3 (M = In, Cr, Ga, Fe, Al) and M(NO3)2 (M = Ni, Ca, Co, Mn, Cu, Fe, Cd, Pb, Mg, 0.06 mmol) were dissolved in 3.0 mL

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buffer. 40.5 μL of each metal (2x10-2 M) was added into 3.0 mL bis-tris buffer to make 9 equiv.

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40.5 μL (2x10-2 M) of Zn2+ ion was added into the solutions to afford 9 equiv. 9 μL of HMID (1x10-2 M) was added into the blended solutions. The solutions were well mixed and

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fluorescence spectra taken.

For Cu2+ ion, DMSO (100 L) was employed to dissolve compound HMID (2.5x10-1 mg,

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1x10-3 mmol). Stock solutions of AgNO3, KNO3, NaNO3, M(NO3)3 (M = In, Cr, Ga, Fe, Al) and M(NO3)2 (M = Zn, Ni, Hg, Pb, Mn, Co, Ca, Fe, Cd, Mg, 0.06 mmol) were dissolved in 3 mL MeCN. 2.4 μL of each metal (2x10-2 M) was added into 3.0 mL MeCN/buffer solution

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(95:5; v/v) to make 1.6 equiv. 2.4 μL (2x10-2 M) of Cu2+ ion was added into the solutions

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prepared above to 1.6 equiv. 3 μL of HMID solution (1x10-2 M) was added into the blended solutions. The solutions were well mixed and UV-vis spectra taken.

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2.7 pH effect

By mixing HCl in bis-tris buffer and NaOH solution, buffers having different pH values (2-

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12) was made. With the desired pH solutions, DMSO (0.1 mL) was used to dissolve sensor HMID (2.5x10-1 mg, 1x10-3 mmol), and then its final concentration of 30 μM was obtained by adding 9 μL of the stock solution (1x10-2 M) into 2.991 mL of bis-tris buffer. 40.5 μL of the Zn(NO3)2 (2x10-2 M) dissolved in bis-tris buffer was transferred to HMID (3x10-2 mM). The solutions were well mixed and fluorescence spectra taken. 2.8 Imaging tests in live cells HeLa cells were kept in solution including penicillin (100 U/mL), 100 mg/mL GIBCO, 5

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bovine serum (10%), and DMEM. The cells were bred with 5.0 % CO2 at 36 °C in a humidified atmosphere. Cells were transferred to six-well plates with a density of 1.5x106 cells/1.0 mL and incubated at 37 °C for 1 d. Cells were treated with HMID (4x10-2 M) for 20 min. Then, varied concentrations of zinc nitrate were added. Imaging tests were achieved with a microscope (ex = 357 (± 22) nm and em = 447 (± 30) nm).

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2.9 Colorimetric test kit Sensor HMID (249.2 mg, 1x10-3 mol) was dissolved in MeCN/DMSO (9:1, 20.0 mL). The

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HMID-test kits were obtained by immersing filter papers into compound HMID (5x10-2 M)

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and dried in air. AgNO3, KNO3, NaNO3, M(NO3)3 (M = Cr, In, Fe, Ga, Al) and M(NO3)2 (M = Fe, Pb, Cd, Ni, Mg, Co, Zn, Hg, Mn, Ca, Cu, 0.3 μmol) were dissolved in MeCN/buffer

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solution (95:5, 10.0 mL). Test kits obtained were added into the solutions including various metal, and then dried.

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2.10 Calculations

Gaussian 03 program [40] was employed for all calculations at the B3LYP level [41,42] with

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TD-DFT. The 6-31G** basis set [43,44] was employed for the main group elements and ECP [45,46] done for Cu and Zn. There was no imaginary vibration frequency to the optimized

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geometries of HMID-Zn2+, HMID, and HMID-Cu2+ species, indicating that the geometries presented local minima. The Cossi and Barone’s CPCM were used to deal with the solvent effect of water in the calculations [47,48]. The GaussSum 2.1 [49] was applied to measure the

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contributions of molecular orbitals of electronic transitions.

3. Results and discussion Compound HMID was afforded by the combination of 1-aminohydantoin hydrochloride and 3-methoxysalicylaldehyde (74% yield; Scheme 1), and fully analyzed by elemental analysis, 13C NMR, 1H NMR FT-IR spectrometry and ESI-mass.

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Scheme 1. Synthesis of HMID.

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3.1 Fluorescent studies of HMID to Zn2+

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The fluorescent emission change was measured with different kinds of cations in buffer solution to examine the fluorescent behavior of HMID. As displayed in Fig. 1, HMID

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exhibited little fluorescence band at 479 nm (ex = 380 nm). On the addition of different kinds of cations like K+, Cu2+, Ca2+, Na+, Co2+, Cd2+, Mn2+, Fe2+, Pb2+, Ni2+, Mg2+, Cr3+, Al3+, Fe3+,

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Ga3+ and In3+ with their nitrate salts, HMID showed either no or a little variation in the fluorescence. However, the addition of Zn2+ displayed a great enhancement of fluorescence intensity of 479 nm. Therefore, HMID may be applied as a fluorescent sensor for the selective

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detection of Zn2+.

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Fig. 1 Fluorescent spectral changes of HMID (30 M) with different metal ions (9 equiv) like K+, Na+, Cd2+, Zn2+, Ca2+, Co2+, Cu2+, Fe2+, Pb2+, Ni2+, Mg2+, Mn2+, Al3+, Cr3+, Fe3+, Ga3+ and In3+ (ex = 380 nm).

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To understand the sensing process of HMID to Zn2+, fluorescent titration of HMID with

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Zn2+ was achieved (Fig. 2). The emission of HMID at 479 nm substantially increased until the concentration of Zn2+ reached up to 9 equiv. The photophysical character of HMID was also investigated with UV-vis spectrometer (Fig. S1). On the addition of Zn2+ into HMID, the

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absorbance of 275 nm and 325 nm decreased and the new band of 250 nm, 310 nm and 370 nm increased until the concentration of Zn2+ reached up to 10 equiv. There were three defined

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isosbestic points at 258 nm, 319 nm and 336 nm, signifying that a single product was afforded from the reaction of HMID with Zn2+.

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1, [1+Ga ,Cd ,Cu , Fe , Fe , Cr , Mg ,

Flu. Int. at 479nm

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Fig. 2 Fluorescent spectra of HMID (30 μM) with the change of concentrations of Zn2+. Inset: Fluorescent intensity at 479 nm vs. the equiv of Zn2+ added.

The binding of HMID and Zn2+ showed a 1:1 complexation with Job plot analysis (Fig. S2) [50]. Formation of HMID-Zn2+ was evidenced by ESI-mass test (Fig. 3). Positive-ion

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spectrum exhibited that the peak of m/z: 390.00 was suggestive to [HMID(-H+) + Zn2+ + DMSO]+ (calcd, 390.01). For further information on the formation of HMID-Zn2+ complex,

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FT-IR study was conducted (Figs. S3 and S4). The band at 3510 cm-1 associated with O-H of the phenol group of HMID disappeared after the reaction of HMID with Zn2+, indicating that the hydroxyl group might be deprotonated. In addition, the band at 1703 cm-1 associated with the carbonyl group of HMID was shifted to 1596 cm-1, implying that the oxygen atom of the carbonyl group might coordinate to Zn2+. Based on the analysis of ESI-mass, FT-IR and Job plot, the 1:1 binding of HMID with Zn2+ was suggested, as displayed in Scheme 2. With fluorescence titration, the binding constant (K) of 4.5 × 103 M-1 for the HMID-Zn2+ was given by a Benesi-Hildebrand equation (Fig. S5) [51]. The K value is in the scope of those (1 ~ 1.0 9

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× 1012) for previously announced Zn2+ sensors [52–54]. Detection limit (DL) of HMID for Zn2+ was calculated to be 11.9 μM based on 3/k (Fig. S6) [55]. The value was lower than the

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standard (76 μM) recommended by WHO [56].

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Fig. 3 Positive-ion ESI-Mass of HMID (0.1 mM) with the addition of Zn2+ (1.0 equiv).

Scheme 2. Proposed structures of HMID-M2+ complexes (M = Zn or Cu).

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The competitive tests were achieved in the presence of Zn2+ (9 equiv) mixed with other cations (9 equiv; K+, Cu2+, Ca2+, Cd2+, Co2+, Fe2+, Na+, Ni2+, Mg2+, Mn2+, Ga3+, Pb2+, Al3+, Fe3+, Cr3+ and In3+) to confirm the practicable applicability of HMID as a fluorescent probe (Fig. S7). Among the metal ions, Cu2+, Cr3+ and Co2+ almost interfered with the interaction between HMID and Zn2+, and Fe2+, Fe3+ and Ni2+ quenched more than half of the fluorescence intensity obtained with Zn2+ alone. For biological application, the pH effect of HMID-Zn2+ was analyzed

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in buffers with pH of 2 to 12 (Fig. S8). Compound HMID with Zn2+ displayed a significant fluorescence enhancement between pH 7.0 and 9.0. Thus, HMID could clearly detect Zn2+

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through the fluorescence change over the physiologically relevant pH range (7.0-8.4) [57].

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To investigate the reversibility of sensor HMID to Zn2+, the mixed solution of HMID and Zn2+ was treated with ethylenediaminetetraacetic acid (EDTA, 9 equiv; Fig. S9). Fluorescence

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intensity of 479 nm was instantly quenched by adding of EDTA. On addition of Zn2+ into the solution, the emission intensity was returned. The emission spectra were nearly reversible

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even after several repeated procedures with the consecutive addition of Zn2+ and EDTA. The imaging tests were achieved in live cells to check the possibility of HMID to sense Zn2+ in live matrices (Fig. 4). HeLa cells were incubated with HMID for 20 min and then

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exposed to varied amounts of Zn2+ (0 - 200 μM). The presence of both HMID and Zn2+ in

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HeLa cells exhibited fluorescence-on, but those cells which were cultured without Zn2+ or HMID showed no fluorescence. These results validated that HMID could be a satisfactory

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probe for Zn2+ in living cells.

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Fig. 4 Fluorescent responses of HMID to Zn2+ in HeLa cells. Cells were preincubated with HMID for 20 min before addition of varied concentrations of Zn2+. Conditions: [Zn(II)] = 0,

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100, and 200 μM; [HMID] = 40 μM. The scale bar is 50 μm.

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3.2 Colorimetric studies of HMID to Cu2+ The colorimetric sensing possibility of HMID was studied with a wide range of cations in

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MeCN/bis-tris buffer (95:5; Fig. 5). Addition of 1.6 equiv of the cations (Co2+, Ag+, K+, Na+, Zn2+, Ca2+, Cd2+, Fe2+, Ni2+, Mg2+, Mn2+, Pb2+, Hg2+, Cr3+, Fe3+, Al3+, In3+ and Ga3+) had no serious change on the color and absorbance. By contrast, Cu2+ showed a change in color (from

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colorless to pink) with a distinct spectral change. These results illustrated that HMID may operate as a “naked-eye” probe for Cu2+.

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0.2 2+

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Fig. 5 (a) Absorption changes of HMID (10 with 1.6 equiv of a broad range of cations in MeCN/bis-tris buffer solution (95:5). (b) Color changes of HMID (10 upon addition of a broad range of cations (1.6 equiv).

UV-vis titration was achieved to comprehend the binding interaction between HMID and

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Cu2+ (Fig. 6). On addition of Cu2+ to HMID, the absorbance of 260 nm increased and the absorption band of 290 nm decreased constantly. In addition, a new distinct band of 503 nm steadily increased and reached a maximum at 1.6 equiv of Cu2+ with an obvious isosbestic point (270 nm). It meant that a species was produced from HMID upon binding to Cu2+.

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Fig. 6 Absorption spectra of chemosensor HMID (10 μM) with varied concentrations of Cu2+ (0-2.0 equiv). Inset: the absorbance (503 nm) vs. the equiv of Cu2+ added.

The binding ratio of Cu2+ and HMID was found to be a 1:1 through Job plot (Fig. S10)

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[50]. To validate the binding ratio of HMID-Cu2+, a positive-ion mass analysis was achieved (Fig. 7). The addition of Cu2+ (1 equiv) into HMID indicated the generation of [HMID(-H+) + Cu2+ + DMSO]+ [m/z: 389.00; calcd, 389.01]. In addition, FT-IR spectrum for HMID-Cu2+

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complex showed that the hydroxyl group peak of HMID at 3510 cm-1 disappeared and the peak of the carbonyl group of HMID at 1703 cm-1 was shifted to 1598 cm-1 (Fig. S11). These

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results suggested that the oxygen of the carbonyl group of HMID might coordinate to Cu2+, with the deprotonation of the hydroxyl group. The results of ESI-mass and FT-IR, Job plot led us to suggest the possible structure of HMID-Cu2+ (Scheme 2). With UV-vis titration of HMID with Cu2+, the binding constant (K) of 3.3 x 102 M-1 for the HMID-Cu2+ complex was given by the non-linear fitting method (Fig. S12) [58,59]. Detection limit (3σ/k) of HMID for Cu2+ was turned out to be 0.91 μM (Fig. S13) [55].

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Fig. 7 Positive-ion ESI-Mass of HMID (0.1 mM) with the addition of Cu2+ (1.0 equiv).

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The competition experiments were conducted for investigating the preferential selectivity

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for Cu2+ with various metal ions (Fig. 8). HMID was treated with Cu2+ in the presence of interfering cations (Ag+, K+, Na+, Hg2+, Zn2+, Ca2+, Co2+, Cd2+, Ni2+, Mg2+, Pb2+, Mn2+, Al3+, Fe2+, Cr3+, Fe3+, Ga3+ and In3+). Most interfering cations did not display any interference for

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the detection of Cu2+ by HMID, except for Ag+, Hg2+ and Fe2+. They interfered with about 50, 30 and 90% of the absorbance obtained with Cu2+ alone. This result validated that HMID may

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be employed as a colorimetric probe for effective detection of Cu2+. To check the practicable application of HMID, test kits were obtained by soaking filter papers into HMID and then dried in an oven. The test kits were applied to recognize Cu2+ among a wide range of cations (Fig. 9). When diverse cations were added into the test kits coated with HMID, an absolute color change was shown only for Cu2+. The test kits coated with HMID could be used for sensing Cu2+ below the WHO standard.

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2+                   Cu 3+ 3+ 3+ 2+ 2+ 2+ 3+ 2+ 3+ 2+ + 2+ 2+ + + 2+ 2+ 2+ Al Ga In Zn Cd Fe Fe Mg Cr Hg Ag Co Ni Na K Ca Mn Pb

Fig. 8 (a) Competitive selectivity of HMID (10 μM) to Cu2+ (1.6 equiv) with a broad range of cations (1.6 equiv). (b) Color changes of HMID (10  with the addition of Cu2+ (1.6 equiv)

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with and without 1.6 equiv of a broad range of cations.

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Fig. 9 Photographs of filter papers coated with HMID in presence of different types of cations (30 μM).

3.3 Calculations for Zn2+ and Cu2+ Theoretical calculations were conducted with comparison to the experimental tests to

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comprehend the detection mechanisms of HMID for Zn2+ and Cu2+. Based on Job plot and ESI-mass tests, the 1:1 stoichiometry of HMID to Zn2+ and Cu2+ was applied to all theoretical

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calculations, respectively. Energy-minimized forms for HMID, HMID-Zn2+ and HMID-Cu2+ were provided by applying density functional theory. The forms with the important structural

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properties of HMID, HMID-Zn2+ and HMID-Cu2+ are shown in Fig. 10. The absorptions to the singlet excited states of HMID, HMID-Zn2+ and HMID-Cu2+ were investigated via TD-

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DFT calculations (Figs. S14, S15 and S16). For HMID, HOMO → LUMO transition was characterized to be ICT transition, based on the MO contribution of the lowest excited state

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(340.51 nm, Fig. S14). For HMID-Zn2+, the MO contribution of the lowest excited state was primarily suggestive to HOMO → LUMO+1 transition with predominant ICT transition (411.81 nm, Fig. S15). There were no apparent variations for the electronic transitions

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between HMID and HMID-Zn2+. Only, the hypochromic shift (340.51 to 411.81 nm) was

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observed upon chelating of HMID with Zn2+, which was matched with the UV-vis data of HMID and HMID-Zn2+. Therefore, the detection mechanism of HMID to Zn2+ could be described by the rigidity of the structure, indicating the CHEF effect [60]. For HMID-Cu2+,

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pink color of HMID-Cu2+ stemmed from the 5th lowest excited state. This excited state was primarily calculated to HOMO (α) → LUMO (α) and HOMO (β) → LUMO+1 (β), which

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could be characterized by ICT transition (427.71 nm, Fig. S16). Compared with the electronic transitions of HMID, the complexation between HMID and Cu2+ might cause the enhancement of ICT transitions, which resulted in the hypochromic shift (340.51 to 427.71 nm) with color change of HMID. In addition, the energy transfer occurred from HMID to the d orbitals, and paramagnetic property of Cu2+ might generate non-radiative decay of the excited state. Hence, fluorescence change of HMID toward Cu2+ wasn’t observed. Thus, contrary to the fluorescence enhancement of HMID with Zn2+, HMID-Cu2+ showed no enhancement of the fluorescence [61]. 17

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Fig. 10 Energy-minimized structures of (a) HMID, (b) HMID-Zn2+ and (c) HMID-Cu2+.

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4. Conclusion We presented a novel dual-functional fluorescent and colorimetric chemosensor HMID, given by the combination of 1-aminohydantoin hydrochloride and 3-methoxysalicylaldehyde. HMID can operate as a great efficient fluorescence sensor for detection of Zn2+. Binding of HMID and Zn2+ was reversible with a suitable reagent like EDTA. Detection limit (11.9 μM)

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for Zn2+ was below the guideline (76.0 μM) of WHO. Significantly, HMID could be applied to image Zn2+ in live cells, which proved its value in the practicable application. Moreover,

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HMID can selectively detect Cu2+ by a pronounced change of color from colorless to pink. In particular, HMID could be successfully applied to detect Cu2+ by an easy test kit. Therefore,

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these results present that HMID can operate as both a new fluorescence chemosensor for Zn2+

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and a “naked-eye” one for Cu2+.

Acknowledgements National

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2017R1A2B3002585) is greatly acknowledged.

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(NRF)

(2018R1A2B6001686

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Graphical abstract

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Highlights ► New Schiff-base chemosensor HMID was developed for detection of Zn2+ and Cu2+. ► HMID can detect Zn2+ through “turn on” fluorescence in living cells. ► HMID was used to detect Cu2+ from colorless to pink though ICT mechanism. ► Chemosensor HMID could be used as a practical visible colorimetric test kit for Cu2+.

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► Sensing mechanisms for Zn2+ and Cu2+ by HMID were explained by DFT calculations.

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