Accepted Manuscript

A novel fluorescent probe based on the flexible dipicolylamine: recognizing zinc(Ⅱ) in

aqueous solution and imaging in living cell

Na Zhang, Xiaohe Tian, Jun Zheng, Xiuzhen Zhang, Weiju Zhu, Yupeng Tian, Qiyong
Zhu, Hongping Zhou

PII: S0143-7208(15)00366-6
DOI: 10.1016/j.dyepig.2015.09.019
Reference: DYPI 4930

To appear in: Dyes and Pigments

Received Date: 11 August 2015

Revised Date: 14 September 2015
Accepted Date: 16 September 2015

Please cite this article as: Zhang N, Tian X, Zheng J, Zhang X, Zhu W, Tian Y, Zhu Q, Zhou H, A novel
fluorescent probe based on the flexible dipicolylamine: recognizing zinc(Ⅱ) in aqueous solution and
imaging in living cell, Dyes and Pigments (2015), doi: 10.1016/j.dyepig.2015.09.019.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT

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2 A novel fluorescent probe based on the flexible dipicolylamine: recognizing

3 zinc( ) in aqueous solution and imaging in living cell
4
5 Na Zhanga, Xiaohe Tiana, Jun Zhengc, Xiuzhen Zhangc, Weiju Zhua, Yupeng Tiana,
6 Qiyong Zhub, * and Hongping Zhoua, *
7

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a
8 College of Chemistry and Chemical Engineering, Anhui University and Key Labotatory of
9 Functional Inorganic Materials Chemistry of Anhui Province, 230601, Hefei, P.R. China
b
10 Department of Chemistry, Huainan Normal College, Huainan, 232001 P.R. China

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c
11 Center of Modern Experimental Technology, Anhui University, Hefei 230039, P.R. China
12

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13 Corresponding author. Fax: +86-551-63861279; Tel: +86-551-63861279
14 E-mail: zhpzhp@263.net
15

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16 Abstract
A novel fluorescent probe for Zn2+ has been designed and synthesized based on a
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18 dipicolylamine group connected by a benzimidazole amide backbone. A comprehensive

19 analysis on recognition ability of the probe towards metal ions was conducted by ultraviolet
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20 absorption and fluorescence emission spectra. Results demonstrated that the probe showed
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21 selective and sensitive fluorescence turn-on signaling behavior toward Zn2+ in HEPES
22 buffered (pH =7.4) aqueous solution. The detection limit of this probe was calculated to be
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23 5.10 ×10-8 M for Zn2+. Fluorescent imaging inside HepG2 cells indicated potential
24 application of the probe for detecting Zn2+ ions in living cell imaging.
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25
26 Keywords: DPA; Probe; Recognition abilities; Zinc ion; Fluorescence spectra; Cell
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27 imaging
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28

29 1. Introduction
30 The development of probes for highly selective and sensitive detection of biologically
31 and environmentally important heavy and transition metal (HTM) ions has attracted great
32 attention owing to the essential roles they play in chemical, biological and environmental
33 processes [1–3]. After iron, zinc as the most abundant transition metal in living organisms
34 is involved in gene transcription, signal transmission, and mammalian reproduction [4, 5].

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1 Disruption of Zn2+ concentration in cells may be associated with a variety of diseases such
2 as Alzheimer’s disease, epilepsy, and infantile diarrhoea [6, 7]. Zinc ions are colorless in
3 aqueous solutions, do not have unpaired electrons, and remain in the plus two oxidation
4 state. It is difficult to image the concentration of zinc ions in biological environments.
5 However, it has been discovered that some unique molecules in the presence of Zn2+

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6 fluoresce, which can be monitored and quantified [8]. Despite having many good and even
7 commercial fluorescent probes to facilitate the study of this silent metal ion in biological

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8 systems, chemists are continuously endeavoring to design new probes and improve their
9 sensitivity, selectivity, and reliability in order to satisfy various needs due to the wide

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10 existence of Zn2+ in organisms. Therefore, developing reliable fluorescent probes for Zn2+
11 detection has attracted considerable attention in recent years.

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12 Up to now, a variety of fluorescent Zn2+ probes have been discussed based on various
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13 fluorophores [9, 10]. However, most of the reported fluorescent probes were applied in
14 pure organic or organic-water mixed solvents during the function processes, which
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15 inevitably hinder their real applications. Therefore, it is still challenging to develop

16 effective fluorescence “turn-on” probes with high selectivity, sensitivity, rapid and
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17 ratiometric responses to Zn2+ in aqueous solution.

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18 In this current study the DPA (bis (2-pyridylmethyl) amine) moiety was adopted as a
19 zinc chelator because it is able to afford an excellent selectivity for Zn2+ over other metal
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20 ions [11]. In addition, the amino nitrogen of the DPA group is a good candidate as an
21 electron donor in either photoinduced electron transfer or photoinduced charge transfer
(PET or PCT) probes [12]. In order to modulate the electronic structures and thereby the
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23 spectral properties, DPA has been bonded to a variety of fluorophores such as

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24 naphthalimide [13], rhodamine [14], quinoline [15,16], coumarin [17,18], benzoxazole [19].
25 Triphenylamine benzimidazole as a common fluorophore signaling unit, is widely used in
26 coordination chemistry due to its ability to bind an array of d- and f-block elements [20].
27 Herein, we designed and synthesized a novel fluorescent turn-on probe for Zn2+, which
28 has a DPA subunit to coordinate with Zn2+ and a triphenylamine benzimidazole group as
29 fluorophore (Scheme 1). We have intentionally increased the carbon chain length by the
30 presence of an amide group between the fluorophore and zinc( ) binding domain to
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1 change the fluorescence response to zinc(Ⅱ). Target probe L exhibits highly sensitive,
2 selective, lower detection limits (Table 1), and rapid fluorescence response for zinc(Ⅱ) in
3 aqueous solution.
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5 Table 1
6 Detection limits of other fluorescence probes (1-3[a]) and probe Lb for Zn(II)
7
Fluorescence probes Detection limits
1[21] 7.89 10-7 M
2[22] 8.30 10-7 M
3[23] 6.21 10-7 M
4[24] 6.10 10-7 M
Lb 5.10 10-8 M
[a]
Fluorescence probes have been reported for Zn(II); b Fluorescence probe we have
designed and synthesized in this paper.
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9 2. Experimental
10 2.1. Materials and instruments
11 Triphenylamine, 4-Nitro-o-phenylenediamine, Chloroacetyl chloride, Bis(2-
12 pyridylmethyl)amine and solvents were purchased from commercial suppliers and used as
13 received. Solvents used in spectra experiments were spectrometric grade. Solutions of Hg2+
14 and Mn2+ were prepared from their chloride salts; solutions of Ag+, Al3+, Ba2+, Ca2+, Cd2+,
15 Co2+, Cr3+, Cu2+, Fe3+, K+, La3+, Li+, Mg2+, Na+, Ni2+, Pb2+ and Zn2+ were prepared from
16 their nitrate salts. HEPES buffer solutions (20 mM, pH 7.4) were prepared in water.
17 The chemical structures of these intermediates and target compound were confirmed by
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18 H NMR, 13C NMR, IR and MS. 1H NMR and 13C NMR spectra were recorded on a Bruker
19 Avance 400 MHz and 100 NMR spectrometer using dimethyl sulfoxide-d6 (DMSO-d6) as
20 solvent. Chemical shifts are reported in parts per million (ppm) relative to internal TMS (0
21 ppm) and coupling constants in Hz. Splitting patterns are described as singlet (s), doublet
22 (d), triplet (t), quartet(q), or multiplet (m). IR spectra were recorded with an FT-IR
23 spectrometer (KBr discs) in the 4000-400 cm-1 region. The mass spectra were measured
24 with a LTQ Orbitrap XL. The one-photon absorption (OPA) spectra were recorded on a
25 UV-265 spectrophotometer. The one-photon excited fluorescence (OPEF) spectra

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1 measurements were performed using a Hitachi F-7000 fluorescence spectrophotometer. All

2 the pH measurements were carried out on a Mettler-Toledo Delta 320 pH meter.
3 Bioimaging of the probe was performed on a Zeiss LSM 710 META upright confocal laser
4 scanning microscope using 40× magnification water-dipping lenses for monolayer cultures.
5 Image data acquisition and processing was performed using Zeiss LSM Image Browser,
6 Zeiss LSM Image Expert and Image J.
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10 Scheme 1. Synthetic route of probe L.
11 2.2. Synthesis of M1
12 5-amino-2-triphenylaminebenzimidazole (M1) was prepared by procedures in the literature
13 [25].
14 2.3. Synthesis of M2
15 Synthesis of 2-chloro-N-(2-triphenylaminebenzimidazole-5-yl)acetamide (M2).
16 M1 (100.00 mg, 0.27 mmol), potassium carbonate (40.00 mg, 0.30 mmol) in
17 dichloromethane solution (30 mL) was stirred for 20 min in an ice bath . Then the
18 chloroacetyl chloride (36.00 mg, 0.32mmol) in dichloromethane solution (15 mL) was
19 added drop-wise to the reaction mixture, and the solution was stirred at room temperature
20 for 12 h [21].The resulting reaction mixture was removed under reduced pressure. The
21 residue was washed thoroughly with water and then collected by vacuum filtration and
22 dried to give the crude product, which was recrystallized in ethyl acetate and petroleum
23 ether to gain yellow power. Yield: 0.10 g (85.12%). Mp:>300 0C. 1H NMR:(400 MHz,
24 DMSO-d6) δ (ppm): 10.75 (s, 1H), 8.27 (s, 1H), 8.08 (s, 2H), 7.71 (d, J = 8.00 Hz, 1H),
25 7.54 (s, 1H), 7.44 (t, J = 8.00 Hz, 4H), 7.25 (d, J = 8.00 Hz, 2H) , 7.21 (d, J = 8.00 Hz, 5H),

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1 7.04 (d, J = 8.00 Hz, 2H), 4.33 (s, 2H). 13C NMR (100MHz, DMSO-d6) δ (ppm): 164.97,
2 151.47, 149.63, 148.81, 145.46, 136.30, 130.07, 129.14, 126.20, 125.45, 119.02, 117.75,
3 116.50, 113.99, 103.18, 43.54. FT-IR (KBr, cm−1): 3383.83 (w), 3056.73 (w), 2922.04 (w),
4 1682.08 (w), 1633.90 (w), 1610.83 (m), 1589.63 (vs), 1488.60 (vs), 1472.20 (s), 1335.73
5 (s), 1299.62 (m), 1264.04 (m), 1203.35 (m), 758.49 (m), 698.17 (s). ESI-MS: found:

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6 [M+H]+ 453.1452; molecular formula C27H21ClN4O requires [M+H]+ 453.1404.
7 2.4 Synthesis of L

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8 Synthesis of
9 2-(bis(pyridine-2-methy)amino)-N-(2-triphenylaminebenzimidazole-5-yl)acetamide (L).

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10 M2 (250.00 mg, 0.55 mmol) with diisopropyl ethyl amine (364.90 µL, 2.20 mmol),
11 potassium iodide (92.00 mg, 0.55 mmol) in acetonitrile (60 mL) were stirred for 30 min,

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12 then dipicolylamine (198.10 µL, 1.10 mmol) was added dropwise under a nitrogen
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13 atmosphere. The reaction mixture was refluxed for 24 h [26]. The mixture was then cooled
14 to room temperature, filtered to remove inorganic impurities, and concentrated under
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15 reduced pressure to give a brown, thick oil, which was purified by thin layer
16 chromatography (TLC) (CH2Cl2/CH3OH = 100 : 2) to afford L (Scheme 1). Yield: 0.14 g
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17 (25.20%). Mp: 125 0C. 1H NMR: (400 MHz, DMSO-d6) δ (ppm): 10.66 (s, 1H), 8.61 (d, J
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18 = 4.00 Hz, 2H), 8.12 (s, 1H), 8.01 (d, J = 8.00 Hz, 2H), 7.78 (t, J = 8.00 Hz, 2H), 7.54 (d, J
19 = 8.00 Hz, 1H), 7.48 (d, J = 8.00 Hz, 2H), 7.38 (t, J = 6.00 Hz, 5H), 7.31 (t, J = 6.00 Hz,
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20 2H), 7.13 (t, J = 10.00 Hz, 6H), 7.05 (d, J = 8.00 Hz, 2H), 4.42 (s, 1H), 3.96 (s, 4H), 3.48 (s,
13
21 2H). C NMR (100 MHz, DMSO-d6) δ (ppm): 169.33, 158.85, 152.43, 151.71, 149.50,
149.16, 147.07, 137.83, 137.24, 134.25, 130.23, 128.00, 125.37, 124.42, 124.11, 123.71,
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23 123.61, 123.57, 122.94, 122.11, 59.93, 58.40. FT-IR (KBr, cm−1): 3422.98 (s), 3187.49 (s),
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24 2923.39 (s), 2851.67 (m), 2360.75 (w), 1592.43 (vs), 1540.96 (m), 1487.82 (vs), 1435.66
25 (s), 1400.14 (m), 1331.90 (m), 1278.12 (m), 1193.72 (w), 757.93 (m), 697.11 (m). MS
26 (APCI): found: [M+H]+ 616.2932; molecular formula C39H33N7O requires [M+H]+
27 616.2932.

28 3. Results and discussion

29 3.1. Spectroscopic properties

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1 The fluorescence selectivity of L to various metals was measured in HEPES buffer (20
2 mM, pH =7.4) at 25 oC (λex = 365 nm). Stock solutions (10 mM) of the nitrate salts (or
3 chlorate salts) of Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, La3+, Li+,
4 Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ were prepared respectively in distilled water. Probe
5 L was dissolved in N, N-dimethylformamide (DMF) to give the stock solution (1 mM). The

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6 prepared stock solution of the metal ions and probe L were directly used in the
7 spectroscopic measurement. Probe L exhibited a major absorption band centered at 365 nm

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8 (Fig. S7). To obtain insight into the sensing properties of L toward metal ions, the emission
9 changes were examined with different metal ions above all (Fig. 1).

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12 Fig. 1. Fluorescence spectra of L (10 µM) in the presence of various metal ions (10 µM) in
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13 aqueous solution (HEPES 20 mM, pH 7.4). Inset: Fluorescence color changes of L solution
14 before and after addition of Zn2+ under UV lamp.
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16 As shown in Fig. 1 a 37 nm red-shift (from 435 to 472 nm) were observed upon the
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17 addition of Zn2+ and Cd2+ to the aqueous solution of L, respectively. Fluorescence spectra
18 depicted that only Zn2+ results in a pronounced fluorescence enhancement at 472 nm, of
19 which, the fluorescence color of its solution changes from blue to green, which can be
20 identified by ultraviolet lamp. Compared with Zn2+, Cd2+ induced an similar emission
21 red-shift, but this fluorescence intensity has not obvious change, which can also
22 discriminate these two ions qualitatively. The results indicate that probe L can readily
23 distinguish Zn2+ from biologically and environmentally relevant metal ions by the red-shift
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1 and enhanced fluorescence. In addition, the intensity of L completely quenched with the
2 addition of Cu2+, which can be used as a ON-OFF probe for Cu2+.

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3

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4 Fig. 2. Job’s plot for the determination of the stoichiometry of L and Zn2+ in aqueous
5 solution (HEPES 20 mM, pH 7.4) .The total concentration of L and Zn2+ was 10 µM.
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7 In order to understand the binding stoichiometry of L-Zn2+ complexes. Job’s plot
8 experiments were carried out. By keeping the sum of the initial concentration of the zinc
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9 ion and L at 10 µM and the molar ratio of zinc changing from 0 to 1. The fluorescent
10 intensities of L-Zn2+ complexes in different molar ratio were determined, respectively. A
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11 plot of fluorescence intensity versus the molar ratio is provided in Fig. 2. It shows that
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12 fluorescent intensity goes through a maximum at a molar ratio of about 0.5, indicating a 1:1
13 stoichiometry of the Zn2+ to L in the complexes.
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14 To further understanding the interactions between L and Zn2+, The fluorescence titration
15 of the Zn2+ ion was carried out using a solution of L (10 µM) in aqueous HEPES buffered
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16 solution (pH =7.4). Upon the addition of increasing concentrations of Zn2+, the
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17 fluorescence intensity in the 390-610 nm range showed a linear enhancement. The titration
18 reaction curve showed a steady and smooth increase until a plateau was reached (4 equiv of
19 Zn2+) with 5-fold increase at the plateau. Meanwhile, the maximum emission wavelength
20 undergoes a dramatic red shift from 432 to 472 nm (Fig. 3). All of this supports our
21 expectation that probe L could serve as a sensitive fluorescent switcher as a probe for Zn2+.

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2 Fig. 3. Fluorescence spectra of L (10 µM) on addition of different amount of Zn2+ in

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3 aqueous solution (HEPES 20 mM, pH 7.4).
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5 From the titration profile, the binding constant (Ka) of L and Zn2+ was evaluated to be
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6 5.32 ×104 M-1 according to nonlinear least-squares fitting method based on 1:1 binding
7 equation (R = 0.9809) (Fig. 4a). From the changes in Zn2+ dependent fluorescence intensity
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8 (Fig. 4b), the detection limit was estimated to be 5.1×10-8 M based on the equation DL=
9 K×SD/S, where K = 3, SD is the standard deviation of the blank solution, and S is the slope
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10 of the calibration curve [27-29].

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14 Fig. 4. (a) Benesi-Hildebrand plot of L-Zn2+ complexes in aqueous solution (HEPES 20

15 mM, pH 7.4); (b) Calibration curve of L-Zn2+ in aqueous solution (HEPES 20 mM, pH
16 7.4).
17 Reversibility is a prerequisite in developing novel probes for practical application. The

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1 reversibility of the recognition process of L was performed by adding Na2EDTA, which is

2 a Zn2+ binding agent. Because of the high stability constant of the EDTA-Zn2+ complex, it
3 was anticipated that addition of EDTA will sequester Zn2+ from the metal complex and thus
4 liberate the free L [30]. With this intention, excess of EDTA was added to the Zn2+ complex
5 of L. The fluorescence of the solution decreased instantaneously upon the addition of

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6 EDTA, whereas re-addition of excess Zn2+ could recover the fluorescence signal (Fig. 5),
7 suggesting that the binding of L with Zn2+ is chemically reversible.

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10 Fig. 5. Fluorescence spectra of L (10 µM) upon the addition of 2 equivalent of Zn(NO3)3 in
aqueous solution (HEPES 20 mM, pH 7.4). EDTA (2 equiv) was added to L-Zn2+ mixture
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12 to show the reversible binding nature of Zn2+ with L.

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14 To study the influence of other metal ions on Zn2+ binding with probe L, this research
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15 performed competitive experiments by adding 1.0 equiv. of Zn2+ to L in the presence of 1.0
16 equiv of other metal ions in HEPES buffer (pH =7.4). Based on the data in Fig.6, we can
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17 get the conclusion that L could selectively detect Zn2+ even in the presence of Ag+, Al3+,
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18 Ba2+, Ca2+, Cd2+, Cr3+, Fe3+, Hg2+, K+, La3+, Li+, Mg2+, Mn2+, Na+, Ni2+, and Pb2+. Among
19 other transition metal cations, only Cu2+ compromised the Zn2+-induced fluorescence
20 enhancement of L, which may be owing to the its paramagnetic nature [23]. We also have
21 tested Job’s plot, fluorescence titration, detection limit, binding constant and competitive
22 experiments towards Cu2+ (Fig. S10-13). To some extent, probe L can detect Cu2+
23 simultaneously, considering the practical application in cell living, we only evaluate
24 recognition ability of the probe L to Zn2+ mainly.

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4 Fig. 6. The fluorescence response of probe L (10 µM) upon addition of various metal ions

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5 (10 µM) in and without the presence of Zn2+ (10 µM) in aqueous solution (HEPES 20 mM,

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6 pH 7.4).
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8 For practical application, the pH working range of the probe L was evaluated (Fig. S8).
9 As expected in the pH range of 6-13, L showed very stable fluorescence, indicating that the
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10 probe could be used under common environmental and physiological condition. We also
11 investigated the time course of the response of L (10 µM) to 1 equiv. of Zn2+ in HEPES
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12 buffer (pH =7.4) solution. The interaction of L with Zn2+ was completed in about 3 minutes
(Fig. S9). Therefore, this system could be used for real-time tracking of Zn2+.
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15 3.2. Proposed interaction mechanism of L to Zn2+

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17 Fig. 7. 1H NMR spectrum of L (DMSO-d6) in the absence (I) and presence (II) of 1 equiv
18 of Zn2+.

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1 To get further insight into the binding mode of L and Zn2+, the 1H NMR analysis of
2 probe L in DMSO-d6 in the presence of 1.0 equiv. of Zn2+ in D2O was recorded and the
3 result was shown in Fig. 7. The amide NH (Hc) signal appeared at 10.65 ppm in free L
4 (Fig.7(I)), which disappeared upon the addition of 1.0 equiv. of Zn2+ (Fig.7 (II)). The
5 Zn2+-induced disappearance of the amide NH proton may be attributed to the

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6 transformation of the amide group to imidic acid upon binding with Zn2+, which also
7 indicated that the negatively charged oxygen atom in deprotonated imidic acid upon

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8 binding with Zn2+ [31, 32]. However, the imidazole NH (Hd) signal at 4.42 ppm probably
9 exchanged with trace D2O [33, 34]. The signal of protons (He) neighbouring pyridine N

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10 atom at 8.60 ppm are shifted downfield to 8.71 ppm upon addition of Zn2+. Meanwhile, the
11 methylene protons (Ha) adjacent to amide carbonyl group signaling at 3.49 ppm downfield

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12 shifted to 3.88 ppm after addition of Zn2+. Concomitantly, the singlet peak of methylene
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13 protons (Hb) in DPA moiety at 3.96 ppm was shifted downfield and split into two
14 non-equivalent groups at 4.49 ppm and 4.42 ppm with a large coupling constant of 16 Hz,
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15 indicating that the free rotations of sigma bonds in DPA moiety are suppressed after
16 coordination with Zn2+. Accordingly, the structure of L-Zn2+ is proposed in Fig. 7(II), in
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17 which Zn2+ may coordinate with the amide oxygen and DPA nitrogen atoms [35].
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18 3.3 Cell imaging

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20 Fig. 8. Images of HepG2 cells incubated with probe L (top) or L with Zn2+ (bottom): (a)
21 fluorescent images; (b) Bright field image of the HepG2 cells; (c) Overlay image of (a) and
22 (b).
23 To further assess the sensing performance of L to Zn2+ in biological samples, fluorescent

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1 imaging inside HepG2 cells was monitored by fluorescence microscopy using a Zeiss
2 LSM710 META upright confocal scanning microscope. HepG2 cells as the testing
3 candidates were cultured and stained with DMSO-containing L (10 µM, DMSO : water =
4 1 : 100) for 2 h at 37 °C, the cells exhibited very faint fluorescence. However, when HepG2
5 cells were pretreated with 10 µM Zn2+, then incubated with probe L for 2 h and washed

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6 with PBS buffer, an intense response was observed, as shown in Fig. 8. The results
7 demonstrate the bioimaging application of the mixtures of probe L with Zn2+ by labeling

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8 HepG2 cells.

9 4. Conclusion

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10 In summary, we have developed a new triphenylamine substituted
11 benzimidazole-based fluorescent probe for sensing Zn2+. It exhibits a fluorescence response
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toward Zn2+ in HEPES buffer (pH =7.4) solution with high sensitivity and selectivity.
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13 Moreover, the microscopy imaging experiments show that the new probe is cell permeable
14 and can be used for imaging Zn2+ in living cells.
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15 Acknowledgements
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16 This work was supported by the Doctoral Program Foundation of the Ministry of
17 Education of China (20113401110004), the National Natural Science Foundation of China
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18 (21271003, 51472002, 51432001 and 21404001), the 211 Project of Anhui University,
19 Higher Education Revitalization Plan Talent Project of (2013).
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Highlights
1. A novel fluorescent benzimidazole derived probe was designed and

synthesized.

2. The probe showed selective and sensitive recognition toward Zn2+.

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3. The detection limit of the probe was 5.1×10-8 M for Zn2+.

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4. The probe can be used in live cell imaging.

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A novel fluorescent probe based on the flexible dipicolylamine:

recognizing zinc ( ) in aqueous solution and imaging in living cell

Na Zhanga, Xiaohe Tiana, Jun Zhengc, Xiuzhen Zhangc, Weiju Zhua, Yupeng Tiana,
Qiyong Zhub, * and Hongping Zhoua, *

a
College of Chemistry and Chemical Engineering, Anhui University and Key Labotatory of

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Functional Inorganic Materials Chemistry of Anhui Province, 230601, Hefei, P.R. China
b
Department of Chemistry, Huainan Normal College, Huainan, 232001 P.R. China
c
Center of Modern Experimental Technology, Anhui University, Hefei 230039, P.R. China

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Corresponding author. Fax: +86-551-63861279; Tel: +86-551-63861279
E-mail: zhpzhp@263.net

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Fig. S1. The 1H-NMR of M2

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Fig. S2. The 13C-NMR of M2
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Fig. S3. The ESI of M2

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Fig. S4 The 1H-NMR of L
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Fig. S5 The 13C-NMR of L

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Fig. S6 The APCI of L.
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Fig. S7. UV-vis spectra of L (10 µM) in the presence of various metal ions (10 µM) in
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aqueous solution (HEPES 20 mM, pH 7.4).

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Fig. S8. The fluorescence intensity changes of L (10 µM) solution at different pH
conditions

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Fig. S9. The time course of the response of L (10 µM) to 1 equiv. of Zn2+ in aqueous
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solution (HEPES 20 mM, pH 7.4).

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Fig. S10. Job’s plot for the determination of the stoichiometry of L and Cu2+ in
aqueous solution (HEPES 20 mM, pH 7.4) .The total concentration of L and Cu2+ was
10 µM.
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Fig. S11. Fluorescence spectra of L (10 µM) on addition of different amount of Cu2+

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in aqueous solution (HEPES 20 mM, pH 7.4).

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Fig. S12. (a) Benesi-Hildebrand plot of L-Cu2+ complexes in aqueous solution

(HEPES 20 mM, pH 7.4); (b) Calibration curve of L-Cu2+ in aqueous solution
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(HEPES 20 mM, pH 7.4).

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Fig. S13. The fluorescence response of probe L (10 µM) upon addition of various
metal ions (10 µM) in and without the presence of Cu2+ (10 µM) in aqueous solution
(HEPES 20 mM, pH 7.4).