A small-molecule and resumable two-photon fluorescent probe for Zn2+ based on a coumarin Schiff-base

Accepted Manuscript Title: A small-molecule and resumable two-photon fluorescent probe for Zn2+ based on a coumarin Schiff-base Author: Jing-can Qin Long Fan Zheng-yin Yang PII: DOI: Reference:

S0925-4005(16)30031-4 http://dx.doi.org/doi:10.1016/j.snb.2016.01.031 SNB 19540

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

19-10-2015 30-12-2015 8-1-2016

Please cite this article as: Jing-can Qin, Long Fan, Zheng-yin Yang, A small-molecule and resumable two-photon fluorescent probe for Zn2+ based on a coumarin Schiff-base, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.01.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A small-molecule and resumable two-photon fluorescent probe for Zn2+ based on a coumarin Schiff-base Jing -can Qin, Long Fan, Zheng-yin Yang* College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, P.R. China **Corresponding author. Tel.: +86 931 8913515; Fax: +86 931 8912582; e-mail: [email protected] (Z.Y. Yang)

Graphical abstract:

Highlights 

A simple and resumable two-photon probe for Zn2+ is designed and synthesized.



The reason are attributed to the restricted -C=N isomerization and the inhibited (PET).



It has lower detection limit.

1

Abstract In

this

study,

a

simple

one-photon

and

two-photon

probe,

7-(4’-(diethylamino)-2’-hydroxybenzylideneimino)-4-methyl coumarin, for Zn2+ is designed and synthesized. The sensor shows high selectivity towards Zn2+ ions with significant fluorescence intensity enhanced at 500 nm, which might be attributed to the formation of 2:1 stoichiometric L-Zn complex resulting in the restricted -C=N isomerization and the inhibited photo-induced electron transfer. More importantly, the reversibility of the recognition process of HL is performed by adding a Zn2+ bonding agent Na2EDTA.

Keywords: fluorescent sensor; two-photon; Zn2+; -C=N isomerization /photo-induced electron transfer; reversibility.

1. Introduction Following iron, Zinc is the second most abundant transition metal ion in the human body [1-2], In addition, as an essential element, it is an important component of more than 300 enzymes and in greater number of other proteins, and plays an indispensable role in a variety of biological process as well as gene transcription, regulation of metalloenzymes, neural signal transmission, superoxide dismutase, cytochrome coxidase [3-9]. Thus, Minute quantity of Zn2+ is beneficial for the people’s health, but at higher concentrations, it exhibits toxicity in that it causes some neurodegenerative diseases such as Alzheimers, Parkinsons, Menkes and familial 2

amyotropic lateral sclerosis [10-15]. In order to protect the human health, it is crucial to develop several effective and convenient tools to detection of the concentration levels of Zn2+. In the last decade, the development of fluorescent sensor for the detection of metal ions has currently attracted significant interest because of its high sensitivity, selectivity, rapidity and easy operational procedure [16-25], the reported Zn2+-selective fluorescent sensor also is becoming increasingly more common, However, the majority of these probes are based on one-photon excitation (OPE) in which the short excitation wavelength may result in photobleaching of probes and damage in cells and tissues. Two-photon fluorescent probes (TFP) that the fluorescence is triggered by two-photon excitation are still few in number. Compare with conventional one-photon excitation, Two-photon excitation (TPE) can show a variety of advantages such as reducing photodamage to biosamples, increasing tissue penetration, and minimizing background fluorescence [26-30]. Taking this in to account, we have reported a small-molecule and resumable two-photon

fluorescent

probe

(HL)

for

Zn2+,

which

was

prepared

by

4-diethylaminosalicylaldehyde and 7-amino-4-methyl coumarin. The free sensor was almost non-fluorescent (quantum yield in DMF-H2O (v/v, 9:1), Ф=0.0221) owing to the -C=N isomerization process and photo-induced electron transfer (PET) process. Upon the addition of Zn2+, the sensor shows significant enhancement of fluorescence intensity at 500 nm (quantum yield in DMF-H2O (v/v, 9:1), Ф=0.1651) which is because that the formation of a 2:1 stoichiometric L-Zn complex restrict -C=N 3

isomerization and inhibit the photo-induced electron transfer (PET). 2. Experimental 2.1. Apparatus and reagents All chemicals were obtained from commercial suppliers and used without further purification. 1H NMR spectra were measured on the JNM-ECS 400MHz instruments using TMS as an internal standard. ESI-MS were determined on a Bruker esquire 6000 spectrometer. UV-Vis absorption spectra were determined on a Shimadzu UV-240 spectrophotometer. Fluorescence spectra with both one-photon excitation (OPE) and two-photon excitation (TPE) were carried out using the Hitachi RF-4500 fluorescence spectrometer and Edinburgh Instrument FLS920, respectively. 2.2. Analysis Stock solutions of various cations (5 mM) were prepared using nitrate salts. A stock solution of HL (5 mM) was prepared. The solution of HL was then diluted to 50 μM. In titration experiments, each time a 2 mL solution of HL (50 μM) was filled in the quartz optical cell of 1 cm optical path length, and the ions stock solution were added into the quartz optical cell gradually by using a pipette. In selectivity experiments, the test samples were prepared by placing appropriate amounts of ions stock into 2 mL solution of HL (50 μM). For fluorescence measurements, the excitation slit width was 3 nm, the emission slit width was 3 nm. 2.3. Calculation The detection limit was calculated based on the fluorescence titration according to the following equation: detection limit 3σ/κ. where σ was the standard deviation of blank measurements, and κ was the slope between intensity versus sample 4

concentration [31] The binding constant K was obtained from the plot of linear regression of log [(F- Fmin)/(Fmax - F)] versus log [M] in equation, where the intercept was log K. Fmin, Fmax, and F were the fluorescence intensity in the absence of Zn2+, in presence of saturated Zn2+, and the fluorescence intensity of the [L-Zn] complex at time intervals. [M] was the concentration of free metal ions which could be assumed equal to its total concentration [32].

2.4. Synthesis 4-methyl-7-aminocoumarin (AMC) (Fig.S1) was synthesized according to the method reported [33-34]. The synthetic route of the sensor was shown in Scheme 1. An ethanol solution (20 mL) of 7-amino-4-methyl coumarin (1mmol, 0.203g) was added to another ethanol (20 mL) containing 4-diethylaminosalicylaldehyde (1 mmol, 0.193 g), then the solution was refluxing for 12 h under stirring. After cooling to room temperature, the mixture was filtered and dried, recrystallized from DMF and ethanol. Yield: 65%; mp: 220-221°C. 1HNMR (Fig. S 2a): (400 MHz; DMSO-d6): δ= 1.08 (t, J=7.0 Hz, 3H9, 3H12), δ= 2.41 (q, J=7.0 Hz，2H10, 2H11), δ= 2.39 (d, J=1.2 Hz, 3H2), 6.02 (d, J=2.4 Hz, H13), 6.27 (d, J=1.2 Hz, H1), 6.33 (dd, J=2.4 Hz，J=8.9 Hz, H8), 7.28 (dd, J=2.1 Hz，J=8.5Hz，H4), 7.30 ~7.33 (m, H3, H7), 7.72 (d, J=8.5 Hz, H5), 8.78 (s, H6), 13.32 (s, H14). 13CNMR (100 MHz; DMSO-d6) (Fig. S 2b): 30.44, 30.54, 5

55.56,98.06,98.25,104.16,106.40,106.63,107.61,110.64,110.82,114.06,114.67,114.90, 121.34,128.02,128.22. EI-MS (Fig. S3): m/z 351.149 [M+H]+, IR (KBr, cm-1) (Fig. S4): 2972.27, 1723 46, 1631.04, 1612.83. Elemental analysis: C21H22N2O3, Found: C, 71.85; H, 6.41; N, 7.87. Calcd: C, 71.98; H, 6.33; N, 7.99.

3. Results and discussion 3.1 Selectivity and competition As shown in Fig. 1. The selectivity of HL was investigated upon addition of several metal ions such as Na+, Ag+, K+, Mg2+, Ca2+, Pb2+, Cr3+, Mn2+, Fe2+, Ni2+, Co2+, Fe3+, Cu2+, Ba2+, Cd2+, Hg2+, Zn2+ and Al3+. The free sensor showed weak fluorescence emission at 500 nm (λex =380 nm) with a low fluorescence quantum yield (Ф=0.0221). Upon the addition of various metal ions to solution of the sensor, the sensor did not give any observable response for other relevant metal ions except for Zn2+, the addition of Zn2+ brought remarkable enhancement in the fluorescence intensity at 500 nm with a high fluorescence quantum yield (Ф=0.1651). In addition, to check the practical applicability of HL as a selective fluorescent sensor for Zn2+, it was crucial to realize the selectivity of the sensor in the presence of other competing metal ions. As shown in Fig. 2, the sensor HL +Zn2+ with other metal ions showed no significant variation except in the case of Cu2+, M3+ (Al3+, Fe3+, Cr3+). The fluorescence of HL was almost completely quenched in the presence of Cu2+, which was attributed to its inherent to the magnetic property. As for M3+, this was because the strong Lewis acidity of trivalent cations resulting in the hydrolytic

6

cleavage of the imine-bond [35-37]. Furthermore, the sensor could be reversed back by the addition of Na2EDTA (Fig. 3), which is quite useful for developing the resumable fluorescent sensor for Zn2+ in the presence of most competing metal ions. 3.2. Titration analysis To understand the binding ability of HL and Zn2+, a quantitative investigation of HL with Zn2+ was performed by one-photon fluorescence titration. The spectra of HL are constantly changing as a function of the concentration of Zn2+ as shown in Fig. 4. With the increasing concentration of Zn2+, the fluorescence intensity reached a maximum in the presence of 0.5 equiv. of Zn2+, indicating the 2:1 binding of HL and Zn2+, which was consistent with the result from UV-Vis titration (Fig. S5) and Job′s plot. Moreover, the association constant (log K) and the detection limit were calculated to be 6.04 (Fig.S6) and 2.59×10-6 M (Fig.S7) respectively. Two-photon fluorescence titration was also carried out to evaluate the coordinated condition under the long-wave excitation. As shown in Fig. 5, with the addition of Zn2+ to HL, the sensor showed more than 12-fold enhancement of fluorescence intensity at about 500 nm. When the concentration of Zn2+ reached 25 mM, the fluorescence reinforcement no longer appeared which also confirmed the formation of a 2:1 stoichiometric L-Zn complex. The association constant (log K) was calculated to be 6.37 (Fig. S8), which was near that from one-photon fluorescence titration and the detection limit for Zn2+ reached at 2.72×10-6 M (Fig. S9) Additionally, we proposed the phenomenon that the addition of Zn2+ resulted in the fluorescence enhancement at about 500 nm whether one-photon excitation or 7

two-photon excitation may be attributed to the restricted -C=N isomerization, More specifically, before addition of Zn2+, the -C=N isomerization was the predominant decay processd of the excited states for coumarin derivative with an unbridged C=N structure so that the sensor showed weak fluorescence. however, up addition of Zn2+, the binding of the nitrogen atom of the –C=N with Zn2+ restricted C=N isomerization in the excited states, as result, the fluorescence occurred remarkably at about 500 nm as shown in Scheme 2 [38-40]. In addition，part of the reason was attributed to the chelation of the nitrogen atom of the -C=N group with Zn2+ which resulted in the efficient inhibition for the PET process of the -C=N group [41]. 3.3 Two-photon absorption cross section (δ) In order to understand the property of two-photon fluorescent sensor, we try to study the absorption cross section of the sensor in the absence of Zn2+ and in the presence of Zn2+ respectively. As shown in Fig.6, up on the addition of Zn2+, they had ca 10-fold improvement in the absorption cross section which can be ascribed to the increased degree of rigidity and conjunction in metal complex. Two-photon excitation fluorescence spectra were measured using a Steady-state and Life time Fluorescence Spectrometer (FLS920, Edinburgh Instruments). Two-photon absorption cross section (δ) at different excitation wavelengths were determined by comparing their two-photon excitation fluorescence with that of fluoresce [42], according to the following equation: δ= δref Φref / Φcref / cnref /nF/Fref

(1)

In Eq. (1), the subscript ref stands for the reference molecule. δ was the 8

two-photon absorption cross section value, c was the concentration of solution, n is the refractive index of the solution, F was the two-photon excitation fluorescence integral intensities of the solution emitted at the exciting wavelength, and Φ was the fluorescence quantum yield. The δref value of reference was taken from the literature [43] 3.4 The complexation of HL with Zn2+ In order to further validate the conjugation of L-Zn, it is necessary to Job’s plot and spectra studies for HL with Zn2+ except for the above experiments. The total concentration of HL and Zn2+ was 50 μM. XZn = ([Zn2+]/[Zn2+] + [HL]). As shown in Fig. 7, the maximum point appeared at a mole fraction of 0.33, which indicated that it was a 2:1 stoichiometry of the binding mode of HL and Zn2+. It also was further evidenced by one-photon fluorescence titration, two-photon fluorescence titration, UV–Vis titration and EI-MS (Fig.S10) (a peak at m/z 763.27 was assigned to [2HL +Zn2+-H]+). On the other hand, 1H NMR titration (Fig.S11), the spectral differences was depicted in Fig. 8. The proton peak at 8.76 ppm assignable to -CH=N of HL was shifted downfield towards 9.18 ppm owing to Zn2+ chelate nitrogen of -C=N. Moreover, the addition of Zn2+ to HL also caused the loss of the -OH proton (at 13.32 ppm) with subsequent binding to electron deficient Zn2+. Furthermore, new signals for the aryl protons appeared compared with those in the spectrum of the parent ligand HL. Thus, based on the research and analysis above, they suggested that Zn2+ was coordinated with the imine nitrogen atom, the hydroxyl oxygen atom and forms 2:1 ligand-metal complex. 9

4.

Conclusions In summary, we have designed and synthesized a simple and resumable

two-photon fluorescent probe which shows a high selectivity towards Zn2+ ions in DMF-H2O (v/v, 9:1). The sensor shows significant enhancement of fluorescence intensity at about 500 nm in the presence of Zn2+ (λex = 380 nm and 760 nm), owing to the restricted -C=N isomerization and the inhibited the PET process. In addition because of its lower detection limit, the sensor should find potential applications to detect micromolar concentrations of Zn2+ in both biological systems and the environment.

Acknowledgments This work is supported by the National Natural Science Foundation of China (81171337). Gansu NSF (1308RJZA115).

References [1] W. Maret, Zinc biochemistry, physiology, and homeostasis—recent insights and current trends, Biometals 14 (2001) 187-190. [2] R.K. Pathak, V.K. Hinge, A. Rai, D. Panda, C.P. Rao, Imino-phenolic-pyridyl con-jugates of Calix[4] arene (L1 and L2) as primary fluorescence switch-on sensorsfor Zn2+ in solution and in HeLa cells and the recognition of pyrophosphate and ATP by [ZnL2], Inorg. Chem. 51 (2012) 4994-5005. [3] X. Xie, T.G. Smart, A physiological role for endogenous zinc in rat 1 0

hippocampalsynaptic neurotransmission, Nature. 349 (1991) 521. [4] B. L. Vallee, K. H. Falchuk, The biochemical basis of zinc physiology, Physiological Reviews. 73 (1993) 79-118. [5] K. Li, A. Tong, A new fluorescent chemosensor for Zn2+with facile synthesis: “turn-on” response in water at neutral pH and its application for live cellimaging, Sens. Actuators B 184 (2013) 248-253. [6] Q. Ai, The role of zinc in caspase activation and apoptotic cell death, BioMetals. 14 (2001) 315-330. [7] M. Shellaiah, Y.H. Wu, H.C. Lin, Simple pyridylsalicylimine-based fluorescence turn-on sensors for distinct detections of Zn2+, Al3+, and OH−ions in mixed aqueous media, Analyst 138 (2013) 2931-2942. [8] R. McRae, P. Bagchi, S. Sumalekshmy, C. J. Fahrni, In situ imaging of metals in cells and tissues, Chem Rev. 109 (2009) 4780-4827. [9] L. J. Tang, M. J. Cai, P. Zhou, J. Zhao, K. L. Zhong, S. H. Hou, Y. J. Bian, A highly selective and ratiometric fluorescent sensor for relay recognition of zinc(II) and sulfide ions based on modulation of excited-state intramolecular proton transfer, RSC Adv. 3 (2013) 16802-16809. [10] A.I. Bush, Metals neuroscience, Current Opinion in Chemical Biology. 4 (2000) 184-191. [11] A.I. Bush, Copper, -amyloid, and Alzheimer’s disease: tapping a sensitive connection, Trends in Neuroscience. 26 (2003) 207-214. [12] M.P. Cuajungco, G.J. Lees, The biochemical basis of zinc physiology, Brain Res. 1 1

23 (1997) 219-236. [13] T. Jin, J. Lu, M. Nordberg, Toxicokinetics and biochemistry of cadmium with special emphasis on the role of metallothionein, Neurotoxicology 19 (1998)529-535. [14] J. Li, V.N. Uversky, A. L. Fink. Effect of familial parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human a-synuclein, Biochem. 40 (2001) 1604-1613. [15] A. Takeda, Zinc homeostasis and functions of zinc in the brain, Biometals 14 (2001) 343-351. [16] M.R.B. Binet, R. Ma, C.W. McLeod, R.K. Poole, Detection and characterization of zinc and cadmium-binding proteins in Escherichia coli by gel electrophore-sis and laser ablation-inductively coupled plasma-mass spectrometry, Anal.Biochem. 318 (2003) 30-38. [17] M. Shamsipur, M. Yousefi, M. Hosseini, M.R. Ganjali, H. Sharghi, H. Naeimi, A Schiff Base Complex of Zn(II) as a neutral carrier for highly selective pvc membrane sensors for the sulfate ion, Anal. Chem. 73 (2001) 2869-2874. [18] V.K. Gupta, A.K. Jain, P. Kumar, S. Agarwal, G. Maheshwari, Chromium (III)selective sensor based on tri-o-thymotide in PVC matrix, Sens. Actuator B 113(2006) 182-186. [19] P.K. Tewari, A.K. Singh, Thiosalicylic acid-immobilized amberlite XAD-2: metal sorption behaviour and applications in estimation of metal ions by flame atomicabsorption spectrometry, Analyst 125 (2000) 2350-2355. 1 2

[20] B.J. Sanghavi, W. Harhue, J.L. Chávez, C.F. Chou, N.S. Swami, Electrokinetic pre-concentration and detection of neuropeptides at patterned graphene-modified electrodes in a nanochannel, Anal. Chem. 85 (2014) 4120-4125. [21] X. Y. Zhou, P. X. Li, Z. H. Shi, X. L. Tang, C.Y. Chen, W. S. Liu, A Highly Selective Fluorescent Sensor for Distinguishing Cadmium from Zinc Ions Based on a Quinoline Platform, Inorg. Chem. 51 (2012) 9226-9231. [22] W. H. Hsieh, C. F. Wan, D. J. Liao, A. T. Wu, A turn-on Schiff base fluorescence sensor for zinc ion, Tetrahedron Lett. 53 (2012) 5848-5851. [23] M.M. Yu, Z.X. Li, L.H. Wei, D.H. Wei, M.S. Tang, A 1,8-naphthyridine-based fluorescent chemodosimeter for the rapid detection of Zn2+and Cu2+, Organ. Lett.10 (2008) 5115-5118. [24] Z. Xu, J. Yoon, D.R. Spring, Fluorescent chemosensors for Zn2+, Chem. Soc. Rev.39 (2010) 1996–2006 [25] Y. Weng, Z. Chen, F. Wang, L. Xue, H. Jiang, High sensitive determination of zinc with novel water-soluble small molecular fluorescent sensor, Anal. Chim. Acta 647 (2009) 215-218 [26] W. Zhang, P. Li, F. Yang, X. Hu, C. Sun, W. Zhang, D. Chen, B. Tang, Dynamic and reversible fluorescence imaging of superoxide anion fluctuations in live cells and in vivo, J. Am. Chem. Soc. 135 (2013) 14956-14959. [27] H. Yu, Y. Xiao, L. Jin, A lysosome-targetable and two-photon fluorescent probe for monitoring endogenous and exogenous nitric oxide in living cells, J. Am. Chem. Soc. 134 (2012) 17486-17489. 1 3

[28]Q.Q. Wu, Z.F. Xiao, X.J. Du, Q.H. Song, A novel ratiometric two-photon fluorescent probe for the detection of biothiols in solution and imaging of living cells, Chem. Asian J.

8 (2013) 2564-2568.

[29] P. Li, W. Zhang, K. Li, X. Liu, H. Xiao, W. Zhang, B. Tang, Mitochondria-targeted reaction-based two-photon fluorescent probe for imaging of superoxide anion in live cells and in vivo, Anal. Chem. 85 (2013) 9877-9881. [30] L. Li, J. Ge, H. Wu, Q.-H. Xu, S.Q. Yao, Organelle-specific detection of phosphatase activities with two-photon fluorogenic probes in cells and tissues, J. Am. Chem. Soc. 134 (2012) 12157-1216 [31] C. R. Lohani, J. M. Kim, S.Y. Chung, J. Yoon, K. H. Lee, Colorimetric and fluorescent sensing of pyrophosphate in 100% aqueous solution by a system comprised of rhodamine B compound and Al3+ complex, Analyst.135 (2010) 2079-2084 [32] H. A. Benesi, J. H. Hildebrand, A Spectrophotometric Investigation of the Interaction of Iodine with Aromatic Hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703-2707. [33] J..C Qin, L.Fan, B.D. Wang, Z.Y. Yang, T.R. Li, The design of a simple fluorescent chemosensor for Al3+/Zn2+ via two different approaches, Anal. Methods. 7 (2015) 716-722. [34] G. Behl, M. Sikka, A. Chhikara, M. Chopra, PEG-coumarin based biocompatible self-assembled fluorescent nanoaggregates synthesized via click reactions and studies of aggregation behavior J. Colloid. Interface. Sci. 416 (2014) 151-160. 1 4

[35] M. Venkateswarulu, Trinetra Mukherjee,b Subhrakanti Mukherjee,

Rik Rani

Koner, Turn-on trivalent cation selective chemodosimetric probe to image native cellular iron pools, Dalton Trans. 43 (2014) 5269-5273 [36] X.J Chen,X..Y Shen, E.Guan, Y. Liu, A.J. Qin, J.Z. Sun, B. Z. Tang, A pyridinyl-functionalized tetraphenylethylene fluorogen for specific sensing of trivalent cations Chem. Commun.49 (2013) 1503-1505. [37] S. Samanta , S. Goswami b, A. Ramesh, G. Das, A new chemodosimetric probe for the selective detection of trivalent cations in aqueous medium and live cells, J. Photoch. Photobio. A: 310 (2015) 45-51 [38] J. S. Wu, W. M. Liu, X. Q. Zhuang, F.Wang, P. F. Wang, S. L. Tao, X. H. Zhang, S. K. Wu, S. T. Lee, Fluorescence Turn On of Coumarin Derivatives by Metal Cations: A New Signaling Mechanism Based on C=N Isomerization, Org. let. 9 (2007) 33-36. [39] S. Toyota, Rotational Isomerism Involving Acetylene Carbon, Chem. Rev. 2010, 110, 5398-5424 [40] D. Ray and P. K. Bharadwaj, A Coumarin-Derived Fluorescence Probe Selective for Magnesium, Inorg. Chem. 47 (2008) 2252-2254. [41] H. S. Jung, K.C. Ko, J.H. Lee, S.H. Kim, S.Bhuniya, J.Y. Lee, Y. Kim, S. J. Kim, J.S. Kim, Rationally Designed Fluorescence Turn-On Sensors: A New Design Strategy Basedon Orbital Control, Inorg. Chem. 49 (2010) 8552-8557

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[42] M. A. Albota, C. Xu, and W.W. Webb, Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm, Appl.Opt. 37 (1998) 7352-7356. [43] Y.M. Li, H.B Chong, X.M Meng, S. X.Wang, M.Z. Zhu, Q. X. Guo, A novel quinoline-based two-photon fluorescent probe for detecting Cd2+ in vitro and in vivo, DaltonTrans. 41(2012)6189-6194.

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Biographies Jingcan Qin received her bachelor's degree in 2012 in chemical from Shandong University of Technology, PR China. He is currently studying for degree of master at College of Chemistry and Chemical Engi-neering, Lanzhou University, PR China. Her work include fluorescent sensors and contrast agent .Long Fan received her bachelor's degree in 2012 in chemical from Hexi University, PR China. He is currently studying for degree of master at College of Chemistry and Chemical Engi-neering, Lanzhou University, PR China. Her work is studying the development of fluorescent sensors. Zhengyin Yang received his Ph.D. degree from Lanzhou University, PR China, in 1997. He is a professor of Inorganic Chemistry in College of Chemistry and Chemical Engi-neering,

Lanzhou University. His current research interests include spectrum

analysis and bioinorganic chemistry.

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Fig.1 Fluorescence spectra of HL (50 μM) upon the addition of metal salts (50 μM) of Na+, K+, Ag+, Mg2+, Ca2+, Pb2+, Cr3+, Mn2+, Fe2+, Ni2+, Co2+, Fe3+, Cu2+, Ba2+, Cd2+, , Al3+and Zn2+ in DMF-H2O (v/v, 9:1) (λex=380 nm, slit widths: 3 nm /3 nm).

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Fig.2 Fluorescence intensity of HL and its complexation with Zn2+ in the presence of various metal ions in DMF-H2O (v/v, 9:1), Red bar: HL (50 μM) and HL with 1.0 equiv. of Li, Na+, K+, Ca2+, Mg2+, Cr3+, Pb2+, Mn2+, Fe2+, Ni2+, Co2+, Fe3+, Cu2+, Ba2+, Cd2+and Al3+ stated. Blue bar: 50μM of HL and 1 equiv. of Zn2+ ; 50 μM of HL and 1.0 equiv. of Zn2+ with 1.0 equiv. of metal ions stated in DMF (λex=380 nm, slit widths: 3 nm /3 nm).

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Fig.3 The reversibility of HL (50 μM) reacting with Zn2+ (50 μM). (λex= 380 nm, slit widths:3 nm /3 nm)

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Fig. 4 Fluorescence spectra of HL (50 μM) in DMF-H2O (v/v, 9:1) upon the addition of Zn2+ (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50μM.) (λex= 380 nm, slit widths: 3 nm /3 nm) nm. Inset: color of HL (left) and HL + Zn2+ (right) system under UV lamp.

2 1

Fig. 5 Two-photon fluorescence spectra of HL (50 μM) in DMF-H2O (v/v, 9:1) upon the addition of Zn2+ (0, 5, 10, 15, 20, 25, 30μM.) (λex= 760 nm, slit widths: 0.9 nm /0.9 nm)

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Fig. 6 The two-photon the absorption cross section of the sensor in the presence of Zn2+ and in the absence of Zn2+ in DMF-H2O (v/v, 9:1)

2 3

Fig.7 Job’s plot for determining the stoichiometry of the sensor HL and Zn2+ in DMF-H2O (v/v, 9:1) (XZn= ([Zn2+]/([ Zn2+] +[ HL]), the total concentration of HL and Zn2+ was 50 μM (λex=380nm, slit widths: 3 nm /3 nm)

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Fig. 8 1H NMR spectrum: (I) HL; (II) HL and Zn2+

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Scheme 1. Reagents and conditions: (a) NaHCO3, ethylchloroformate, diethylether, RT, 4h; (b) ethylacetoacetate, H2SO4 (70%), RT, 18 h; (c) H2SO4 and glacial acetic acid (1:1), reflux, 5h; (d) EtOH, reflux, 10-12 h.

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Scheme 2. Proposed mechanism for detection of Zn2+ by HL.

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