A novel dual-channel Schiff base fluorescent chemo-sensor for Zn2+ and Ca2+ recognition: Synthesis, mechanism and application

Article abstract of DOI:10.1016/j.dyepig.2019.107614

A novel multifunctional chemo-sensor (RHCS-NH₂), incorporating Schiff base, rhodamine B hydrazide, hydrazine hydrate group into triazine, was successfully designed and synthesized. It was found that the RHCS-NH₂ sensor displays immediate multi-response toward Ca²⁺ in CH₃CN-H₂O (v/v = 9/1, Tris-HCl pH 7.0) and Zn²⁺ in DMF-H₂O (95/5, v/v). Under the optimized conditions, the linear range of identifing Ca²⁺ ions are from 0.01 to 10.0 μM in CH₃CN-H₂O (v/v = 9/1, Tris-HCl pH 7.0), 0.03–10.0 μM for Zn²⁺ ions in DMF-H₂O (95/5, v/v), and the detection limit were measured to be 0.10 nM for Ca²⁺ and 0.14 nM for Zn²⁺, respectively. The reaction mechanism between RHCS-NH₂ and metal ions was investigated in detail based on fluorescence titration, Job's plots and ¹H NMR titration curve, ESI-MS measurement and theoretical calculation respectively. Simultaneously, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays and inverted fluorescence microscopy imaging also demonstrated that the probe has well cell membrane permeability and hypotoxicity, indicating its potential applicability in vitro.

Full text of DOI:10.1016/j.dyepig.2019.107614

Accepted Manuscript
2+
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A novel dual-channel Schiff base fluorescent chemo-sensor for Zn and Ca
recognition: Synthesis, mechanism and application
Gang Zhao, Binyuan Guo, Gang Wei, Shanyi Guang, Zhengye Gu, Hongyao Xu
PII:
S0143-7208(19)30890-3
DOI:
https://doi.org/10.1016/j.dyepig.2019.107614
Article Number: 107614
Reference:
DYPI 107614
To appear in: Dyes and Pigments
Received Date: 21 April 2019
Revised Date: 1 June 2019
Accepted Date: 2 June 2019
Please cite this article as: Zhao G, Guo B, Wei G, Guang S, Gu Z, Xu H, A novel dual-channel Schiff
2+
2+
base fluorescent chemo-sensor for Zn and Ca recognition: Synthesis, mechanism and application,
Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107614.
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A novel dual-channel Schiff base fluorescent chemo-sensor for Zn and
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Ca recognition: synthesis, mechanism and application
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Gang Zhao , Binyuan Guo , Gang Wei , Shanyi Guang , Zhengye Gu , Hongyao Xu
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Chemistry, Chemical Engineering and Biotechnology & State Key Laboratory for Modification of Chemical
Fibers and Polymers, Donghua University, Shanghai 201620, China
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Research Center for Analysis and Measurement, College of Materials Sciences and Engineering, Donghua
University, Shanghai 201620, China
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*Corresponding Authors: hongyaoxu@163.com, syg@dhu.edu.cn,
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ABSTRACT: A novel multifunctional chemo-sensor (RHCS-NH ), incorporating Schiff base,
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rhodamine B hydrazide, hydrazine hydrate group into triazine, was successfully designed and
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synthesized. It was found that the RHCS-NH sensor displays immediate multi-response toward Ca
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in CH CN-H O (v/v=9/1, Tris-HCl pH 7.0) and Zn in DMF-H O (95/5, v/v). Under the optimized
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conditions, the linear range of identifing Ca ions are from 0.01 to 10.0 µM in CH CN-H O
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(v/v=9/1, Tris-HCl pH 7.0), 0.03~10.0 µM for Zn ions in DMF-H O (95/5, v/v), and the detection
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limit were measured to be 0.10 nM for Ca and 0.14 nM for Zn , respectively. The reaction
mechanism between RHCS-NH and metal ions was investigated in detail based on fluorescence
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titration, Job’s plots and H NMR titration curve, ESI-MS measurement and theoretical calculation
respectively. Simultaneously, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT)
assays and inverted fluorescence microscopy imaging also demonstrated that the probe has well cell
membrane permeability and hypotoxicity, indicating its potential applicability in vitro.
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Keywords: multifunctional, Zn , Ca , Schiff base, in vitro, fluorescence sensor
1. Introduction
Development of fluorescent probes or chemo-sensors for selective recognition of metal ions is of
considerable interest because of its practical applications in the field of chemistry, medicine, biology
and environment[1-11]Error! Bookmark not defined.. Among various chemo-sensors, the design of
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novel sensors for quantitative and selective detection of biologically important cations, such as Ca
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and Zn which is of extreme abundant and important ions in the course of human physiology. In

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previous publications, most probes often play a role as single ion receptor such as Ca , which is the
intracellular divalent cation with the largest concentration variations and evolved in variety of
biological phenomena and often serve as a second messenger in signaling pathway[12, 13]. For
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example, the intracellular Ca concentration is related to physiological responses in obesity, as well
as immune responses and pathological responses in Alzheimer’s disease[14, 15]. While, for the zinc
ion as the abundant metal ion for supporting life, its insufficient or excessive intake in body will
result in a booming amounts of disease such as Parkinson’s disease, Friedreich’s ataxia, and
Alzheimer’s disease[16-23].
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Therefore, the design and synthesis of highly selective and sensitive Ca and Zn chemo-sensors
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has been of great interest. Zhang, et al.[24], developed an efficient “Turn-on” fluorescent probe for
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detection of Ca ions, the probe exhibited a rapid signal response time (within 25 s), a good linearity
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range and a lower detection limit (2.70×10 M). Kaur et al [25]., developed a highly selective probe
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based on imine linkage for Zn , the probe displays high selectivity over other competitive ions with
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high detection limit of 3.21 10 M. Karakaya, et al [26] reported a novel dual channel responsive
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zinc (II) probe, which can be utilized for both visual and turn-off fluorometric detection of Zn ions
in aqueous acetonitrile solution.
The research of multi-ion probe has always been a challenge owing to their significant advantage
such high efficiency and fast speed[27-29]. Recently, our group[24] has developed a multifunctional
rhodamine derivative combining triazine, Schiff base and phenolic hydroxyl group for turn-on
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fluorescent responses to Zn and Bi in CH CN-water (95/5, V/V), with a detection limit of 3.0
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nmol·L for Zn and 8.6 nmol·L for Bi . Hou, et al [30] reported an efficient single
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fluorescein-based sensor for selective colorimetric and fluorometric dual sensing of Cu and Al
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even in living cells with the detection limits of 1.47×10 mol·L and 7.32×10 mol·L
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respectively. However, simultaneous recognition of Ca and Zn were rarely reported.
Herein, a novel fluorescent chemo-sensor RHCS-NH for simultaneous detection of Ca and Zn
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with good selectivity and high sensitivity based on a dual-channel method in different solvent system
were designed and characterized. The probe performance and detailed recognition mechanism was
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investigated by fluorescence titration, Job’s plots and H NMR titration curve, ESI-MS measurement
and theoretical calculation. Simultaneously, it has been applied to the detection and analysis in
environment and living biological cells, showing a good prospect of practical application.

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2. Experimental
2.1 Materials and instrumentation
All the materials for synthesis (spectroscopic grade and analytical grade) were obtained from
commercial suppliers and used without further purification. Rhodamine B was obtained from Taitan
Reagent Co. Ltd. Hydrazine hydrate and cyanuric chloride were acquired from J&K Scientific Ltd
and used as received. 4-Amino hydrazone salicylaldehyde benzohydrazide was prepared in view of
the reported method in the literature [31]. All reactions were monitored through TCL (thin-layer
chromatography). Double deionized water was employed throughout the experiment.
Tetrahydrofuran absolute redistilled through sodium and benzophenone. Unless otherwise noted, all
reactions were performed under a nitrogen atmosphere.
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FTIR spectra of the target probe molecules in KBr disc were obtained using a Nicolet 8700
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spectrometer at room temperature in the 4000~400 cm region. H/ C NMR spectra using TMS as
an internal standard were collected on a Bruker AVANCE/DMX600 and DMSO-d as solvent.
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Elemental analysis was obtained using a PE-2400CH Elemental analytic apparatus. ESI-MS
spectrum was collected on a Finnigan DECAX-30000 LCQ Deca spectrometer. UV-vis spectra were
recorded on Lambda 35 UV/vis spectrophotometer using a 1-cm quartz cell. Fluorescence emission
spectra was collected using a PE 55 fluorescence spectrophotometer. The biological imaging was
recorded on an Olympus FV-1000 fluorescence microscope.
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Scheme. 1 Synthetic route of RHCS-NH sensor
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2.2 Synthesis of the sensors
The general synthetic process was illustrated in Scheme 1. The rhodamine hydrazide (compound 3)
was prepared according to the reported literature in previous[32]. 4-Amino hydrazone
salicylaldehyde benzohydrazide was synthesized on the basis of the reported method in the literature
with a slight modification[31].
2.2.1 Synthesis procedure of compound
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Compound 2 was first synthesized on the basis of the literature method [33]. A solution of
compound 1 (1.030 g, 4.0 mmol) and sodium bicarbonate (0.660 g, 8.0 mmol) in dry acetone (20 mL)
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under a dry nitrogen atmosphere was stirred at 0~5 C for ~10 min before adding (0.730 g, 4.0 mmol)
of cyanuric chloride. The resulting mixture was permitted to slowly cool to room temperature while
being stirred. Completion of the reaction was confirmed through TLC (silica gel, ethyl
acetate/petroleum ether 1:4), and the principal the orange precipitate was filtered-off, washed with
cold acetone and deionized water successively, and dried in vacuum to obtain compound 2 as an
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organic solid. Yield: 84%: Mp= 200-212 C; FTIR (KBr): ν=3442cm (OH); 3285 cm (NH); 1701
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cm (C=O); 3048 cm , 1609 cm , 1540 cm (Ar-H); 1521 cm , 1434 cm , 1379 cm (triazine ring).
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H NMR (600 MHz, DMSO, δ/ppm): 5.71 (2H, s), 7.73-7.82 (4H, J=6.8 Hz, m), 7.94-8.01 (4H,
J=7.0 Hz, m), 10.91 (1H, s), 11.35 (1H, s).
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2.2.2 Synthesis procedure of compound 4
The compound 4 was prepared through the reaction of compound 3 with rhodamine hydrazide in
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absolute tetrahydrofuran (THF). Rhodamine hydrazide (0.77 g, 1.48 mmol), K CO (0.612 g, 4.44
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mmol) and compound 2 (0.60 g, 1.48 mmol) were dissolved in 30 mL of dry THF and reflux 18 h
under stirring. After cooling to room temperature, the excess solvent was removed under the reduced
pressure. Completion of the reaction was confirmed by TLC (silica gel, dichloromethane and ethanol
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10:1), and the desired product was obtained as a white powder. Yield: 64%. Mp= 230-250 C; FTIR
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(KBr): v = 3435 cm (NH ); 2957 cm , 2926 cm (CH ); 1724 cm , 1620 cm (C=O); 1603 cm ,
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1566 cm , 1521 cm (C H ) ; 1271 cm (C-N). H NMR (600 MHz, DMSO, 298 K, δ/ppm) :
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1.07-1.09 (t, 12 H, J = 16.0 Hz, CH ), 2.45 (s, 1H , Ar-CH), 3.36 (q, 8 H, J = 8.0 Hz, CH ), 4.30 (s, 1
3
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H, OH), 6.95 (d ,1 H, J = 1.2 Hz, ArH), 6.92 ( s, 4H ), 7.05 (s, 3 H, ArH), 7.39-7.42 (d, 2 H, J = 2.0

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Hz, ArH), 7.41-7.42 (s, 2 H, ArH), 7.43-7.41 (d, 2 H, J = 3.0 Hz, ArH), 7.69-7.70 (s, 2 H, ArH) ,
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7.71-7.72 (s , 2 H, ArH), 9.02 (s, 2 H). C NMR (150 MHz, DMSO, 298 K, δ/ppm): 12.8, 44.3, 95.5,
108.04, 121.31, 122.94, 123.12, 123.21, 123.90, 126.31, 126.46, 128.229, 128.53, 129.08, 129.68,
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132.80, 148.90, 153.25, 166.61, 169.67, 170.61, 171.57, 188.39. MS (ESI), m/z: 829.35 [4+2H] ;
Anal. Calcd. for 4: C, 65.33; H, 5.73; N, 16.93. Found: C, 65.40; H, 5.69; N, 16.84.
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2.2.3 Synthesis procedure of target RHSC-NH sensor
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The sensor RHSC-NH was prepared by the reaction of compound 4 (0.802 g, 0.97 mmol),
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hydrazine hydrate (0.050 mmol, 0.97 mmol) and K CO (0.276 g, 2.0 mmol) dissolved in 50 mL of
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dry THF and refluxed for 24 h under dry N atmosphere. After cooling to room temperature, the
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excess solvent was removed under the reduced pressure. Completion of the reaction was confirmed
by TLC (silica gel, dichloromethane and ethanol 20:1), and the desired product was obtained as a
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white powder. Yield: 40%. Mp= 245-260 C. FTIR (KBr ): v = 3440 cm (NH ); 2920 cm (CH );
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1705 cm , 1615 cm (C=O); 1615 cm , 1510 cm (Ar-H); 1510 cm , 1100 cm (C-N); 1405 cm
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(O-H); 802 cm (C-H). H NMR (600 MHz, DMSO, 298 K, δ/ppm): 6.56 (d, 1 H), 6.75-6.95 (s, 4H),
7.35 (s, 1 H), 7.39-7.45 (m, J=7.5 Hz, 1 H), 7.50-7.62 (s, 2 H), 7.70-7.75 (m, J=6.8 Hz, 4 H),
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7.91-7.95 (s, 2 H ), 8.90 (s , 1 H) , 11.50 (s , 2H ). C NMR (150 MHz, DMSO, 298 K, δ/ppm): 12.5,
43.8, 96.5, 109.34, 121.67, 121.94, 123.53, 123.87, 123.98, 125.78, 126.67, 128.56, 128.43, 129.43,
129.54, 132.65, 148.32, 153.43, 166.62, 168.98, 170.66, 171.58, 188.54. MS (ESI), m/z: 845.35
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[RHSC-NH +Na ]; Anal. Calcd. for RHSC-NH : C, 65.68; H, 6.12; N, 20.42. Found: C, 65.75; H,
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6.23; N, 20.36.
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2.3. Procedures of metal ions
Various nitrate and chloride salts of CaCl , BaCl ·2H O, ZnCl , MnCl ·4H O, CoCl ·6H O,
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Al(NO ) ·9H O, FeCl ·6H O, CuCl ·2H O, NiCl ·6H O, Pb(NO ) , CdCl ·2.5H O, SnCl ·2H O,
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MgSO were dissolved in distilled water (100 mL) to obtain 1.0×10 M stock solutions. Probe
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RHCS-NH stock solution (1.0×10 M) were obtained in DMF and CH CN respectively.
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All the measurements were performed on the basis of the following procedure. To 10.0 mL
volumetric flasks including different amounts of metal ions, the appropriate amounts to the solution
of chemo-sensor were diametrically added by a micropipette, then diluted with deionized water to
volume. All stock solutions were obtained at room temperature, vigorously shaken for 30 s and
waited for 10 min before detection. In the selectively experiments, the test samples were prepared by

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means of adding 0.1 mL stock solution of metal ions and 0.1 mL stock solution of RHSC-NH using
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double deionized water to constant volume, respectively. Then the mixture solutions were diluted to
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10.0 mL with deionized water. During the titration experiments, 0.1 mL stock solution of Zn /Ca
in 10.0 mL volumetric flasks and was diluted with deionized water.
2.4 MTT assay
The viability of HeLa cells under different treatments were assessed by means of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell (10000/well) were
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cultured into a 96-well plate with 100 mL medium and culturing for 24 h at 37 C in humidified
environment containing 5% CO , followed by washing with 100 mL/well phosphate buffered saline
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(PBS buffer). The cells were then treated with different concentrations of sensors RHCS-NH for
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another 24 h. After each treatment, the wells were washed with PBS (pH 7.4) and 20 mL MTT (0.5
mg/mL) and 180 mL medium was added to each well and incubated for an additional 4 h. The
absorbance was measured at 490 nm using a microplate reader. Cell viability was expressed as a
percentage of the cell survival rate. All treatments were repeated 3 times.
2.5 Theoretical calculation
The HOMO-LUMO energy gap for molecule RHCS-NH and its complexed were calculated by
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Density Functional Theory (DFT) calculations at the B3LYP/6-31G/LANL2DZ level of Gaussian 09
program. The DFT based B3LYP/6-31G model was carried out only C, N, O atoms whereas
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LANL2DZ for metal ions (Ca and Zn ). The binding mode of RHCS-NH with Ca , Zn were
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further confirmed by the calculation results using DFT.
2.6 Cell culture and fluorescence imaging
Hela cells were cultured in DMEM (Gibco) Medium. All media supplemented with 10% fetal
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bovine serum (FBS) and 100 IU mL-1 penicillinstreptomycin. The cells were maintained at 37 C in
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95% humidity and 5% CO atmosphere. The cells were seeded with 100 µM Ca and Zn for 30
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min, and then washed three times with 10% FBS butter and further seeded with 100 µM RHCS-NH2
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for 30 min at 37 C respectively. The cells were washed three times with FBS and carried out
confocal fluorescent imaging.
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3. Results and discussion

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3.1 Synthesis of RHCS-NH2
The Schiff based derivation as the recognition group are effectively incorporated into the active
cyanuric chloride to stoichiometrically prepare the objective fluorescent sensor (RHSC-NH2,
Scheme 1) by simple thermodynamic control method. All molecular structures have been confirmed
to be consistent with their molecular structures by the NMR spectra, ESI-MS analyses (Supporting
information: Figure S1~S2, Figure S7~S11).
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3.2 Spectral response to metal ions and selectivity measurement as colorimetric and fluorescent probe
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The absorption and fluorescence behavior response of the sensor RHCS-NH towards various
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metal ions was investigated CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0 using Tris-HCl buffer
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solution and in DMF/H O (95/5, v/v). No noticeable change was observed in the absorption spectrum
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of RHCS-NH2 in the presence of Ba , Mg , Cu , Fe , Mn , Ni , Pb , Sn , Al , Co and Cd ,
respectively, indicating that the lactam form of RHCS-NH is retained in the solution. However,
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upon addition of Ca and Zn , a new absorption peak emerged for Ca at 377 nm in CH CN/H O
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accompanying with a significant color change from colorless to green, and for Zn at 370 nm in
DMF/H O (95/5, v/v) with an obvious color changes from colorless to blue as shown in Figure 1b
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and Figure 1d, respectively. The absorption peak at ~370 nm should be attributed to the
characteristic peak for metal ion complex of Schiff base. It clearly indicates that the sensor
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RHCS-NH is very selective toward Zn and Ca based on colorimetric measurement, respectively
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To further explore fluorescent recognition ability of sensor RHCS-NH2, the selectivity of
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RHCS-NH toward Ca in CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0) and toward Zn in
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DMF-H O (95/5, v/v) solution were carried out by recording the fluorescence spectra. RHCS-NH2
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didn’t show any fluorescence when was exited at 377 nm and 370 nm. In existence of various metal
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(Figure 1c) but Ca , no obvious fluorescence was also observed in CH CN-H O (v/v=9/1, 10.0 mM
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Tris-HCl pH 7.0) solution. The strong green fluorescence intensity was observed in DMF-H O (95/5,
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v/v) when adding the Zn ion with fluorescence emission peak at 470 nm when was exited at 370
nm (Figure 1d and Figure S3b). The results demonstrated that RHCS-NH could be used for
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selective identification the Ca and Zn in the difference solution.
For evaluating selectivity of RHCS-NH , the competition experiments were carried out. Their
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fluorescence emission peak intensity of RHCS-NH with various ion (blue bar) and containing Ca
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or Zn (red bar) at same time are showed in Figure 1e and 1f, these results exhibited that other ion to
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Ca or Zn shows almost negligible interference. That is，the RHCS-NH in CH CN-H O (v/v=9/1,
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10.0 mM Tris-HCl pH 7.0) toward Ca and in DMF-H O (95/5, v/v) for Zn display very well
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selectivity. Simultaneously, it is found that no obvious fluorescence intensity change was observed
even being tested over a period of 2 h, suggesting that the solution of sensor is very stable.
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In order to determinate the practicality of receptor RHCS-NH for Ca in CH CN-H O (v/v=9/1,
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10.0 mM Tris-HCl pH 7.0) medium, Zn in DMF-H O (95/5, v/v) system. As a probe, a real-time
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detection was necessary. The kinetic characteristic of the reaction system was studied. The results
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were shown in Figure 1g and 1h. It could be found that the fluorescence intensity of the sensor
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rapidly increased to their maximum value within 9 min at peak wavelength 446 nm for Ca in
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CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0) solution, 32 min at 470 nm for Zn in DMF-H O
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(95/5, v/v) solution, respectively, as shown in Figure 1g and 1h. Hence, RHCS-NH was a very well
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selective probe, it could be used in real-time detection of Zn and Ca in the environmental analysis.

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Figure. 1 UV-vis absorption spectrum of (a) RHCS-NH (10.0 µM) in CH CN-H O (v/v=9/1, 10.0
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mM Tris-HCl pH 7.0) (b) RHCS-NH (10.0 µM) in DMF/H O (95/5, v/v) with the presence of 1.0
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7
8
9
0
1
2
3
4
equiv. of different metal ions; Fluorescence spectra of RHCS-NH (10.0 µM) upon addition of
2
2
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
different metal ions salts (1.0 equiv.) of Ca , Zn , Ba , Mg , Cu , Fe , Mn , Ni , Pb , Sn ,
3
+
2+
2+
Al , Co and Cd (c) in CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0); (d) in DMF/H O (95/5,
3
2
2
v/v), Changes of fluorescence intensity of in the presence of 1.0 equiv.; (e) Competitive selectively
2
+
of RHCS-NH (10.0 µM) towards Ca (1.0 equiv.) in the presence of other metal ions (1.0 equiv.) in
2
CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0) solution, λ =446 nm; (f) Competitive selectively
3
2
em
2
+
of RHCS-NH (10.0 µM) towards Zn (1.0 equiv.) in the presence of other metal ions (1.0 equiv.) in
2
2
+
DMF-H O (95/5, v/v) solutions, λ =470 nm. (g) Ca as the function time (0~25 min) in
2
em
2
+
CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0, λ =446 nm), (h) Zn as the function time (0~40
3
2
ex
1
1
1
1
1
min) in DMF/H O (v/v=95/5, λ =470 nm); (i) (j)The fluorescent color changes in the presence of
2 ex
different metal ions with a hand-held 365 nm UV lamp.
3.3 Sensitivity measurement of RHCS-NH Fluorescence probe
2
3.3.1 Fluorescence titration measurement
1
1
5
6
2
+
Figure. 2 Fluorescence spectra of RHCS-NH (10.0 µM) obtained during the titration by (a) Ca
2
1
7
(0~30.0 µM) in CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0), λ =377 nm, λ =446 nm; (b)
3 2 ex em

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9
0
1
2
3
4
5
6
7
8
9
0
Zn (0~50.0 µM) in DMF-H O (95/5, v/v), λ =370 nm, λ =470 nm. The fluorescence intensity of
2 ex em
2
+
2+
Ca concentration at 446 nm in CH CN, and Zn concentration at 470 nm in DMF solution,
3
respectively; (c) Fluorescence intensity at 446 nm for RHCS-NH as a function of the concentration
2
2
+
of Ca ion in CH CN (v/v=9/1, 10.0 mM Tris-HCl pH 7.0) solutions; (d) Fluorescence intensity at
3
2
+
470 nm for RHCS-NH as a function of the concentration of Zn ion in DMF-H O (95/5, v/v)
2
2
solutions; The concentrations of their metal ions are 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0
µM, respectively.
In order to investigate recognition sensitivity of sensor RHCS-NH as a practical ion-selective
2
2
+
2+
fluorescence probe for Ca and Zn , the fluorescence titration experiments of RHCS-NH were
2
2
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
carried out systematically. As shown in Figure 2a, with the concentration increase of Ca , a
remarkable improvement of the emission intensity at 446 nm occurred. When the concentration of
2
+
Ca ions increased up to 1.0 equiv., the fluorescence intensity of RHCS-NH has shown negligible
2
change and almost reaches to saturation, indicating the formation of 1:1 complex between
2
+
RHCS-NH and Ca (Figure 2a) with the fluorescence quantum yield of 0.25. Similar results were
2
2
+
2+
also observed in Zn ion recognition (Figure 2b), when the concentration of Zn ions increased up
to 1.0 equiv., the fluorescence intensity of probe RHCS-NH also enhanced significantly. Upon
2
2
+
addition of Zn ion, the fluorescence intensity of probe RHCS-NH has shown negligible change
2
and almost reaches to saturation, hinting that the formation of 1:1 chelation between RHCS-NH and
2
2
+
Zn (Figure 2b) with the fluorescence quantum yield of 0.24.
2
+
2+
3.3.2 Detection sensitivity of RHCS-NH toward Zn and Ca ions
2
2
+
2+
For fluorescent detection of Ca and Zn , a linear relationship between RHCS-NH and
2
2
+
2+
2+
Ca /Zn concentration was displayed in the range of 0.01~10.0 µM for Ca in CH CN-H O
3
2
2
+
(v/v=9/1, 10.0 mM Tris-HCl pH 7.0), 0.3~10.0 µM for Zn in DMF-H O (95/5, v/v) and with a
2
2
+
correlation coefficient (R) of 0.996 and 0.998, respectively. The regression equation is F/F (Ca in
0
−
6
−1
CH CN-H O, v/v=9/1, 10.0 mM Tris-HCl pH 7.0, 446 nm) = 21.7560+1.93457c (10 mol·L ),
3
2
2
+
−6
−1
F/F (Zn in DMF-H O, 470 nm) =0.8553+0.78438 c (10 mol·L ). On the basis of the definition
0
2
of detection limit, three times of average deviation of relative fluorescence intensity (F/F ) of the
0
2
+
2+
intensity at 470 nm for Zn in CH CN-H O, 450 nm for Ca in DMF-H O (95/5, v/v) in 20 blank
3
2
2
2
+
samples with metal ions, the limit detection of RHCS-NH -Ca in CH CN-H O (v/v=9/1, 10.0 mM
2
3
2

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8
−
1
2+
−
8
−
1
1
2
3
4
5
Tris-HCl pH 7.0) is up to 1.0×10 mol L , RHCS-NH -Zn is up to 1.4×10 mol L , respectively
2
on the basis of 3d/S. Where d is the standard deviation of the blank solution, S is the slope of the
calibration curve (Figure 2c and Figure 2d) [34, 35].
2
+
2+
3.4 Sensing mechanism of RHCS-NH for Ca and Zn
2
6
7
2
+
Figure. 3 Job’s plot of RHSC-NH and (a) Ca in CH CN-H O (v/v=9/1, 10.0 mM Tris-HCl pH 7.0)
2
3
2
2
+
8
9
0
1
2
solution, λ =446 nm, (b) Zn in DMF-H O (95/5, v/v) solution, λ =470 nm. The total
em
2
em
1
concentration of RHCS-NH and the metal ions were 10.0 µM; Partial H NMR spectra of (c) probe
2
2
+
1
1
1
RHCS-NH (5.0 mM) and increasing concentration of Ca in CH CN/D O (v/v=9/1, 10.0 mM
2 3 2
2
+
Tris-HCl pH 7.0) solution, (d) probe RHCS-NH (5.0 mM) and increasing concentration of Zn in
2
2
+
2+
DMF/D O solution (v/v=95/5), (e) The coordination mechanism of RHCS-NH for Ca and Zn .
2
2

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2
Based on Stern-Volmer theory[36]，for confirming the probe mechanism of RHCS-NH toward
2
2
+
2+
Ca and Zn , the Job’s plot experiments were carried out. The fluorescence intensity of RHCS-NH
2
next to maximum when the molar ratio of third complexes system was 0.5, revealing 1:1 ratio for
two system complexes. Thus, Job’s plot experiments were made based on the total concentration of
RHCS-NH and the metal ions were 10.0 µM.
2
In the light of the fluorescence titration experiments (Figure 2a and 2b) and the Job’s plot
experiment (Figure 3a and 3b), the binding stoichiometry calculated by Benesi-Hildebrand
5
-1
2+
method[37] was 1.55×10 M for RHCS-NH and Ca in CH CN-H O (v/v=9/1, 10.0 mM
2
3
2
5
-1
2+
Tris-HCl pH 7.0) solution, 1.05×10 M for RHCS-NH and Zn in DMF-H O (95/5, v/v) solution,
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
respectively (Figure S4a and S4b).
To further verify the assumptive mechanism, the FTIR spectra were obtained (Figure S5a and
-1
S5b). The peak at 1705 cm belonging to carbonyl absorption of RHCS-NH , shifted to low
2
-1
2+
2+
-1
frequency (near 1620 cm ) after chelating with Zn or Ca ions. The peaks at 3440, 1405 cm ,
which are ascribed to the hydroxyl absorption of RHCS-NH (Ar-OH), shifted to low frequency with
2
2
+
2+
a weaker band intensity when the system was added with Zn or Ca ions. Moreover, the peak of
bending vibration (Ar-OH) shows blue shift and the peak intensity becomes stronger, displaying that
2
+
2+
the amide carbonyl oxygen of RHCS-NH is included in the coordination of Zn /Ca ions. Hence,
2
on the basis of the above results, it could be inferred that stronger interactions between metal ions
and the amide carbonyl and hydroxyl groups of Schiff based have happened.
In order to further explore the probing mechanism for the different association behavior of sensor
2
+
2+
1
toward Ca /Zn and complexation, H NMR titration experiments using sensor RHCS-NH were
2
2
+
conducted under the same conditions. For the RHCS-NH +Ca complex in CH CN-d /D O (9/1, v/v)
2
3
3
2
2
2
3
4
solution (Figure 3c and Figure 3d). RHCS-NH have two significant signals at 9.00 (position H )
2
a
and 6.17 (position H ) ppm, being ascribed to Ar-OH and N=CH groups respectively (Figure 3c).
b
2
+
2
2
2
2
2
5
6
7
8
9
The hydroxyl (-OH) (Ha) proton of Schiff moiety is located at 9.00 ppm in the absence of Ca in
2
+
CH CN-d /H O solution; upon addition of Ca ion, the -OH proton peak shifted downfield (9.30
3
3
2
ppm), which is ascribed to the reduction of the electron-cloud density of the Schiff-rhodamine
according to the coordination of meal ions. Meanwhile, the proton of benzene ring next to Schiff
base group shifts to downfield from δ 7.27 to δ 7.31 by means of δ 0.04 ppm, while the proton for

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+
1
2
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4
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9
0
1
2
3
4
5
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7
8
9
benzene-ring of rhodamine unit shows no change, indicating that Ca was complexed with O atom
in C=O group, N atom in C=N group and O atom in Ar-OH group as shown in Figure 3e. Compared
with the CH CN-d /H O solution system, similar phenomena were observed in DMF-d /H O system
3
3
2
7
2
2
+
for Zn as shown in Figure 3c and Figure 3d. It could be found that the proton of hydroxyl (-OH)
2
+
2+
group disappeared upon the addition of metal ions (Zn or Ca ), which may be ascribed to
2
+
deprotonation and the formation of metal coordination Zn complex. Namely, the deprotonated
oxygen group is participated in the complex process, which strongly promotes the ICT process from
the donor Schiff group to the acceptor Schiff-rhodamine moieties along with a red-shift of
fluorescence spectra. The upfield shift could be attributed to a through-bond effect[38, 39]. Therefore,
2
+
2+
1
1
1
1
1
1
1
1
1
1
a binding model for the probe complexation mechanism RHCS-NH +Ca and RHCS-NH +Zn was
2 2
2
+
2+
proposed, as shown in Figure 3e. Namely, the recognition ability of sensor toward Ca or Zn is
attributed to Stable five-membered condensed ring formation between metal ion and O or N of Schiff
base receptor in sensor molecules.
Additionally, ESI-MS analysis spectra were also carried out and the results were shown in Figure
S9~Figure S11. The peaks at 934.93 and 959.91 (the calculated values are 934.96 and 959.96 can be
2
+
+
2+
+
assigned to [RHCS-NH +Ca +2Cl+H ] and [RHCS-NH +Zn +2Cl+H ]. These results further
2
2
2
+
support a 1:1 stoichiometry for the RHCS-NH -Ca in the CH CN-H O (v/v=9/1, 10.0 mM
2
3
2
2
+
Tris-HCl pH 7.0) system, RHCS-NH -Zn in the DMF-H O (95/5, v/v) medium. The ESI-MS
2
2
2
+
results further sustain proposed complexation mechanism of the RHCS-NH -Ca
and
2
2
+
2
2
0
1
RHCS-NH -Zn as showed in Figure 3e.
2
Theoretical calculations (DFT) were performed for further verifying the linking mode between the
2
+
2+
2
2
2
2
2
2
2
2
3
3
2
3
4
5
6
7
8
9
0
1
probe molecule and Ca or Zn . The structures and energy levels of RHCS-NH before and after
2
2
+
2+
binding with Ca or Zn were displayed in Figure 4, which were optimized based on the
2
+
B3LYP/LANL2DZ in the Gaussian 09 [40]. From the optimized structure of RHCS-NH -Ca
2
2
+
complex (Figure 4a), it can be found that the distance of Ca to N atom in O atom in C=O group,
2
+
C=N group and O atom in –OH group were 2.25 Å, 2.58 Å and 2.42 Å, the distances of Zn to O
atom in C=O group, N atom in C=N group and O atom in -OH group were1.84 Å, 2.07 Å and 2.19 Å,
respectively, which meet the prerequisite to form stable coordination bonds totally. The same
2
+
2+
conclusion could be drawn from the optimized structure of RHCS-NH -Ca and RHCS-NH -Zn
2
2
2
+
2+
complex, respectively (Figure 4a and Figure 4b). Without Ca or Zn ions, RHCS-NH is spread
2
Schiff groups, whose electronic cloud in LUMO mainly distributes on the Schiff moiety. Once

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+
2+
1
2
addition of Ca or Zn , the electronic cloud in LUMO is distributed in the Schiff moiety. The
2
+
energy gaps (
△ E) between HOMO and LUMO for RHCS-NH and RHCS-NH -Ca or
2 2
2
+
3
4
5
6
7
8
RHCS-NH -Zn are 4.2166 eV, 3.5042 eV, and 2.4785 eV, respectively, suggesting that the binding
2
2
+
2+
of Ca or Zn to RHCS-NH decreases the HOMO-LUMO energy gap of the complex and
2
stabilizes the system. Thus, these results show a favorable complexation interaction according to
proposed coordination. structure based on experimental results. The theoretical calculation results are
well consistent with that from experimental.
9
0
1
1
1
1
Figure. 4 Conformation of optimized by density functional theory calculations at B3LYP/6-31G
2
+
2+
1
2
3
level of theory. (a) RHCS-NH -Ca ; (b) RHCS-NH -Zn ; (c) The optimized structures and Frontier
2 2
2
+
2+
molecular orbitals of FHCS (Left), RHCS-NH -Ca (Middle) and RHCS-NH -Zn (Right)
2
2
optimized at B3LYP/LANL2DZ level of theory.
1
1
1
1
4
5
6
7
3.6 Real sample analysis
To further investigated the feasibility, three kinds of environmental water samples from the Tap
water, mineral water and lake water were used to verify the application of probe in environmental

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9
0
1
analytical chemistry. For these, 10.0 µM FHCS standard solution was prepared in CH CN/H O
3
2
2
+
2+
(v/v=9/1) and EtOH/H O (v/v=99/5). Then Ca (3.0, 4.0 and 5.0 µM) and Zn (3.0, 4.0 and 5.0 µM)
2
samples with various concentrations were added and mixed adequately, whose fluorescent spectra
were recorded and repeated 5 times. The resultant metal ion contents calculated from the regression
equations above were given in Table 1 and Table 2. From Table, it is easy to find that the recoveries
2
+
2+
and relative standard deviations (R.S.D.) for sensing Ca or Zn are all satisfactory, i.e.,
2
+
2+
98.2%~102.1% and ≤5.0 % for Ca , 95.5%~104.1% and ≤6.8 % for Zn respectively. The analytic
results well confirm the reliability and practicality for the present RHCS-NH to dual-channel
2
2
+
2+
simultaneously recognize Ca and Zn .
1
1
a
Table 1. Fluorescent determination results for environment samples (n=5)
2
+
–6
2+
–6
Samples
Ca Spiked (10 M)
Ca Found (10 M)
Recovery (%)
100.6
R.S.D （. %）
Tap water 1
Mineral water
Lake water 1
3.0
4.0
5.0
3.02
4.08
4.91
2.8
5.0
4.2
102.1
98.2
1
1
2
3
a) λ =377 nm, cRSHT-NH2=10.0 µM, V
3
:V
2
=9:1
ex
CH CN H O
Table 2. Fluorescent determination results for environment samples (n=5)
2
+
–6
2+
–6
Samples
Zn Spiked (10 M)
Zn Found (10 M)
Recovery (%)
95.5
R.S.D （. %）
Tap water 1
Mineral water
Lake water 1
3.0
4.0
5.0
2.87
4.16
4.96
3.4
6.8
5.2
104.1
99.2
1
1
1
1
1
1
2
4
5
6
7
8
9
0
b) λ =370 nm, cRSHT-NH2=10.0 µM, VDMF:V
2
=99:5
H O
ex
3.7 Cell imaging studies
2
+
2+
To further test the practical application of RHCS-NH chemical sensor for Zn and Ca in
2
o
biological assay, HeLa cells were cultured with sensor RHCS-NH (10.0 µM) for 0.5 h at 25 C,
2
nearly no fluorescence could be observed (Figure 5b). In contrast, a bright fluorescence was
2
+
2+
o
observed for those cells seeded with Zn and Ca (10.0 µM) for 0.5 h at 37 C, followed by means

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2
3
4
5
of incubation with RHCS-NH under similar conditions (Figure 5e, 5f), bright blue fluorescence for
2
2
+
2+
Zn and dark green fluorescence for Ca could be observed in the HeLa cells (Figure 5e, 5f). The
cell morphology remained in good condition after sensor RHCS-NH got inside the cells, hinting
2
good cytocompatibility and low toxicity of the sensor. As a result, it could be used as in the detection
2
+
2+
of Zn and Ca in vitro.
6
7
Figure. 5 Confocal fluorescence images of HeLa cells. (a-c) HeLa cells cultured with probe (10.0
µM) for 0.5 h; (d-f) HeLa cells pretreated with probe (10.0 µM) for 0.5 h, then incubated with 50.0
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
+
µM Ca ions for 0.5 h; (g-i) HeLa cells cultured with probe (10.0 µM) for 0.5 h, then cultured with
2
+
1
1
1
1
1
1
1
1
1
1
2
2
50.0 µM Zn ions for 0.5 h. (a, d, g): Bright field images; (b, e, h): Fluorescence images; (c, f, i):
Merged images.
4. Conclusions
2+
In summary, a new dual-channel chemo-sensor RHCS-NH for simultaneous detection of Ca and
2
2
+
Zn was designed and prepared. Their structures (Intermediates and target molecule) were
2
+
characterized. The high sensitivity and good selectivity for Ca in CH CN-H O (v/v=9/1, 10.0 mM
3
2
2
+
Tris-HCl pH 7.0) and Zn in DMF-H O (95/5, v/v) system were achieved by strong fluorescence
2
enhancement (turn-on) method, respectively, which have the detection limit of the sensor RHCS-NH2
2
+
2+
toward Ca and Zn are 0.10 nM and 0.14 nM, respectively. The detailed recognition mechanism
1
was investigated by fluorescence titration, Job’s plots and H NMR titration curve, ESI-MS
measurement and theoretical calculation, respectively. Additionally, the experiment for real water

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sample and cell imaging shows good cytocompatibility and low toxicity, hinting that it could be used
2
+
2+
as in the detection of Zn and Ca in environment and vitro.
Acknowledgements
This work is financially supported from the Natural Science Foundation of China (No. 21771036
and 21671037). Thank Yizhe Chen from Shanghai Pinghe Bilingual School for his Participation and
contribution.
Reference
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Highlights:
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A new dual-channel Schiff based materials (RHCS-NH ) was designed and prepared >The sensor
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RHCS-NH can be applied as a fluorescent probe for the detection of Zn ions and Ca >The sensor
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can be applied in environment and living biological cells for detect Zn and Ca > The recognition
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ability of sensor toward Ca or Zn is attributed to Stable five-membered condensed ring formation
between metal ion and O or N of Schiff base receptor in sensor molecules.>