Coumarin-based colorimetric-fluorescent sensors for the sequential detection of Zn2+ ion and phosphate anions and applications in cell imaging

Journal Pre-proof Coumarin-based colorimetric-fluorescent sensors for the sequential detection of Zn2+ ion and phosphate anions and applications in cell imaging

Jiaxin Fu, Kun Yao, Bai Li, Huihui Mei, Yongxin Chang, Kuoxi Xu PII:

S1386-1425(19)31180-1

DOI:

https://doi.org/10.1016/j.saa.2019.117790

Reference:

SAA 117790

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

16 August 2019

Revised date:

6 November 2019

Accepted date:

11 November 2019

Please cite this article as: J. Fu, K. Yao, B. Li, et al., Coumarin-based colorimetricfluorescent sensors for the sequential detection of Zn2+ ion and phosphate anions and applications in cell imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2019), https://doi.org/10.1016/j.saa.2019.117790

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© 2019 Published by Elsevier.

Journal Pre-proof Coumarin-based colorimetric-fluorescent sensors for the sequential detection of Zn2+ ion and phosphate anions and applications in cell imaging Jiaxin Fu, KunYao, Bai Li, Huihui Mei, Yongxin Chang, Kuoxi Xu College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, Henan, China

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Abstract: Two novel coumarin based fluorescent sensors CHP and CHS have been synthesized for the sequential detection of Zn2+ ion and phosphate anion (PA) in DMF/HEPES buffer medium (1/5 v/v, 10 mM, pH=7.4). On the addition of Zn2+ ion to the solution of CHP or CHS resulted in a pronounced fluorescence enhancement, accompanying noticeable color change (under UV or daylight), while there was hardly obvious change with other competing metal ions co-existing. The detection limits (DL) of CHP and CHS towards Zn2+ were separately determined as 1.03×10−7 (R2 = 0.9886) and 1.87×10−7 (R2 = 0.9902). The PET binding processes were affirmed by spectroscopic techniques, HRMS experiments and theoretical calculations. Subsequently, the CHP-Zn2+ or CHS-Zn2+ complexes showed high selectivity fluorescence quenching toward PA by snatching Zn2+ ion from its complex and the binding processes were reversible. DLs were calculated as 2.07×10−7 M (R2 = 0.9928) and 2.63×10−7 M (R2 = 0.9954), respectively. Furthermore, the cell imaging experiments demonstrated that the sensors were capable of detecting of Zn2+ and PA in vitro cells.

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1. Introduction

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Keywords: fluorescent sensor; coumarin; Zn2+; phosphate anion; cell imaging

As we all know that appropriate levels of metal ions and anions play an

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indispensable role in the tissue structures and physiological responses in living organisms [1-3]. The development of detecting various metal ions and anions in environment and biological system has rapidly increased in chemical and biological technology [4, 5]. Some standard instrumental techniques like ion chromatography (IC) [6], atomic absorption spectrometry (AAS) [7], inductively coupled plasma mass spectrometry (IPC-MS) [8] and liquid chromatography [9] are expensive and not suitable for many applications. It becomes important to find alternative ways for the analyte determination. The fluorescence spectroscopy is an alternative method due to its advantages, such as low cost, simple operation, high selectivity and selectivity, real-time detection [10-14]. Hence, it is significant to design fluorescence sensors for detecting analyte, especially “naked eye” detection sensors. 

Corresponding author. Tel.:/Fax: +86 37123881589. E-mail addresses: [email protected]. 1

Journal Pre-proof Zinc (Zn2+) is one of the essential trace metals in living creatures [15]. Appropriate content of zinc plays an indispensable role in the organization and physiological response of organisms [16-19]. Excessive intake of Zn2+ in the human body can lead to prostate cancer, Alzheimer's disease, and neurological diseases [20, 21]. But the lack of Zn2+ in the human body can lead to cognitive impairment, immune dysfunction, diarrhea and death, especially for children under 5 years of age [22-24]. On the other hand, phosphate anions maintain the stability of the body's environment and the normal functioning of cellular functions in the human body, including cell

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metabolism and energy transfer [25, 26]. Excessive or insufficient phosphate in body

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fluids can cause diseases such as muscle weakness, impaired white blood cell function, rickets, rickets, etc. [27-30]. According to the strong covalent interaction of the

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targeted anions with metal ions, we can select the appropriate metal complexes to

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detect the anions indirectly. It is a strategy to detect anions [31]. Zn2+ complex is a common sensor for phosphate anions, mainly because phosphate is a reversible

lP

inhibitor of Zn2+ in prostate tissue [32].

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In present study, two sensors based coumarin were synthesized simply, which contained -C=N- to enhance the ability of binding metal ions and the system of

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conjugation. As a result, free sensor displayed weak fluorescence due to -C=Nisomerization, while specific metal ions combined, isomerization process was crippled so that fluorescence released. The literatures [33-39] towards Zn2+ recently published were taken to compare with this study (Table 1.), which started with media, association constant (Ka), detection limit (DL) and applications. Firstly, monitoring the guest in pure aqueous solution has always been the goal of the researchers, and some media still remain in the pure organic solvent phase, and we have chosen other less toxic aqueous media. Moreover, Ka and DL have been reached the upper middle level. Finally, our sensors were applied by more methods to prove their practical abilities.

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Journal Pre-proof Table 1. Comparison of the properties of CHP and CHS with recently reported Zn2+ sensors Entr y

Probe

Media

DMSO-

1

HEPES

2

MeOH

Ka

DL (M)

Application

Reference

/

6.9×10-7

Cell imaging

33

2.0×105

2.4×10-6

/

34

(M-1)

Anion

HEPES

3.97×10

of

3

recognition; 5

5.6×10

-8

Detection

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35

DNA

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MeOH-

HEPES

DMF-

5

HEPES

na

6

buffer

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7

1.47×10-5

lP

H2O

1.68×105

8

DMSO

2.1×10-8

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MeOH-

4

-p

Amplification

3.9×104

1.1×10-7

6.2×10-8

Cell imaging

On-Site Analysis

Cell imaging

36

37

38

Anion 1.39×10

5

-7

1.40×10

recognition;

39

Cell imaging 3.33×104

1.03×10-7

DMF-

Logic function; On-site analysis Anion

HEPES 8.11×103

1.87×10-7

This work

recognition; Cell imaging

2. Experimental 2.1 Materials and instrumentations The chemicals used in the experiment are all marketed products (analytical grade) without secondary purification. 1H NMR and

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C NMR data were recorded on a

Bruker AV-400 MHz NMR spectrometer using DMSO-d6 solvent. FT-IR spectroscopy 3

Journal Pre-proof was performed using a Nicolet 670 FT-IR infrared spectrophotometer using KBr pelleting. Mass spectrometry data was recorded by a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Fluorescence spectra and UV absorption spectra were determined by F-7000FL fluorescence spectrophotometer and Hitachi U1900 UV spectrophotometer, respectively. Melting point was determinated by MEL-TEMP melting point apparatus. The intermediate 7-hydroxycoumarin hydrazone was synthesized according to the literatures [40].

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2.2 Synthesis of sensor CHP and CHS The synthesis of sensors CHP and CHS are depicted in Scheme 1. Compound

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7-hydroxycoumarin hydrazine (1.02 g, 5 mmol) was dissolved in 20 mL of ethanol

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and heated to reflux. Then a solution of 2-pyridinecarboxaldehyde (0.80 g, 7.5 mmol) dissolved in 10 mL of ethanol was gradually dropped into the flask. Stir at 65 °C for

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10 h under the protection of nitrogen until a pale yellow solid precipitated. Then the

lP

sediment was filtered and washed with ethanol to give 1.12 g of CHP (Yield: 76 ％, m.p. 263.56-264.23 ℃), 1H NMR δ 12.44 (s, 1H), 9.28 (s, 1H), 8.87 (s, 1H), 8.75 (d,

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J=4 Hz, 2H), 8.20 (d, J = 12Hz, 1H), 8.06 (d, J = 16 Hz, 1H), 7.97 (t, J = 16 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 20 Hz, 1H), 7.04 (d, J = 12 Hz, 1H), 6.40 (d, 13

C NMR δ 164.10, 162.84, 159.72, 154.76, 152.44, 150.55,

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J=12Hz, 1H) (Fig. S1);

145.09, 137.61, 133.86, 126.40, 122.43, 114.26, 112.82, 111.73, 105.59 (Fig. S2); IR (KBr): 3430, 2426, 1724, 1612, 1235, 1117, 992, 780 cm−1 (Fig. S3); HRMS: calcd for: [CHP+H+]: 294.0873; Found: 294.0878 (Fig. S4). The same way to get 1.05 g of CHS as a yellow solid, Yield: 68％, m.p. 246.35-247.36 ℃, 1H NMR δ 12.57 (s, 1H), 10.90 (s, 1H), 9.31 (s, 1H), 9.10 (s, 1H), 8.05 (d, J = 12 Hz, 1H), 7.77 (t, J = 20 Hz, 2H), 7.44 (t, J = 20 Hz, 1H), 7.00 (q, J = 28 Hz, 3H), 6.38 (d, J = 12 Hz, 1H) (Fig. S5);

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C NMR δ 163.78, 162.76, 159.73,

158.42, 154.59, 145.10, 134.16, 133.49, 130.95, 120.12, 118.63, 117.12, 114.21, 112.77, 111.73 (Fig. S6); IR (KBr): 3448, 3085, 1738, 1618, 1483, 1235, 1108, 991, 752, 447 cm−1 (Fig. S7); HRMS: calcd for: [CHS+H+]: 309.0870; Found: 309.0872 (Fig. S8). 4

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Scheme 1. Synthesis of compound CHP and CHS.

2.3 Spectroscopic study Compound CHP or CHS were formulated into a 0.1 M solution in DMF. Then it

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was diluted to 3.33×10−5 with a mixed solution of HEPES buffer solution (pH=7.4)

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and DMF solution (1:5 v/v, 10 mM, pH=7.4) before the spectral experiment. The stock solutions of various metal ions (Na+, K+, Zn2+, Mn2+, Ni2+, Ba2+, Pb2+, Hg2+,

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Fe2+, Cd2+, Al3+, Ca2+, Ag2+, Cr3+, Mg2+, Cu2+, Fe3+ and Co2+ ions) were used for their

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nitrates. Anions (F−, Cl−, Br−, I−, SO42−, SO32−, SH−, S2−, NO3−, CO32−, CH3COO−, HCO4−, HSO3−, SCN−, HPO42−) stock solutions were used their sodium salt in

lP

distilled water to configure 1.00×10−3 M. (Due to inorganic phosphate anion (PA) are present with the ratio H2PO4−/HPO42− of 1/4 in human physiology pH [41, 42],

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HPO42− is taken as the research analyte in this study). The spectral changes of the

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mixed solutions of sensors CHP or CHS with various ions were studied by fluorescence and UV absorption spectroscopy at room temperature. The fluorescence emission of CHP and CHS were recorded with excitation at 420 nm.

2.4 MTT assay

The cytotoxicity of CHP or CHS was researched by MTT assay according to reported manner. The PC12 cells were grown Dulbecco's Modified Eagle's medium with containing fetal bovine serum (10 %) at 37 °C and 5% CO2. Then the cells were wished washed with PBS 3 times and incubated with various concentrations of CHP and CHS(0, 20, 40, 60, 80, 100 μM) in 96-well plates at 37 °C for 1 h, respectively. Subsequently, the solution (10 mg mL-1 in PBS buffer) were added in each well plats and incubated at 37 °C for 5 h. The formazan was solubilized in DMSO and the fluorescence of cells was tested enzyme-linked immunosorbent assay reader. 5

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3. Result and discussion 3.1 Spectral characteristics of CHP and CHS toward Zn2+ The selectivity was first investigated of CHP and CHS to different metal ions in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4) (Fig. 1). The compound CHP showed weak fluorescence emission maybe due to the photoinduced electron transfer (PET) process [43] between the imine group and the coumarin group. Then addition of

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different metal ions (3.0 equiv. of Na+, K+, Zn2+, Mn2+, Ni2+, Ba2+, Pb2+, Hg2+, Fe2+, Cd2+, Al3+, Ca2+, Ag2+, Cr3+, Mg2+, Cu2+, Fe3+ and Co2+ ions) to the solution of CHP,

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only Zn2+ caused obvious fluorescent enhancement at 533 nm with the color change

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from colorless to yellow. Also for CHS, the fluorescence emission at 510 nm was enhancement in the presence of Zn2+ with the change of color from colorless to yellow.

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In contrast, the fluorescence spectra of the sensors CHP and CHS did not change

lP

significantly in the presence of other metal ions. These results indicated that the sensors CHP and CHS could be used as fluorescent sensors with a “turn-on”

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fluorescence change for Zn2+ accompanied by “naked eye recognition”. The ability was also explored of sensors CHP and CHS to detect Zn2+ in the

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presence of other metal ions. When 3.0 equiv. of Zn2+ and 3.0 equiv. of other metal ions were added into the sensor CHP or CHS, most of these metal ions did not lead to obviously changes in the detection of Zn2+ (Fig. S9). In general, CHP and CHS have good anti-interference performance for detecting Zn2+ in DMF/HEPES buffered solution (5:1 v/v, 10 mM, pH=7.4).

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(B)

Zn2+

300

Fluorescence Intensity(a.u.)

Fluorescence Intensity(a.u.)

(A)

250

200

150

CHP and Li+, Na+, K+, Ag+, Ba2+ Mn2+, Ni2+, Cr3+, Al3+, Cd2+, Ca2+

100

Pb2+, Hg2+, Co2+, Fe3+, Fe2+, Cu2+ Mg2+

50

0 450

500

550

600

650

250

Zn2+

200

150

100

CHS and Li+, Na+, K+, Ag+, Ba2+ Mn2+, Ni2+, Cr3+, Al3+, Cd2+, Ca2+ Pb2+, Hg2+, Co2+, Fe3+, Fe2+, Cu2+ Mg2+

50

0

700

450

500

Wavelength(nm)

550

600

650

Wavelength(nm)

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Fig. 1. Fluorescent spectra of (A) CHP and (B) CHS (3.33×10−5 M) with various metal ions (3.0 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4). Inset: photograph of fluorescence change of CHP or CHS after addition of Zn2+.

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The UV absorption spectra of CHP and CHS with Zn2+ were explored. The Fig. 2 showed that free CHP displayed a sharp and clear absorption band at 320 nm, this was

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due to π-π* charge transfer in the sensor molecule. Upon gradual addition of Zn2+, the

lP

intensity of the UV absorption peak at 320 nm of the mixed solution gradually weakened, the UV absorption peak at 420 nm was gradually enhanced. And a distinct

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isosbestic point at 344 nm was appeared. In addition, the mixed solution of sensor CHP and Zn2+ exhibited bright yellow fluorescence under 365 nm UV light (see inset

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of Fig. 2), which was caused by the chelation-enhanced fluorescence (CHEF) [44] effect between Zn2+ and CHP. In the same way, absorption peak of CHS displayed a sharp band at 316 nm, after addition of Zn2+ to the solution of CHS, the band at 316 nm was gradually diminished and red-shifted to 350 nm. However, the absorption peak of CHS around 420 nm was gradually enhanced with a new isosbestic point at 346 nm (Fig. S10). The illustration also showed CHS and Zn2+ mixed solution showing bright green fluorescence under 365 nm UV light (see inset of Fig. S10). All the results indicated CHP and CHS with Zn2+ interact to form stable complex, respectively.

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1.6 1.4

Absorbance

1.2 1.0 0.8 0.6 0.4 0.2 0.0 300

400

500

Wavelength(nm)

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Fig. 2. UV–vis spectra of CHP (3.33×10−5 M) upon addition of Zn2+ (0-2.8 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4). Inset: photograph of daylight change of CHP after addition of Zn2+.

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The fluorescence properties of CHP and CHS with Zn2+ were further investigated by fluorescence titration experiments, respectively. As shown in Fig. 3A, upon

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gradual addition of 0 to 2.8 equiv. Zn2+ into the solution of CHP, the fluorescence

lP

emission at 533 nm was increased gradually, which meanly attributed to the PET and –C=N– isomerization processes were restrained [45]. Alike, upon addition of 0 to 2.6

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equiv. Zn2+ to the solution of CHS, a new fluorescence emission at 510 nm was appeared and enhanced approximate 215-fold (Fig. 3B). The 1:1 complex ratio

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between CHP and CHS with Zn2+ were obtained by job's plot (Fig.S11, Fig. S12) [46] and HR-MS experiments. The mass spectra were showed that the peak at m/z 418.9976 and 371.0036 was classified as [CHP+Zn2++NO3−] (Fig. S13) and [CHS+Zn2+-H+] (Fig. S14), respectively. Base on the fluorescence titrations data, the association constants of CHP-Zn2+ and CHS-Zn2+ were estimated to be 3.33×104 M−1 and 8.11×103 M−1 by using the Benesi-Hildebrand plot [47] analysis, respectively (Fig. 4). Moreover, the detection limit (DL = 3/slope) [48] of CHP for Zn2+ and CHS for Zn2+ were calculated to be 1.03×10−7 M (R2 = 0.9886) and 1.87×10−7 M (R2 = 0.9902), respectively (Fig. S15), which were much lower than the limit by WHO [49].

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(B) 2.8 eq. Zn

250

200

Fluorescence Intensity(a.u.)

Fluorescence Intensity(a.u.)

(A) 300 2+

200

150

100

50

2.6 equiv. Zn2+ 150

100

50

0

0 450

500

550

600

650

700

450

500

550

600

650

Wavelength(nm)

Wavelength(nm)

0.025

Intercept Slope

y = a + b*x No Weighting 1.23016E-5

0.035

Value Standard Error 0.00248 1.59942E-4 7.43833E-8 1.79332E-9

0.030

0.99597 0.99165

Intercept Slope

B

Value Standard Error 0.00193 1.86211E-4 2.37992E-7 4.20174E-9

-p

B

Equation Weight Residual Sum of Squares Pearson's r Adj. R-Square

(B)0.040

0.99538 0.99021

0.025

0.015

1/(I-I0)

1/(I-I0)

0.020

y = a + b*x No Weighting 4.2246E-6

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Equation Weight Residual Sum of Squares Pearson's r Adj. R-Square

(A)

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Fig. 3. (A) Fluorescence spectra of CHP (3.33×10−5 M) upon addition of Zn2+ (0-2.8 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4); (B) Fluorescence spectra of CHS (3.33×10−5 M) upon addition of Zn2+ (0-2.6 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4).

0.020

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0.010

0.015 0.010

0

50000

100000

150000

lP

0.005

200000

1/[Zn2+]

250000

300000

0.005 0

20000

40000

60000

80000 100000 120000 140000 160000

1/[Zn2+]

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Fig. 4. Benesi-Hildebrand plot for (A) CHP and (B) CHS with Zn2+ in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4) solution.

3.2 Effect of pH

In order to investigate the application value of sensors CHP and CHS in identifying Zn2+ in actual samples and biological systems, we conducted the identification of sensors CHP and CHS towards Zn2+ in the range of pH 2-12 (Fig. S16), respectively. When the pH <5 in an acidic environment, CHP and CHS were showed negligible fluorescence intensity to Zn2+, this may due to the deprotonated of coumarin was inhibited. The complexes of CHP-Zn2+ and CHS-Zn2+ maintained a good fluorescence emission at pH 7-11. These results indicated that CHP and CHS had strong fluorescence enhancement response for Zn2+ under weak alkaline conditions.

3.3 Photographs of CHP and CHS after immersion with various concentrations of Zn2+ 9

Journal Pre-proof The“naked eye” fluorescence sensors are paid much attention due to their specific fluorescence response toward the analyte. In view of the obvious fluorescence changes of CHP and CHS with Zn2+, thus, we wondered whether the relative low detection limit of CHP and CHS toward Zn2+ could be captured by the “naked eye”. Hence, various concentrations solutions of Zn2+ were mixed with CHP and CHS and took photos of the fluorescence changes of mixed solutions in visible light and 365 nm UV light, respectively. As shown in Fig. 5A, upon addition of Zn2+ in CHP solution, the color was gradually changed from colorless to yellow, and the

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fluorescence of mixed solutions were gradually enhanced. The fluorescence change

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was very obvious and could be easily observed by the“naked eye”. And the“naked eye” detection limit of CHP toward Zn2+ is 1.00×10−6 M under 365 nm UV light. As

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shown in Fig. 5B, the color and fluorescence of mixed solutions were also changed

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step and step with gradually added Zn2+ ion. The“naked eye” detection limit of CHS

lP

toward Zn2+ is also 1.00×10−6 M by the fluorescence response under 365 nm UV light. This experiment proved that, in the actual sample, the concentration range of the guest

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sample.

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to be tested in the sample can be inferred by comparison with the color of the standard

Fig. 5. (A) photographs of CHP in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4) solutions upon addition various concentrations (10−6-10−1 M) of Zn2+, above: color changes, below: fluorescence changes; (B) photographs of CHS in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4) solutions upon addition various concentrations (10−6-10−1 M) of Zn2+, above: color changes, below: fluorescence changes.

3.4 1H NMR titrations of CHP and CHS toward Zn2+ The complexation mode of CHP-Zn2+ and CHS-Zn2+ complexes were investigated by 1H NMR spectroscopy titrations. As shown in Fig 6, upon gradual addition 1.00 10

Journal Pre-proof equiv. of Zn2+ into the solution of CHP, the 1H NMR spectrum signals significant change. The proton peak (H1) at 9.28 ppm was shifted to 9.35 ppm, the proton peak of H2 did not change significantly. The results indicated that the imine nitrogen atom near the pyridine group participated in the complexation with Zn2+. Furthermore, the proton peaks H3 and H4 on the pyridine ring were shifted from 7.99 ppm and 7.56 ppm to 8.04 ppm and 7.63 ppm, respectively. The changes of these signal peaks indicated that the nitrogen atom on the pyridine ring also participated in the bonding with Zn2+. And the phenolic hydroxyl (H5) of coumarin was gradually disappeared

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which manifested the oxygen atom of phenolic was also bonded with Zn2+.Similarly,

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after adding 1.0 equiv. of Zn2+ to the solution of sensor CHS, H6 at 9.31 ppm and H7 at 9.10 ppm were shifted to 9.25 ppm and 9.04 ppm, respectively (Fig.S17). These

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results indicated that the nitrogen atoms on the imine (-C=N-) group participated in

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the complexation with Zn2+. The phenolic hydroxyls of coumarin and benzene were

lP

gradually disappeared with adding Zn2+, these results indicated that the oxygen atoms of the benzene phenolic hydroxyl group and coumarin phenolic hydroxyl participated

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in the complexation with Zn2+.

Fig. 6. 1H NMR spectrum of CHP, CHP+Zn2+ (0.5 equiv.) and CHP+Zn2+ (1.0 equiv.) in DMSO-d6 solution (from bottom to top).

3.5 Theoretical calculations The structures of sensors CHP, CHS and their complexes CHP-Zn2+, CHS-Zn2+ were optimized by the Gaussian 09 software at DFT/B3LYP/6-31G (d, p) level. The theoretical calculations were guided from the binding model which were analyzed by spectroscopy experiments. The optimized structures of CHP and CHP-Zn2+ were 11

Journal Pre-proof showed in Fig. 7 and Fig. S18, the deprotonated O atom of coumarin hydroxyl and two N atoms of pyridine and imine were coordinated with Zn2+, respectively. Furthermore, an accessional coordination bond was shaped between Zn2+ and the O atom of nitrate anion based on this experiment condition and the HRMS analysis. For CHS and CHS-Zn2+, the CHS also used the N atoms of imine and two deprotonated O atoms of phenol hydroxyl and coumarin hydroxyl to coordinate with Zn2+.The distance of the N-Zn2+ and O-Zn2+ were in the range of 1.92-2.22 Å, respectively (Fig 7, Fig. S18). These bond length accord with typical coordination bond distances [50]

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which indicated the synergetic system of CHP-Zn2+ and CHS-Zn2+ were formed.

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The electronic transitions of CHP, CHS and the CHP-Zn2+, CHS-Zn2+ complexes were further calculated by TD-DFT. For CHP and CHS, the HOMO mainly

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distributed in imine group while the LUMO were dispersed the whole CHP and CHS

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molecules. The PET process form electron donor (imine group) to the acceptor (fluorophore) were manifested by transitions of HOMO to LUMO. For CHP-Zn2+ and

lP

CHS-Zn2+ complexes, LUMO were not completely occupied the fluorophore due to

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the metal-ligand electronic transitions, which generated the PET process were restrained and fluorescence emission of CHP and CHS were distinctly enhanced. The

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calculated HOMO–LUMO energy gap of CHP-Zn2+ (2.67 eV) and CHS-Zn2+ (3.38 eV) complexes were low than the gap of CHP (3.73 eV) and CHS (3.66 eV), respectively (Fig 7, Fig. S18), which indicated that the CHP-Zn2+ and CHS-Zn2+ complexes were much stabilized than CHP and CHS, and the formation of the CHP-Zn2+ and CHS-Zn2+ complexes were energetically favorable. The theoretical calculation results were further confirmed the sensing mechanism of CHP and CHS to Zn2+ (Scheme 2 and Scheme 3).

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Fig. 7. (A) The optimized structures and frontier orbitals of CHP and CHP-Zn2+.

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3.6. Spectroscopic studies of CHP-Zn2+ and CHS-Zn2+ complexes toward phosphate anion

lP

In order to study whether the complexes CHP-Zn2+ or CHS-Zn2+ can be used as a secondary anion fluorescent sensor, the fluorescence responses were investigated of

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complexes CHP-Zn2+ and CHS-Zn2+ towards F−, Cl−, Br−, I−, SO42−, SO32−, HS−, S2−, NO3−, CO32−, CH3COO−, S2O82-, HCO3−, ClO−, SCN−, citrate, PA in DMF/HEPES

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buffer (5:1 v/v, 10 mM, pH=7.4) solution. As shown in Fig. 8, the same equiv. of different anions were added into the complexes CHP-Zn2+ and CHS-Zn2+, respectively. Interestingly, the fluorescence spectra of the mixed solutions were not significant change except for PA. When the PA added, the fluorescence of the complexes CHP-Zn2+ and CHS-Zn2+ solutions were all quenched and returned to the same fluorescence intensity as the sensors CHP and CHS. In addition, the color of the mixed solutions also changed from yellow to colorless. These results indicated that the complexes CHP-Zn2+ and CHS-Zn2+ can be used as a “turn-off” type fluorescent sensor to achieve selective detection of PA.

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350

(B) 300

250

Fluorescence Intensity(a.u.)

Fluorescence Intensity(a.u.)

(A)

CHP-Zn2+,F-, Cl-, Br-, I-, SO42-, SO32-, NO3-,CO32-, CH3COO-, HCO3-, HSO4-, ClO-, S2-, HS-, SCN-, S2O82- citrate

200

150

100

250

200

CHS-Zn2+, F-, Cl-, Br-, I-, SO42-, SO32-, NO3-, CO32-, CH3COOHCO3-, HSO4-, ClO-, S2-, SH-, SCN-, S O 2- citrate

150

100

2

8

50

50

0 450

300

HPO42-

HPO42500

550

600

650

700

0 450

500

Wavelength(nm)

550

600

650

Wavelength(nm)

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Fig. 8. (A) Fluorescent spectra of CHP-Zn2+ (3.33×10-5 M) with various anions (3.0 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4). Inset: photograph of color change of CHP-Zn2+ after addition of phosphate anion; (B) Fluorescent spectra of CHS-Zn2+ (3.33×10-5 M) with various anions (3.0 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4). Inset: photograph of fluorescence change of CHS-Zn2+ after addition of phosphate anion.

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The sensing mechanism of CHP-Zn2+ and CHS-Zn2+ toward PA were inspected in

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DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4) solution by UV absorption and fluorescence titration experiments, respectively. Fig. 9 showed, with the increasing

lP

concentrations of PA, the absorption peak of complex CHP-Zn2+ solution at 320 nm

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was gradually enhanced and the peak at 420 nm was simultaneously subdued. Eventually the peak restored to a position consistent with the free CHP UV absorption

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spectrum. Similarly, upon addition of PA into complex solution of CHS-Zn2+, the absorption peak of complex solution of CHS-Zn2+ was also regained to as free CHS (Fig. S19). In addition, the color of complexes solutions of CHP-Zn2+ and CHS-Zn2+ were quenched in the presence of PA (see insert of Fig. 9). Considering the appearance above, PA grab Zn2+ from CHP-Zn2+ or CHS-Zn2+ complexes and make sensors free.

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(A)

1.4

1.2

Absorbance

1.0

0.8

0.6

0.4

0.2

0.0 300

400

500

Wavelength(nm)

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Fig. 9. UV–vis spectra of CHP-Zn2+ (3.33×10−5 M) upon addition of phosphate anion (0-2.8 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4). Inset: photograph of daylight change of CHP-Zn2+ after addition of phosphate anion.

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The fluorescence titrations for CHP-Zn2+ and CHS-Zn2+ with PA were also performed. As shown in Fig. 10, upon gradually increased the concentration of

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HPO42−, the fluorescence emission of solutions of CHP-Zn2+ and CHS-Zn2+ were also

lP

gradually subdued, which indicated that the Zn2+ of the complexes CHP-Zn2+ and CHS-Zn2+ were “captured” by PA to release free sensors CHP and CHS. The DL for

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PA were calculated to be 2.07×10−7 M (R2=0.9928) and 2.63×10−7 M (R2=0.9954) based on fluorescence titrations data with 3/slope formula [51], respectively (Fig.

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S20). The sensing mechanisms of CHP or CHS with Zn2+ and HPO42− were proposed combined with the analysis of above results (Scheme 2 and Scheme 3). 300

250

(B) 200

Fluorescence Intensity(a.u.)

Fluorescence Intensity(a.u.)

(A)

200

150

2.8 eq. HPO42-

100

50

0

150

2.0 eq. HPO42-

100

50

0

450

500

550

600

650

700

450

500

550

600

650

Wavelength(nm)

Wavelength(nm)

Fig. 10. (A) Fluorescence spectra of CHP-Zn2+ (3.33×10−5 M) upon addition of phosphate anion (0-2.8 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4); (B) Fluorescence spectra of CHS-Zn2+ (3.33×10−5 M) upon addition of phosphate anion (0-2.0 equiv.) in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4).

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Scheme 2. Proposed binding and sensing mechanism of CHP with Zn2+ and PA.

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Scheme 3. Proposed binding and sensing mechanism of CHS with Zn2+ and PA.

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3.7 Reversibility studies and logic gates behavior

The reversibility fluorescence response was investigated through alternately adding

lP

Zn2+ and PA into the solutions of sensors CHP and CHS, respectively. Zn2+ was first added to the solutions of sensors CHP or CHS, and the fluorescence of the solutions

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of CHP and CHS were enhanced and exhibited strong yellow and green fluorescence under 365 nm UV-light. After adding PA to the solutions above, the fluorescence

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emission of which was all quenched. When Zn2+ was added more to the solutions, the fluorescence emission of solutions of CHP and CHS at 533 nm and 510 nm were enhanced again due to the competitive combination of Zn2+ and PA. The fluorescence efficiency of the sensors CHP and CHS for identifying Zn2+ and PA was still high after several times (Fig. 11 and Fig. S21). The sensors CHP and CHS could be repeated to be used for detecting Zn2+ and PA.

Fig. 11. Fluorescence spectra changes of CHP after alternating addition of Zn2+ and phosphate 16

Journal Pre-proof anion in DMF/HEPES buffer (5:1 v/v, 10 mM, pH=7.4) solution. Inset: visual fluorescence changes of alternating addition of Zn2+ and phosphate anion.

In view of the photosensitive characteristics between the sensors CHP, CHS with Zn2+ and PA, the abilities of sensors CHP and CHS could be built a combinatorial logic gate that contained specifically logic functions AND & NOT [52]. Zn2+ (IN 1) and PA (IN 2) could be defined as input signals and the fluorescence emission of sensors CHP and CHS at 533 nm and 510 nm were defined as output signals,

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respectively. The existence of Zn2+ or PA was defined as 1, and the absence of Zn2+ or HPO42− was defined as 0. The quenching and enhancement of the fluorescence signals

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of sensors CHP and CHS at 533 nm and 510 nm were defined as 0 and 1 respectively.

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As shown in Scheme 4, the fluorescence enhancement (output 1) of CHP and CHS in the presence of input IN 1, the fluorescence quenching (output 0) of CHP and CHS in

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the presence of both input IN 1 and IN 2. The fluorescent behavior of CHP and CHS

lP

were expired by an INHIBIT logic function. Input

IN 2

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IN 1

output OUT

(PA)

Fluorescence emission

0

0

0

0

1

0

1

0

1

1

1

0

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(Zn2+)

Scheme 4. INHIBIT logic gate for CHP and CHS with two inputs (IN 1=Zn2+, IN 2=HPO42−)

3.8 Cell imaging study Due to the excellent fluorescence response of the sensors CHP and CHS to Zn2+ and their complexes CHP-Zn2+ and CHS-Zn2+ to PA in aqueous buffered solutions. The biological applications of CHP and CHS were further assessed by intracellular 17

Journal Pre-proof imaging test, respectively. Firstly, the cytotoxicity of CHP and CHS were assessed by MTT assays in PC12 cells. As shown in Fig. S22 and Fig. S23, diverse concentrations (0-120 μM) of CHP and CHS were incubated with PC12 cells for 5h at 37 ℃, respectively. When the concentrations of CHP and CHS below of 60 μM had almost no virulence for PC12 cells (Fig. 12 and Fig. S24). PC12 cells were incubated in sterile PBS buffer (pH=7.4) containing 20 μM CHP and CHS for 20 minutes at 37 ℃, respectively. Then the bright field and dark field images of cells were observed under a fluorescence microscope, as shown in Fig. 12A2 and Fig. S24D2, cellular image

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showed no fluorescence. While the cells were further incubated with 40 μM Zn2+ for

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30 minutes at 37 ℃ and washed with PBS buffer three times. As shown in Fig. 12B2 and Fig. S34E2, the green fluorescence of cells was observed under the microscope,

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respectively. In addition, we continued to add 40 μM of PA to the cell culture medium

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containing the complexes CHP-Zn2+ and CHS-Zn2+ and incubated for 20 minutes,

lP

respectively. Then the green fluorescence of cell was quenched (Fig. 12C2 and Fig. S24F2). Overall results indicated that CHP and CHS could be used as sensors to

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monitor Zn2+ and PA in living cells.

Fig. 12. (A1) Bright field image of living PC12 cells treated with CHP (20 μM CHP); (A2) Fluorescence image of A1; (B1) Bright field image of living PC12 cells treated with CHP (20 μM CHP) and Zn2+ (40 μM); (B2) Fluorescence image of B1; (C1) Bright field image of living PC12 cells treated with CHP (20 μM CHP) and Zn2+ (40 μM) again incubated phosphate anion (40 μM); (C2) Fluorescence image of C1.

4. Conclusion In summary, we designed and synthesized two coumarin-based Schiff base 18

Journal Pre-proof fluorescence sensors CHP and CHS which revealed selectivity and sensitivity for Zn2+ with “naked eye” detection in buffered solution. Furthermore, the complexes CHP-Zn2+ and CHS-Zn2+ could also act as sensors for detecting phosphate anion. The binding mechanism of sensors with Zn2+ and phosphate anion were affirmed by spectroscopic technique, HRMS experiment and theoretical calculation. And the sensors CHP and CHS showed “Off−On−Off” fluorescence switching response with Zn2+ and phosphate anion, the INHIBIT logic gate were constructed to monitor fluorescence emission of CHP and CHS with Zn2+ and phosphate anion as chemical

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inputs. Moreover, the abilities of sensors CHP and CHS of detecting intracellular Zn 2+

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and phosphate anion were successfully applied.

Acknowledgements

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We thank the Natural Science Foundation of China (No. U1404207) for financial

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support.

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Journal Pre-proof Sinha, Vanillinyl thioether Schiff base as a turn-on fluorescence sensor to Zn2+, ion with living cell imaging, Sens. Actuators B 228 (2016) 287–294. [50] F. Wang, J.H. Moon, R. Nandhakumar, B.T. Kang, D. Kim, K.M. Kim, J.Y. Lee, J. Yoon, Zn2+-induced conformational changes in a binaphthyl-pyrene derivative monitored by using fluorescence and CD spectroscopy, Chem. Commun. 49 (2013) 7228–7230. [51] U. Fegade, A. Saini, S.K. Sahoo, N. Singh, R. Bendre, A. Kuwar, 2,2 0-(hydrazine-1,2-diylidenedimethylylidene)bis(6-isopropyl-3-methylphenol) based selective dual-channel chemosensor for Cu2+ in semi-aqueous media, RSC

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12866–12876.

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Journal Pre-proof Graphical abstract

Two novel coumarin based fluorescent sensors CHP and CHS have been

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synthesized for the sequential detection of Zn2+ and phosphate anion (PA) in

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DMF/HEPES buffer medium (1/5 v/v, 10 mM, pH=7.4). On the addition of Zn2+ ion to the solution of CHP or CHS resulted in a pronounced fluorescence enhancement,

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accompanying noticeable color change (under UV or daylight), while there was

re

hardly obvious change with other competing metal ions co-existing. The detection limits (DL) of CHP and CHS towards Zn2+ were separately determined as 1.03×10-7

lP

(R2 = 0.9886) and 1.87×10−7 (R2 = 0.9902). The PET binding processes were affirmed by spectroscopic techniques, HRMS experiments and theoretical calculations.

na

Subsequently, the CHP-Zn2+ or CHS-Zn2+ complexes showed high selectivity fluorescence quenching toward PA by snatching Zn2+ ion from its complex and the

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binding processes were reversible. DLs were calculated as 2.07×10−7 M (R2 = 0.9928) and 2.63×10−7 M (R2 = 0.9954), respectively. Furthermore, the cell imaging experiments demonstrated that the sensors were capable of detecting of Zn2+ and PA in vitro cells.

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Journal Pre-proof Highlights 1. Two novel Schiff base fluorescent sensors CHP and CHS were synthesized. 2. The sensors CHP and CHS for the sequentially detected of Zn2+ and phosphate anion in aqueous solutions. 3. The binding modes of sensors CHP and CHS with Zn2+ ion had been well demonstrated by ESI-MS, 1H NMR and DFT calculation. 4. The sensor CPM was almost no toxicity and could be used to monitor Zn2+ and

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phosphate anion in living cells.

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