Cascade OFF-ON-OFF fluorescent probe: Dual detection of Fe3+ ions and thiols

Article abstract of DOI:10.1016/j.jphotochem.2016.05.017

A new novel Rhodamine B-based fluorescent probe was synthesized for the selective cascade signaling of Fe³⁺ and thiols (glutathione, homo-cysteine, cysteine). Non-fluorescent rhodamine derivatives can selectively detect Fe³⁺ over some other metal ions in ethanol-water (4/1, v/v, pH 7.0) media, leading to prominent fluorescence OFF-ON switching. The obtained LDF-Fe³⁺ ensemble can subsequently serve as a sensitive and selective chemosensor for thiols, exhibiting complete signal quenching (fluorescence ON-OFF switching). The practical use of its Fe³⁺ ions ensemble was demonstrated by its application to the detection of thiols in the living cells.

Full text of DOI:10.1016/j.jphotochem.2016.05.017

Accepted Manuscript
Title: Cascade OFF–ON–OFF fluorescent probe: Dual
detection of Fe3+ ions and thiols
Author: Shimin Fan Wenge Yang Jianfeng Hao Hongjie Li
Wangdan Zhao Jian Zhang Yonghong Hu
PII:
S1010-6030(16)30283-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2016.05.017
JPC 10236
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
20-4-2016
23-5-2016
26-5-2016
Please cite this article as: Shimin Fan, Wenge Yang, Jianfeng Hao, Hongjie Li, Wangdan
Zhao, Jian Zhang, Yonghong Hu, Cascade OFF–ON–OFF fluorescent probe: Dual
detection of Fe3+ ions and thiols, Journal of Photochemistry and Photobiology A:
Chemistry http://dx.doi.org/10.1016/j.jphotochem.2016.05.017
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.

Title page
Title:
Cascade OFF–ON–OFF fluorescent probe: dual detection of Fe3+ ions and thiols
Authors:
Shimin Fana ·Wenge Yang()a·Jianfeng Haoa . Hongjie Lia·Wangdan Zhao a ·Jian Zhanga ·Yonghong
Hub
a School of Pharmaceutical Sciences, Nanjing Tech University, No. 30, South Puzhu Road, Nanjing
211816, China
b State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University,
No.200, South Zhongshan Road, Nanjing 210009, China
Corresponding author: Wenge Yang
Tel: +86-25-83587108
Fax: +86-25-83587108
E-mail: wengeyang11@163.com
1

Highlights
> One dual-functional fluorescence probe based on Rhodamine B was designed
> LDF shows a highly selective fluorescent response to trace amounts of Fe3+ ions
> [LDF+Fe3+] ensemble is also further successfully utilized for detection of the thiols
> This system can also be applied in Hela Cells
2

Cascade OFF–ON–OFF fluorescent probe: dual detection of Fe3+ ions and
thiols
Shimin Fana ·Wenge Yang()a·Jianfeng Haoa .Hongjie Lia·Wangdan Zhao a · Jian Zhang a ·Yonghong
Hub
a School of Pharmaceutical Sciences, Nanjing Tech University, No. 30, South Puzhu Road, Nanjing
211816, China
b State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University,
No.200, South Zhongshan Road, Nanjing 210009, China
E-mail: wengeyang11@163.com
Abstract
A new novel Rhodamine B-based fluorescent probe was synthesized for the selective cascade
signaling of Fe3+ and thiols (glutathione, homo-cysteine, cysteine). Non-fluorescent rhodamine
derivatives can selectively detect Fe3+ over some other metal ions in ethanol-water (4/1, v/v, pH 7.0)
media, leading to prominent fluorescence OFF–ON switching. The obtained LDF-Fe3+ ensemble can
subsequently serve as a sensitive and selective chemosensor for thiols, exhibiting complete signal
quenching (fluorescence ON–OFF switching). The practical use of its Fe3+ ions ensemble was
demonstrated by its application to the detection of thiols in the living cells.
Keywords: rhodamine; Fe3+ ions ; thiols; Hela Cells
Introduction:
Development of chemosensors is used for sensing and recognizing of environmentally and
biologically important heavy and transition metal ions. On account of its simplicity, high sensitivity
and instantaneous response, fluorescence is increasingly important for sensing various metal ions and
biological thiols [1-10].
Iron has its own biological significance and it directly involved in the cell function [11-12]. For
example, Fe3+ acts as oxygen carrier in hemoglobin, and plays important roles in enzyme catalysis and
cellular metabolism, a deviation of its concentration from normal levels can lead to several disorders,
including Alzheimer‟s, Huntington‟s, and Parkin-son‟s disease. Furthermore, Fe3+ can cause various
diseases depending on its concentration, such as fibrosis with irreversible damage to various organs
3

and tissue damage. In this regard, the development of chemosensor that can detect Fe3+ is very
important to prevent and solve environmental and health problems induced by Fe3+.
Biological thiols, including glutathione (GSH), cysteine (Cys) and homocystein (Hcy), play
crucial roles in maintaining the appropriate redox status of biological systems [13-14]. The most
abundant cellular thiol is GSH. It is an essential endogenous antioxidant that plays a vital role in
cellular defense against toxins and free radicals. Abnormal levels of GSH can lead to cancer, aging,
heart problems, and other diseases. Anabnormal level of cysteine may cause skin lesions and liver
damage. In addition, homocysteine is a risk factor for Alzheimer‟sand cardiovascular diseases.
Rhodamine derivatives, which are ideal chemosensors due to their particular spirolactam
structures and excellent photophysical properties, offer a „naked-eye‟ straightforward detection
method used extensively in the detection of metal ions. The spirolactam structure of rhodamine
derivatives provides very weak fluorescence strength and UV absorption, while ring-opening of the
spirolactam brings about strong UV absorption and fluorescence enhancement along with an apparent
color change. Based on this mechanism, chemosensors based on rhodamine have been developed
extensively.
Nowadays, although a number of probes have been used for the fluorescence sensing of Fe3+ and
thiols by various workers [15-20], the majority of probes currently available cannot be operated in
water let alone in buffered solution or organic solvents, which limited their practical applications.
Hence, there is a great demand for the development of novel fluorescent chemosensors for Fe3+ which
has good water solubility and be suitable for cell imaging studies. In addtion, relatively few studies
have been devoted to the development of fluorescence sensors which are sensitive to Fe3+ ions and
thiols. It is necessary to create a probe which is capable of simultaneous discrimination of Fe3+ ions
and thiols from others.
Herein, we report a simple cascade “OFF–ON–OFF” fluorescent chemosensor for Fe3+ ions and
thiols based on the equilibrium between spirolactam (non-fluorescent) to the ring open amide
(fluorescent) form of rhodamine chromophore. In general, the chemosensor showed a specific
selectivity toward Fe3+ ions over other common metal ions, leading to prominent fluorescence OFF–
ON switching. The resultant LDF-Fe3+ ensemble could selectively recognize thiols, exhibiting
complete ON–OFF signal quenching.
2. Experimental
2.1 Materials and General methods
Rhodamine B was purchased from Aladdin Reagent Co.,Ltd and used without purification. Organic
solvents were purchased from Shanghai Lingfeng chemical reagent Co., Ltd. were all analytical
.
reagent grades. The salts used in stock solutions of metal ions were NaNO3, CdCl2 H2O,
.
.
.
Co(NO3)2 6H2O, Mg(NO3)2, CuSO4, Zn(NO3)2 2H2O, Pb(NO3)2, Ni(NO3)2 6H2O, AgNO3,
4

.
.
Mn(NO3)2 4H2O, AlCl3 6H2O and FeCl3.6H2O. All of them were of analytical reagent grade and used
without purification. Water used in experiment was double distilled water. The UV-visible spectra
measurements were performed with a UV-visible spectrophotometer (Unico UV-2800H) equipped
with 1.0 cm quartz cells. Fluorescence was recorded on a spectrofluorimeter (Shimadzu RF-5301PC)
equipped with 1.0 cm quartz cell.
2.2 Synthesis of probe
Synthesis of probe LDF is outlined in Scheme1. 2-amino-3', 6'-bis (diethylamino) spiro
[isoindoline-1, 9'-xanthen]-3-one (LDF-1) was first synthesized following literature procedures
(Fig.S1) [21-23].
To a solution of LDF-1(456mg,1mmol ) in glacial acetic acid( 20mL) were added maleic anhydride
(98mg,1mmol) and were heated under reflux for 4 h. The solvent was removed in vacuum to give a
pink solid[24]. The crude product was purified by flash chromatography (petroleum/ ethyl acetate=3:1,
v/v) to afford a yellow solid LDF (322.6mg, yield: 60%). 1HNMR(Fig.S2) (400 MHz, C3D6O) δ 7.97
(m, 1H), 7.69 (dtd, J=28.6, 7.5, 1.1 Hz, 2H), 7.22 (d, J = 7.6 Hz, 1H), 6.86 (s, 2H), 6.65 (d, J = 8.9 Hz,
2H), 6.40 (dd, J = 8.9, 2.6 Hz, 2H), 6.29 (d, J = 2.6 Hz, 2H), 3.39 (q, J = 7.0 Hz, 8H), 1.15 (t, J = 7.0
Hz, 12H).13CNMR(Fig.S3)(100MHz,C3D6O) δ(ppm) 167.27,164.63, 154.78,149.96, 141.00, 134.65,
134.60, 131.89, 129.93, 127.85, 125.86, 123.84, 108.78, 105.40, 97.71, 76.66, 57.73, 44.70, 12.86.
ESI-MS (Fig.S4): calcd for C32H32N4O4 [M+H+] +:537.2424; found: 537.2499.
3. Results and discussion
3.1 Fluorescence OFF–ON sensing for Fe3+
3.1.1 Proposed mechanism
As some literatures reported [25-27], the equilibrium between the non-fluorescent spirolactam form
and the fluorescent ring-open amide form of rhodamine derivatives has been proven to be an efficient
platform for the construction of fluorescent sensors for numerous heavy metal cations due to its large
absorption coefficient and high fluorescence quantum yield. In our present work, this typical ion-
recognition mechanism was utilized for the successive detection of Fe3+ ions, and then Michael
addition of thiols damage the conjugate structure of the probe which led to the absence of the
fluorescence. This mechanism rise fluorescence OFF–ON–OFF switching. The mechanism is shown
in Scheme 2.
3.1.2 Fluorescence titration investigaton
Fluorescence titration of probe LDF was conducted in the solution of ethanol-water (4/1, v/v, pH 7.0)
media. The free LDF itself was almost non-fluorescent due to its ring-closed spirolactam structure.
After the addition of Fe3+, probe LDF exhibited a color change from almost colorless to pink as well
5

as a bright jacinth fluorescence. A strong emission band centered at 581 nm was observed with an
extreme fluorescence enhancement compared to the metal-free LDF.
The relative affinities of Fe3+ toward sensor LDF were evaluated from fluorescence spectroscopic
titration experiments in ethanol-water (4/1, v/v, pH 7.0) media, as shown in Fig.1(A). The
concentration of LDF was maintained at 10μM, while the concentration of Fe3+ was varied between 0
and 60μM. The fluorescence spectra were recorded at an excitation wavelength of 455 nm and
emission wavelength of 525–700 nm. For free LDF, no obvious characteristic emission of rhodamine
derivatives was observed. With increasing concentrations of Fe3+, the fluorometric titration reaction
curve showed a steady and smooth enhancement, which was used as the basis of Fe3+ sensing. The
recognition interaction was completed immediately after the addition of Fe3+.When the concentration
of Fe3+ was greater than 30μM, the fluorescence intensity did not increase any further and a plateau
was reached. The chart in Fig.1 shows that the dependence of fluorescence intensity at 581 nm on Fe3+
concentration. Plotting fluorescence intensity versus Fe3+ concentration (0.1-1 equiv.) afford a good
liner relationship (R2=0.98704) Fig. 1(B).
3.1.3 Selectivity investigation
To get an insight into the selectivity, fluorescence spectrum measurements of the probe LDF were
carried out with the addition of various metal ions. Among the metal ions investigated 2 equiv. Fe3+
selectively enhance the fluorescence intensity of LDF (Fig. 2). While other metal ions, such as, Na+,
K+, Ag+, Mg2+, Cu2+, Cd2+, Pb2+, Zn2+, Mn2+, Co2+ , Ni2+ and Al3+ do not enhance fluorescence
intensity or change fluorescence color under the same spectroscopic condition used for the Fe3+.
In order to further explore the selectivity and anti-interference of the sensor, some competitive
ions were first added to the detection solution, and then Fe3+ ions were added after half an hour. As
shown in Fig. 3, most of the detection systems exhibited minimum interference in the detection of
Fe3+ (20μM each) also did not induce any obvious interference in the fluorescence sensing of Fe3+.
The association constants was 5.827×105 for LDF-Fe3+.
To determine the interaction stoichiometry between sensor LDF and Fe3+, Job's method was
employed using an absorbance intensity at 558 nm as a function of molar fraction of LDF because the
total concentration of LDF and Fe3+ ion was located at 20μM[28]. The maximum absorbance was
observed when the molar fraction of LDF reached 0.50 (Fig. 4.), which was indicative of a 1:
1stoichiometry complexation between LDF and Fe3+.
3.1.4. UV-vis absorption spectra of LDF with Fe3+ ion
The sensing property of LDF was investigated in detail utilizing the Fe3+. As shown in Fig.5. The
absorption spectrum of LDF changes upon Fe3+ addition. Sensor LDF exhibited almost no absorption
peak in the visible wavelength due to the spirolactam form of LDF. However, a new band centered at
6

about 558nm emerged upon the gradual addition of Fe3+ indicating the sensor LDF-Fe3+ complex
formation and Fe3+ induced spirolactam ring-opening processes. Moreover, the titration solution
displayed a characteristic color change of rhodamine derivatives from colorless to pink, indicating that
probe LDF could serve as a “naked-eye” indicator for Fe3+in ethanol-water (4/1, v/v, pH 7.0) media.,
with increasing proportionally and levelled off when the concentration of Fe3+ reached 50uM.
3.1.5 Fluorescence response to pH
Appropriate pH condition for the successful operation of the fluorescence sensing was evaluated
[29-30]. Without the addition of the Fe3+, the ring opening of the Rhodamine B-based sensor LDF
occurred under acidic conditions(PH<5.0) due to protonation, while no fluorescence change was
observed with pH values over 5 (Fig. 6). However, the gradual addition of Fe3+ led to an obvious
fluorescence enhancement over a wide pH range from5.0 to 8.0, which was attributed to the similar
opening of the spirolactam structure. Because the most remarkable Fe3+ induced OFF–ON
fluorescence changes occurred under the physiological pH window, all the fluorescence measurements
were conducted at pH 7.0.
3.2 Fluorescence ON-OFF sensing for thiols
3.2.1 Fluorescence spectra of LDF-Fe3+ with thiols
Interestingly, when the LDF–Fe3+ complex was treated with thiol-containing small molecules such
as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) , the color of the solution changed
from pink to colorless, and the emission was almost completely quenched within 1 min (Fig. 7A),
while other natural amino acids including L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-
glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan and L-valine did not show any
interference to the detection. This observation suggested that the probe LDF could not only act as a
sensor for Fe3+ but also for thiols in a successive manner. Once the Fe3+ ion interacted with the sensor
LDF, the spirolactum ring was opened, yielding a high fluorescence emission. However, when treated
with the thiols, the conjugate structure of maleic anhydride was broken, leading to the absence of
fluorescence. The thiols sensing capability of the obtained complex was further investigated in detail
with fluorescence titration analysis (Fig.8). The titration curve showed an excellent linear decrease
(Fig.7B.) and about 2 equiv. of thiols (compared with LDF) was required to obtain almost completely
fluorescence quenching.
3.2.2 Application in living cells
To test the capability of LDF to image in living cells, Hela Cells were treated with various amounts
of Fe3+ and GSH [31-34]. Initially, Hela Cells were incubated with probe LDF (10μM; Fig. 9B) in
7

PBS buffer containing 1/100 DMSO for 30 minutes at room temperature and then it was treated with
FeCl3.6H2O (10μM, Fig. 9C) for 10 minutes. Their fluorescence images became bright (Fig. 9C and
9D), implying that the intracellular uptake of Fe3+ions complexed with probe LDF yielded pink
fluorescence. Upon further incubation of Hela Cells with various amounts of GSH (20uM) for 10 min,
fluorescence images became dim (Fig. 9E and F), indicating that the uptake of GSH resulted in the
decomplexation of intracellular [LDF + Fe3+] ensemble to fluorescent LDF. Therefore, the off–on–off
fluorescence imaging of probe LDF was accomplished in Hela Cells by the intracellular complexation
/ decomplexation interaction modulated by Fe3+and GSH. Furthermore, these results indicate that
probe LDF is cell membrane permeable and able to response to Fe3+/GSH in the living cells.
Conclusions
In summary, we report the synthesis and characterization of a new probe for the cascade
fluorogenic detection of Fe3+ ion and thiols. The probe LDF showed excellent “OFF–ON”
fluorescence signals with high sensitivity and selectivity in the presence of Fe3+ ion, whereas it
remained silent in the presence of other cations such as Na+, K+, Ag+, Mg2+, Cu2+, Cd2+, Pb2+, Zn2+,
Mn2+, Co2+ , Ni2+. A successive “ON–OFF” fluorescence switching was then observed in the presence
of thiols in ethanol-water (4/1, v/v, pH 7.0) media.
Acknowledgements
This research work was financially supported by Key topics for State Key Laboratory of Materials-
Oriented Chemical Engineering (ZK201304) and Jiangsu Province Agricultural Science And
Technology Innovation Fund Projects (CX(14)2057). We thank the editors and the anonymous
reviewers.
8

Reference
[1] Z.Q.Hu ,X.M.Wang, Y.C.Feng , L.Ding , M.Li , C.S.Lin . A novel colorimetric and fluorescent
chemosensor for acetate ions in aqueous media based on a rhodamine 6G-phenylurea conjugate in the
presence of Fe (III) ions. Chem Commun 2011;47:1622-1624.
[2] Z.Q. Hu , L.L.Sun, Y.Y.Gu, Y.Jiang. A sensitive and selective fluorescent probe for detection of
glutathione in the presence of Cu2+ and its application to biological imaging.Sens. Actuators B: Chem
2015;212: 220–224.
[3] Z.P.Dong, X.Tian, Y.Z.Chen, J.R.Hou, Y.P.Guo, J.Sun. A highly selective fluorescent
chemosensor for Hg2+ based on rhodamine B and its application as a molecular logic gate. Dyes
Pigment 2013;97:324-329.
[4] Y.M.Zhou, J.L.Zhang, L.Zhang, Q.Y.Zhang, T.S.Ma, J.Y.Niu. A rhodamine-based fluorescent
enhancement chemosensor for the detection of Cr3+ in aqueous media. Dyes Pigment 2013; 97:148-
154.
[5] C.Y.Li, C.X.Zou,Y.F.Li, J.L.Tang, C.Weng. A new rhodamine-based fluorescent chemosensor for
Fe3+ and its application in living cell imaging. Dyes Pigm 2014;13:110-115.
[6] S.Y.Ma, Y.Zheng , M.Y.She, W.Sun, B.Yin, P.Liu, S.Y.Zhang, J.L.Li. Design and synthesis of
functionalized rhodamine based probes for specific intracellular fluorescence imaging of Fe3+. Dyes
Pigm 2015;115: 120-126.
[7] H.M.Wu, P.Zhou, J.Wang, L.Zhao, C.Y.Duan. Dansyl-based fluorescent chemosensors for
selective responses of Cr(III).New J. Chem 2009: 33: 653–658.
[8] W.B. Tan, T.H. Leng, G.Q.Lai, Z.F.Li, K. Wang, Y.J.Shen, C.Y. Wang, A novel coumarin-based
fluorescence enhancement and colorimetric probe for Cu2+ via selective hydrolysis reaction. J Photoch
Photobio A.2016;324: 81–86
[9] M.Amjad, Z.Abolghasemi-Fakhri, T.Hallaj. Carbon dots-silver nanoparticles fluorescence
resonance energy transfer system as a novel turn-on fluorescent probe for selective determination of
cysteine. J Photoch Photobio A.2015;309: 8–14
[10] Y. Wang, L.J.Hou, Y.B. Wu, L. L. Shi, Z.B. Shang, W. J. Jin, Alizarin Complexone as a highly
selective ratiometric fluorescentprobe for Al3+detection in semi-aqueous solution.J Photoch Photobio
A. 2014;281:40–46
[11] A.Singh, R.Singh, M.Shellaiah, E.C.Prakash, H.C.Chang, P.Raghunath ,M.C. Lin, H.C.Lin. A
new pyrene-based aggregation induced ratiometric emission probe for selective detections of trivalent
metal ions and its living cell application . Sens Actuator B Chem. 2015;207:338–345.
[12] S.Goswami, K.Aich, S.Das, A.K.Das, D.Sarkar, S.Panja. A red fluorescence “off-on” molecular
switch for selective detection of Al3+, Fe3+and Cr3+:experimental and theoretical studies along with
living cell imaging. Chem Commun. 2013;49:107,39-41.
9

[13] X.F. Hou, X.L. Guo, B. Chen, C.H. Liu, F. Gao, J. Zhao, J.H. Wang. Rhodamine-based
fluorescent probe for highly selective detection of glutathione over cysteine and homocysteine. Sens.
Actuators B: Chem. 2015; 209: 838–845.
[14] D.E.Lynch, M.Cox. Metal complexes of dual-pendant sulfonate bis(indolenine) squaraines. Dyes
Pigm. 2015;118:192-198.
[15] H.S.Jung, J.H.Han, T.Pradhan, S.Kim, S.W.Lee, J.L.Sessler. A cysteine-selective fluorescent
probe for the cellular detection of cysteine. Biomaterials. 2012;33:945-953.
[16] L.Y.Niu, Y.S.Guan, Y.Z.Chen, L.Z.Wu, Q.Z.Yang. BODIPY-Based Ratiometric Fluorescent
Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem.
Soc. 2012; 134: 18928−18931.
[17] H.d. Li, L.Y. Jin, Y.H. Kan, B.Z. Yin. A visual and “turn-on” fluorescent probe for rapid
detection of cysteine over homocysteine and glutathione. Sens. Actuators B: Chem. 2014;196: 546–
554
[18] L.J.Qu, C.X.Yin, F.J.Huo, J.F. Li, J.B. Chao, Y.B. Zhang. A maleimide-based thiol fluorescent
probe and its application for bioimaging. Sens. Actuators B: Chem. 2014; 195: 246–251
[19] Y. Liu, Y.Q. Liu, Z.H.Xu, L.C.Yang, Z.J. Wang, D. Zhang , Y. Ye. A rhodamine-based “turn-on”
fluorescent probe for Fe3+ in aqueous solution and its application in bioimaging. Monatsh Chem.
2016;147:311-317
[20] S. Parihar, V. P. Boricha, R. N. Jadeja. Pyrazolone as a recognition site: Rhodamine 6G-based
fluorescent probe for the selective recognition of Fe3+ in acetonitrile–aqueous solution. Luminescence.
2015; 30: 168–174
[21] S.Lohar, A.Banerjee, A.Sahana, S.Panja, I.Hauli, S.K.Mukhopadhyay, D.Das. Selective
fluorescence and naked eye detection of histidine in aqueous medium via hydrogen bonding assisted
Schiff base condensation. Tetrahedron Lett. 2014;55:174–176.
[22] V.Dujols, F.Ford, A.W.Czarnik. A Long-Wavelength Fluorescent Chemodosimeter Selective for
Cu (II) Ion in Water.J. Am. Chem. Soc. 1997;119:7386–7387.
[23] Q.P.Hu, Y.L.Liu, Z.Q.Li, R.Z.Wen, Y.Gao, Y.L.Bei, Q.Z.Zhu..A new rhodamine -based dual
chemosensor for Al3+ and Cu2+.Tetrahedron Lett. 2014;55:4912–4916.
[24] L.J.Qu, C.X.Yin, F.J.Huo, J.F.Li, J.B.Chao, Y.B.Zhang. A maleimide-based thiol fluorescent
probe and its application for bioimaging. Sens Actuator B Chem. 2014; 195: 246–251.
[25] X.X.Fang, S.F.Zhang, G.Y.Zhao, W.W.Zhang, J.W.Xu, A.M.Ren, C.Q.Wu, W.Yang. The
solvent-dependent binding modes of a rhodamine- azacrown based fluorescent probe for Al3+ and
Fe3+.Dyes Pigm. 2014; 101: 58–66.
[26] Z.Q.Hu, C.S.Lin, X.M.Wang, L.Ding, C.L.Cui, S.F.Liu, H.Y.Lu. Highly sensitive and selective
turn-on fluorescent chemosensor for Pb2+ and Hg2+ based on arhodamine– phenylurea conjugate.
Chem. Commun.2010; 46: 3765-3767
10

[27] H.N.Kim, M.H.Lee, H.J.Kim, J.S.Kim, J.Yoon. A new trend in rhodamine-based chemosensors:
application of spirolactam ring-opening to sensing ions.Chem Soc Rev. 2008; 37:1465-1472.
[28] K.C.Ingham. On the application of Job's method of continuous variation to the stoichiometry of
protein-ligand complexes. Anal Biochem. 1975; 68:660-663.
[29] K.Murai, M.Higuchi, T.Kinoshit, K.Nagata, K.Kato. Design of a nanocarrier with regulated drug
release ability utilizing a reversible conformational transition of a peptide, responsive to slight
changes in pH. Phys. Chem. Chem. Phys. 2013;15: 11454--11460.
[30] D.Aigner, S.A.Freunberger, M.Wilkening, R.Saf, S.M.Borisov, I.Klimant. Enhancing Photo
induced Electron Transfer Efficiency of Fluorescent pH-Probes with Halogenated Phenols. Anal.
Chem. 2014; 86:9293−9300.
[31] S.Erdemir, O.Kocyigit, S. Karakurt. A new perylene bisimide-armed calix [4]-aza-crown as
“turn on” fluorescent sensor for Hg2+ ion and its application to living cells.Sens. Actuators B: Chem.
2015;220:381–388.
[32] H.D.Xiao, J.H.Li, K.T. Wu, G.Yin, Y.W.Quan, R.Y.Wang. A turn-on BODIPY-based fluorescent
probe for Hg(II) and its biological applications.Sens. Actuators B: Chem. 2015;213: 343–350.
[33] C.R.Chen,G.W.Men,W.H.Bu, C.S.Liang, H.C.Sun,S.M.Jiang. A colorimetric and fluorescent
probe for multiple transition metalions (Cu2+, Zn2+and Ni2+): Fast response and high selectivity.
Sens. Actuators B: Chem. 2015;220: 463–471.
[34] Liu YF, Kim G, Jun EJ, Swamy KMK, Kim Y, Kim SJ, Yoon J. Pyrene based fluorescent probes
for detecting endogenous zinc ions in live cells. Dyes Pigm 2015;113: 372-377.
11

List of Scheme and Figures
Scheme 1. Synthetic route for probe LDF
Scheme 2. Proposed mechanism for the recognition of Fe3+ ion and LDF- Fe3+
Figure.1 Fluorescence spectra of LDF (10μM) in the presence of various metal ions (10μM each) in
ethanol-water (4/1, v/v, pH 7.0).
Figure.2 Fluorescence spectral data of 3 mL (10μM) probe LDF at pH=7.0 with 4 equiv metal ions
Fe3+, Al3+, Cr3+, Na+, K+, Ag+,Mg2+, Cu2+, Cd2+, Pb2+, Zn2+, Mn2+, Co2+ and Ni2+ in ethanol-water (4/1,
v/v, pH 7.0).
Figure.3 (A)Fluorescence titrations of LDF (10μM) with Fe3+ ions in ethanol-water (4/1, v/v, pH 7.0).
Inset:fluorescence emission intensity changes with increasing Fe3+ ions. (B)Fluorescence intensity as a
function of Fe3+ concentration.
Figure.4 Job‟s plot obtained for the determination of binding stoichiometry between LDF and Fe3+ in
ethanol-water (4/1, v/v, pH 7.0). The total concentration of LDF and Fe3+ was maintained at 30μM.
Absorbance was measured at 565nm.
Figure.5 UV-vis spectra and absorbance changes (565nm) recorded for LDF (10μM) in ethanol-water
(4/1, v/v, pH 7.0).
Figure.6 Fluorescence intensities recorded for LDF (10μM, ethanol-water 4:1, v/v) at various pH
values in the absence and presence of 4 equiv.Fe3+.
Figure.7 (A)Fluorescence intensity recorded for LDF (10μM) with 4 equiv Fe3+ ions upon gradual
addition of GSH (0-50equiv. for Fe3+) in ethanol-water (4/1, v/v, pH 7.0). The spectra were obtained 1
min after Fe3+ addition.(B)Linear response of fluorescence intensity at 586 nm of the LDF-Fe3+
complex to the GSH concentration changes in ethanol-water (4/1, v/v, pH 7.0). [L] = 10 μM,[Fe3+] =
40 μM. The unit of x is 105mmol/L.
Figure.8 Fluorescence spectra obtained for LDF (10μM) in the presence of Fe3+ (40μM) in ethanol-
water (4/1, v/v, pH 7.0) 10min after the addition of various amino acids (40μM each).
Figure.9 (A) Fluorescence microscope images of Hela cells only, (B) images of cells + LDF (10μM),
(C) images of cells + LDF + Fe3+(1μM), (D)images of cells + LDF + Fe3+(4μM), (E) images of cells
+ [ LDF + Fe3] + GSH(5μM), (F) images of cells + [ LDF + Fe3] + GSH(20μM).
12

Scheme 1. Synthetic route for probe LDF
Scheme 2. Proposed mechanism for the recognition of Fe3+ ion and LDF- Fe3+
13

800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
800
700
600
500
400
300
200
100
0
A
3+
Fe
0
1
2
3
4
5
6
[Fe3+]/L
0
550
600
650
700
Wavelength(nm)
700
B
600
500
400
300
200
100
y=489.5369x+139.9685
R2=0.98704
0.0
0.2
0.4
0.6
0.8
1.0
[Fe3+]/105mMol
Fig.1. (A) Fluorescence titrations of 3mL LDF (10μM) to Fe3+ ions (0,0.1,0.2,0.3,0.4,0.5,
0.6,0.7,0.8,0.9,1,2,3,4,5equiv.) in ethanol-water (4/1, v/v, pH 7.0). (B)Fluorescence intensity as a
function of Fe3+ concentration.
14

800
700
600
500
400
300
200
100
0
Fe3+
LDF,Na+,K+,Ag+,
Mg2+,Cu2+,Cd2+,Pb2+,
Zn2+,Mn2+,Co2+,Ni2+
Al3+
550
600
650
700
Wavelength(nm)
Fig.2. Fluorescence spectra of LDF (10μM) in the presence of various metal ions (20μM each) in
ethanol-water (4/1, v/v, pH 7.0).
probe LDF+other ions
probe LDF+Fe3++other ions
700
600
500
400
300
200
100
0
Fe3+ Al3+ Na+ K+ Ag+ Mg2+Cu2+ Cd2+ Pb2+ Zn2+Mn2+Co2+ Ni2+
Fig.3 Fluorescence spectral data of 3 mL (10μM) probe LDF at pH=7.0 with 2 equiv metal ions Fe3+,
Al3+, Na+, K+, Ag+,Mg2+, Cu2+, Cd2+, Pb2+, Zn2+, Mn2+, Co2+ and Ni2+ in ethanol-water (4/1, v/v, pH
7.0).
15

Fig.4 Job‟s plot obtained for the determination of binding stoichiometry between LDF and Fe3+ in
ethanol-water (4/1, v/v, pH 7.0). The total concentration of LDF and Fe3+ was maintained at 20μM.
Absorbance was measured at 565nm.
0.50
0.5
0.4
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
3+
Fe
0.3
0.2
0.1
0.0
0
1
2
3
4
5
[Fe3+]/L
500
550
600
650
Wavelength(nm)
Fig.5. UV-vis spectra and absorbance changes (565nm) recorded for LDF (10μM) in ethanol-water
(4/1, v/v, pH 7.0).
16

800
700
600
500
400
300
200
100
0
LDF+Fe3+
LDF
2
4
6
8
10
12
PH
Fig.6. Fluorescence intensities recorded for LDF (10μM, ethanol-water 4:1, v/v) at various pH values
in the absence and presence of 2 equiv.Fe3+.
800
800
700
700
600
500
600
400
300
200
500
100
0
400
0
10
20
30
40
50
[GSH]/L
300
200
100
0
GSH
550
600
650
700
Wavelength(nm)
17

800
700
600
500
400
300
200
B
y=-66.2221x+710.8799
R2=0.9901
0
2
4
6
8
[GSH]/105mMol
Fig.7.(A)Fluorescence intensity recorded for LDF (10μM) with 2 equiv Fe3+ ions upon gradual
addition of GSH (0-5 equiv. for Fe3+) in ethanol-water (4/1, v/v, pH 7.0). The spectra were obtained 1
min after Fe3+ addition.(B)Linear response of fluorescence intensity at 586 nm of the LDF-Fe3+
complex to the GSH concentration changes in ethanol-water (4/1, v/v, pH 7.0). [L] = 10 μM,[Fe3+] =
20 μM. The unit of x is 105mmol/L.
800
700
other amino acids
600
500
Cys
400
300
200
Hcy
100
GSH
0
550
600
650
700
Wavelength(nm)
Fig.8.Fluorescence spectra obtained for LDF (10μM) in the presence of Fe3+ (20μM) in ethanol-water
(4/1, v/v, pH 7.0) 10min after the addition of various amino acids (40μM each).
18

Fig.9. (A) Fluorescence microscope images of Hela cells only, (B) images of cells + LDF (10μM), (C)
images of cells + LDF + Fe3+(10μM), (D)images of cells + LDF + Fe3+(20μM), (E) images of cells +
[ LDF + Fe3] + GSH(20μM), (F) images of cells + [ LDF + Fe3] + GSH(40μM).
19