Water-soluble rhodamine-based chemosensor for Fe3+ with high sensitivity, selectivity and anti-interference capacity and its imaging application in living cells

Accepted Manuscript 3+ Water-soluble rhodamine-based chemosensor for Fe with high sensitivity, selectivity and anti-interference capacity and its imaging application in living cells Feng Zhou, Tao-Hua Leng, Ya-Jing Liu, Cheng-Yun Wang, Ping Shi, Wei-Hong Zhu PII:

S0143-7208(17)30188-2

DOI:

10.1016/j.dyepig.2017.03.057

Reference:

DYPI 5884

To appear in:

Dyes and Pigments

Received Date: 29 January 2017 Revised Date:

28 March 2017

Accepted Date: 28 March 2017

Please cite this article as: Zhou F, Leng T-H, Liu Y-J, Wang C-Y, Shi P, Zhu W-H, Water-soluble 3+ rhodamine-based chemosensor for Fe with high sensitivity, selectivity and anti-interference capacity and its imaging application in living cells, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.03.057. 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.

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Graphical Abstract Water-soluble rhodamine-based chemosensor for Fe3+ with

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high sensitivity, selectivity and anti-interference capacity and its imaging application in living cells

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Feng Zhou,a Tao-Hua Leng, b Ya-Jing Liu, c Cheng-Yun Wang,*a Ping Shi*c and Wei-Hong Zhu*a

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A water-soluble rhodamine-based chemosensor RL with high sensitivity, selectivity and anti-interference capacity and low detection limit for Fe3+ was fabricated and successfully used for living cell imaging.

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Water-soluble rhodamine-based chemosensor for Fe3+ with high sensitivity, selectivity and anti-interference capacity and its imaging application in living cells

a

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Feng Zhou a, Tao-Hua Leng b, Ya-Jing Liuc, Cheng-Yun Wang a,∗, Ping Shi c,*, Wei-Hong Zhu a,*

Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced

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Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University & Technology, Shanghai 200237, P. R. China.

National Food Quality Supervision and Inspection Center (Shanghai)/Shanghai Institute of

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b

Quality Inspection and Technical Research, Shanghai 200233, P. R. China. c

State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology,

Abstract

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Shanghai 200237, P. R. China.

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Fe3+ plays a crucial role in many vital cell functions and its detection has attracted considerable attention. In this work, a water-soluble rhodamine-based chemosensor

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RL has been designed and synthesized as an “off-on” chemosensor for Fe3+ detection. Upon the addition of Fe3+, RL displayed obvious color change, quick fluorescence enhancement, high sensitivity and anti-interference capacity over other metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+). The detection limit for Fe3+ was calculated to be 0.28

∗ Corresponding author. Tel./fax: +86-021-64252967; e-mail: [email protected]; [email protected]; [email protected]

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ACCEPTED MANUSCRIPT µM with the binding constant Ka to be 4.67 × 108 M-2. Furthermore, an application of RL in the imaging of HeLa cells exposed to Fe3+ was also successfully demonstrated.

Key words

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Rhodamine dye, Chemosensors, Fe3+, Naked-eye detection, Living cell imaging

1. Introduction

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Selective detection of biologically important metal ions has gained

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tremendous importance because of their important roles in a variety of fundamental biological processes in cells. Among these metal ions, iron is of indispensable importance in the metabolism and energy transfer mechanisms of numerous biological systems. The deficiency or overload of iron can lead to

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damage to cells, tissues and organs as well as occurrence of certain diseases such as anemia and breathing problems or some cancers [1-5]. Therefore, it is extremely

important

for

physiological

concerns

to

develop

efficient

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samples.

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chemosensors with good biocompatibility for detection of iron in biological

Fluorogenic methods in conjunction with suitable chemosensors are

preferable solutions to the measurement of iron in consequence of their features - rapidly performed, non-destructive, highly sensitive and can afford real information on the localization and quantity of the targets of interest [6-11]. So far, there have been many successful fluorescent chemosensors for iron based on the fluorophores such as naphthalimide [12,13], fluoranthene [14], coumarin

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ACCEPTED MANUSCRIPT [15-19], BODIPY [20,21], cyanine [22] and so on. Currently, the main shortcoming of Fe3+ chemosensors is their low water solubility. At the same time, rhodamine dye is also an ideal material to construct chemosensors

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because of their excellent photophysical properties such as high extinction coefficients, excellent quantum yields, high photostability and relatively long emission wavelength in the visible region [23-26]. As a result, considerable

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attention has been paid to the development of rhodamine-based chemosensors

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[27-32], whose designs are primarily based on the fact that its spirolactam form is colorless and non-fluorescent while its ring-opened form induced by the analyte gives rise to pink or purple color and a strong fluorescence emission [23,33,34]. Thus, the chemosensor based on the special optical signal which is

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emitted from rhodamine or its derivatives could be developed for detection of iron [35-40].

Herein, a new water-soluble rhodamine-based colormetric and fluorescent

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chemosensor RL for Fe3+ detection was designed and synthesized, which

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provided high sensitivity, selectivity and anti-interference capacity in DMSO/H2O solution (1:1, v/v, PBS buffer, pH=7.04, 20 mM) towards Fe3+ over other metal ions and might be used for characteristic ‘naked-eye’ detection. The detection limit could reach as low as 0.28 µM. Furthermore, chemosensor RL was successfully applied in the imaging of Fe3+ in HeLa cells without significant cytotoxic effect.

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2. Experimental 2.1 General All the starting materials, reagents and solvents were AR grade purchased

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from J&K Chemical Co. and used without further purification. The salts used to prepare metal ion stock solutions were FeCl3, AlCl3, SnCl·2H2O, PbCl2, KCl, MgCl2, BiCl3, CrCl3·6H2O, MnCl2·4H2O, CaCl2, CoCl2·6H2O, NiCl2·6H2O,

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PdCl2, CuCl2·2H2O, LiCl, BaCl2, AgNO3, NaCl, ZnCl2. CdCl2·2.5H2O, HgCl2.

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Thin-layer chromatography was performed on a HAIYANG silica gel F254 plate. Column chromatography was performed using HAIYANG silica gel. 1

H and 13C NMR spectra were measured on a Bruker AM-400 spectrometer

at room temperature. The chemical shifts were reported in d units (ppm)

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downfield relative to the chemical shift of tetramethylsilane (TMS). The abbreviations br, s, d, t and m denoted broad, singlet, doublet, triplet and multiplet, respectively. Mass spectra (MS) were obtained with a Waters

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Micromass LCT Premier TOF Mass Spectrometer. High-resolution mass

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spectra (HRMS) were acquired under electron ionization conditions with a double-focusing high- resolution instrument. The ultraviolet-visible (UV-vis) spectra were recorded on a Nicolet CARY 100 and the Fluorescence spectra were measured on a CARY Eclipse at room temperature. 2.2 Synthesis 2.2.1

2-amino-3',6'-bis(diethylamino)spiro[iso-indoline-1,9'-xanthen]-3-

one (R1).

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ACCEPTED MANUSCRIPT R1 was synthesized according to the literature[41]. Rhodamine B (1.0 g, 2.09 mmol) was dissolved in absolute ethanol (30 mL), then excess hydrazine hydrate (80 wt%, 6 mL) was added dropwise to the solution. The mixture was

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refluxed in an oil bath for 8 h with vigorous stirring. The solution color changed from dark purple to orange. Under reduced pressure, the solvent was removed mostly and then a large quantity of water was added, and the resulting

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precipitate was filtered and washed by water for several times. After drying in

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vacuo, the light pink solid product was obtained (0.76 g, 80%). 1H NMR (400 MHz, CDCl3) δ 7.97-7.90 (m, 1H), 7.49-7.41 (m, 2H), 7.14-7.07 (m, 1H), 6.41-6.47 (m, 4H), 6.29 (dd, J = 8.8, 2.6 Hz, 2H), 3.61 (s, 2H), 3.34 (q, J = 7.1 Hz, 8H), 1.17 (t, J = 7.0 Hz, 12H).

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2.2.2 2-(((3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthen]-2-yl) imino)methyl)benzonitrile (RL).

R1 (0.46 g, 1.0 mmol) and 2-Formylbenzonitrile (0.13 g, 1.0 mmol) were

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dissolved in absolute ethanol (30 mL). After refluxing for 12 h with vigorous

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stirring, the solvent was removed mostly under reduced pressure. The crude product was purified by column chromatography on a silicagel column (petroleum ether: ethyl acetate = 6:1, v/v) to give RL as light yellow powder (0.48 g, 85%). 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 6.6 Hz, 1H), 7.56-7.43 (m, 4H), 7.27-7.31 (m, 1H), 7.13 (d, J = 7.0 Hz, 1H), 6.49-6.51 (m, 4H), 6.25 (dd, J = 8.9, 2.6 Hz, 2H), 3.37 (q, J = 7.1 Hz, 8H), 1.15 (t, J = 7.0 Hz, 12H); 13C NMR (400 MHz, DMSO) δ 164.27, 152.60, 151.36, 148.49, 140.92, 136.96,

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ACCEPTED MANUSCRIPT 134.40, 133.44, 133.31, 130.46, 128.92, 128.01, 127.46, 124.55, 123.91, 123.22, 116.14, 111.07, 108.08, 104.70, 97.55, 65.53, 43.64, 12.33. HRMS [M+H]+ found: 570.2864; calcd for C36H36N5 O2+: 570.2869.

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2.3 Stock solution preparation Stock solutions (1.0 ×10-2 M for Fe3+ and 5.0×10-2 M for the other metal ions) of the chloride or nitrate salts of Fe3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+,

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Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+ and Hg2+ were

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prepared in deionized water. The stock solution of RL (1.0×10-3 M) was prepared in DMSO. Working solutions of RL were freshly prepared by diluting the high concentrated stock solution to the desired concentration prior to spectroscopic measurements.

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2.4 Absorption and fluorescence studies

All experiments were carried out in DMSO/H2O solution (1:1, v/v, PBS buffer, pH=7.04, 20 mM) unless otherwise specified. In each titration experiment, a 10 µM

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solution of RL was placed in a quartz optical cell with a 1-cm optical path length, and

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the appropriate amount of the metal ion stock solution was added to the quartz optical cell using a micropipette. Spectral data were recorded after 1 min on the addition of the metal ions. In the selectivity and anti-interference experiments, the test samples were prepared by placing an appropriate amount of the metal ion stock solution in 3 mL of the chemosensor RL solution (10 µM). In the fluorescence measurements, excitation was provided at 520 nm (λex =520 nm) and emission spectra were collected from 525 to 720 nm.

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ACCEPTED MANUSCRIPT 2.5 Cell studies Vitro experiments were performed using HeLa cells. HeLa cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 oC.

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Cytotoxicity of RL was determined by the MTT assay. Cells were incubated with different concentrations of RL for 24 h. Cell viability was evaluated by incubating with 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

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for 24h at 37 oC. For fluorescence microscopy images, cells were incubated with 10

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µM Fe3+ for 30 min at 37 oC and washed with PBS for three times, followed by incubation with 10 µM RL at 37 oC for additional 30 min. The incubated cells were washed with PBS for three times again and mounted onto a glass slide. Fluorescence

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images in the living cell were acquired on a Nikon A1 Laser Confocal Microscope.

3. Results and discussion 3.1. Synthesis

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The synthesis of RL was shown in Scheme 1. Compound R1 was prepared in

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80% yield from commercially available Rhodamine B and hydrazine hydrate, as described previously [41]. Compound RL was prepared in 85% yield by the reaction of R1 with 2-Formylbenzonitrile in absolute ethanol for 12 h. The structures of R1 and RL were confirmed by 1H NMR, 13C NMR and HRMS (Supporting Information (SI), Fig. S1-S4). Insert Scheme 1 here 3.2 Response time of RL towards Fe3+

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ACCEPTED MANUSCRIPT The response time of RL towards Fe3+ in the fluorescence intensity was investigated as shown in Fig. 1. With the addition of Fe3+, the fluorescence intensity increased rapidly and reached a maximum without 1 min, which

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indicated that the reaction system was stable after 1 min. These results indicated that RL was a sensitive chemosensor for Fe3+. Therefore, a 1-min reaction time was

sufficiently chelate with the chemosensor.

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Insert Fig.1 here

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selected in subsequent experiments to ensure that the metal ions had enough time to

3.3 pH response

Acid-base titration experiments were performed in DMSO/H2O solution (1:1, v/v) by using PBS buffer solutions of different pH. The fluorescence intensities of RL

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at 589 nm at different pH values (2.02, 3.01, 3.98, 5.00, 6.02, 6.98, 7.04, 8.00, 9.01, 10.03, 11.01 and 11.98, respectively) were recorded as shown in Fig. 2. Without the addition of Fe3+, RL did not emit any fluorescence in the pH range of 4.0-12.0, which

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indicated that the spirolactam form of RL was the predominant species. When the pH

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was adjusted to 2.0, the fluorescence intensity at 589 nm was sharply enhanced due to the strong protonation which led to the ring-opening process of the spirocyclic moiety of Rhodamine (Scheme 2). Upon the addition of Fe3+, RL emitted fluorescence in the pH range of 4.0-11.0 between which RL did not emit any fluorescence without Fe3+, which indicated that the addition of Fe3+ led to the opening of the spirocyclic moiety of Rhodamine. At the same time, there was no obvious fluorescence change in the pH range of 4.0-8.0. However, when the pH was over 8, the fluorescence intensity

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ACCEPTED MANUSCRIPT gradually decreased with the increase of pH so that fluorescence almost completely disappeared when pH reached 12, which might be attributed to the formation of Fe(OH)3 which prevented the combination of Fe3+ and RL [42,43]. For further spectral

Insert Fig.2 here Insert Scheme 2 here

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analysis and potential practical applications, pH value should be adjusted to 7.04.

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3.4 Absorption and fluorescence studies of RL towards Fe3+

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To gain insight into the optical property of RL with Fe3+, UV-vis spectra of RL upon titration with Fe3+ in DMSO/H2O solution were recorded as shown in Fig. 3(a). Without addition of Fe3+ to the solution of RL, almost no absorption above 500 nm could be observed. However, with the addition of the Fe3+ (0-180

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µM, 0-18 equiv of RL), a new absorption band centered at 562 nm with a shoulder peak at 520 nm was observed and gradually increased, resulting in a color change from colorless to pink as shown in the insert picture of Fig. 3(a).

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These phenomena might be ascribed to the formation of the ring-opened form

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of RL after the addition of Fe3+ (Scheme 3). Insert Fig.3 here Insert Scheme 3 here

Fig. 3(b) displayed the fluorescence spectra of RL in the presence of different

amounts of Fe3+. Like most spirocycle Rhodamine B derivatives, the free RL (10 µM) exhibited very weak fluorescence at 589 nm (λex=520 nm). However, the titration of Fe3+ (0-180 µM) led to a significant increase in the fluorescence emission intensity.

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ACCEPTED MANUSCRIPT Over 50-fold fluorescence enhancement was observed under saturation conditions. The insert picture of Fig. 3(b) displayed the fluorescence change of RL on the addition of Fe3+ under a 365 nm ultraviolet lamp.

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3.5 Selectivity and anti-interference capacity To evaluate the selectivity of RL for Fe3+, the optical property of RL towards different metal ions was also studied by UV-vis and fluorescence spectra. All of

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the measurements were performed according to the following process. Test

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metal ions (900 µM, 5 equiv of Fe3+) in place of Fe3+ (180 µM) were added to 3 mL of 10 µM RL in DMSO/H2O solution, and the UV-vis and fluorescence spectra were measured 1 min later. As shown in Fig. 4(a) (b), other metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+,

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Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+) possessed very little absorption and very weak fluorescence. These results indicated that RL exhibited excellent selectivity towards Fe3+, which in fact endowed the chemosensor with “naked eye” and

Insert Fig.4 here

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fluorescence detection of Fe3+ (Fig. 4(c)(d)).

Besides, anti-interference capacity of RL for Fe3+ (180 µM) in the presence of

many other metal ions (900 µM, 5 equiv of Fe3+), including Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+ and Hg2+, was also studied by fluorescence spectra. As shown in Fig. 5, compared with Fe3+, no obvious fluorescence was detected upon the addition of above metal ions (black bars), which indicated that the Fe3+ induced the formation of the strongly

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ACCEPTED MANUSCRIPT fluorescent, ring-opened RL-Fe3+ complex (Scheme 3). When Fe3+ was added into RL solution in the presence of these competitive metal ions, no significant variations were observed compared to the spectra when only Fe3+ exited (red bars). These results

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indicated that the detection of Fe3+ was not interfered by other metal ions, and RL could be used as a selective Fe3+ chemosensor, which might be a result of the simultaneous effect of a series of factors, such as the apposite combination ratio, the

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suitable coordination geometry conformation of the receptor and the radius of the Fe3+

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( Scheme 3) [44]. Insert Fig.5 here 3.6 Detection limit calculations

The detection limit is calculated with the following equation[45]:

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Detection limit= 3SD/S

where SD is the standard deviation of the blank and S is the slope of the calibration curve.

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The detection limit of RL for Fe3+ was also calculated based on the fluorescence

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titration. The fluorescence spectra of free RL (10 µM) were measured 20 times, and the standard deviation of fluorescence intensities was then calculated. To obtain the slope, the ratio of the fluorescence intensity at 589 nm was plotted versus the concentration of Fe3+ (Fig. S5). The detection limit was thus calculated to be 0.28 µM. 3.7 Quantum yield measurements The fluorescence quantum yield is determined by using rhodamine B as a reference with a known ΦR value of 0.89 in ethanol [46,47]. The area of the

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ACCEPTED MANUSCRIPT fluorescence spectra is integrated using the software available in the instrument and the quantum yield is calculated according to the following equation [47]:

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where ΦS and ΦR are the fluorescence quantum yields of the sample and the reference, respectively; FS and FR are the area under the fluorescence spectra of the sample and

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the reference, respectively; AS and AR are the corresponding optical densities of the sample and the reference solution at the wavelength of excitation; ηS and ηR are the

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refractive indexes of the sample and the reference, respectively.

The fluorescence quantum yield of RL was calculated to be 0.27 in ethanol. 3.8 Binding mechanism analysis

To explore the binding mechanism of RL to Fe3+, Job’s plot of fluorescence titration

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of Fe3+ was carried out as shown in Fig. 6(a). Total concentration of RL and Fe3+ was 50 µM and kept constantly when the mole fraction of Fe3+ changed from 0 to 1. Maximum

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emission was observed when Fe3+ mole fraction was between 0.3 and 0.4, indicating that 2:1 stoichiometry should be the most possible binding stoichiometry for RL and

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Fe3+.

Insert Fig.6 here

This binding stoichiometry was further confirmed by Benesi-Hildebrand plot

[48-50]. When assuming a 2:1 stoichiometry between RL and Fe3+, the following equation is obtained:

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ACCEPTED MANUSCRIPT where F0, F, and Fmax are the emission intensities in the absence of, at a series of intermediate concentrations of and at an infinite concentration of Fe3+, respectively; Ka is the binding constant.

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When 1/(F-F0) was plotted as a function of 1/[Fe3+]2, a linear relationship was obtained (y = A + Bx), and Ka was calculated from A/B. As shown in Fig. 6(b), the plot showed a good linear relationship (R2 = 0.9996), which further confirmed the 2:1

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stoichiometry between RL and Fe3+ and the binding constant Ka was calculated to be

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4.67 × 108 M-2, from which it is inferred that RL possessed high affinity towards Fe3+. Besides, MS of RL on the addition of Fe3+ was acquired. As shown in Fig. S6, without the combination of Fe3+, the peak m/z 570.3 corresponded to that of [RL+H]+ (the calculated value was m/z 570.2864; with the combination of RL and Fe3+, a peak

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appears at m/z 398.2, which might be attributed to the three-charged complex [2RL+Fe]3+ (Scheme 3) ( the calculated value was m/z = 1194.4914/3 = 398.1638). These results also confirmed the 2:1 stoichiometry between RL and Fe3+.

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3.9 Cell imaging studies

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Further applicability of RL as a chemosensor for Fe3+ in vitro cell studies was also evaluated. HeLa cells were chosen as the sample. The cells were incubated with RL (10 µM) in the growth medium for 30 min at 37 oC, which led to very weak fluorescence when determined by laser scanning confocal microscopy (Fig. 7(b)); in contrast, a bright fluorescence was detected for those cells incubated with Fe3+ (10 µM) for 30 min at 37 oC, followed by incubation with RL (10 µM) under the same conditions (Fig. 7(e)). The phase-contrast images in the bright fields of the cells (Fig.

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ACCEPTED MANUSCRIPT 7(a) (d)) showed that the cells were viable during the experiments, which indicated that RL might be cell-permeable and non-toxic to the living HeLa cells. Insert Fig.7 here

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To further evaluate the cytotoxicity of RL, cell viability was determined by an MTT assay in HeLa cells incubated with different concentrations of RL (10, 20, 50, 100 µM). As shown in Fig. 8, even at a reasonably high concentration of RL (20 µM)

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for 24 h, the cell viability value still remained more than 80%. These results suggested

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that RL might be suitable to be used as a potential chemosensor for detecting Fe3+ in biological samples.

Insert Fig.8 here

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4. Conclusions

In summary, the synthesis of a water-soluble chemosensor RL was presented, which showed excellent labeling and monitoring of Fe3+ in cellular systems with high

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selectivity, sensitivity and anti-interference capacity. RL displayed an apparent and

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quick response to Fe3+ with significant changes in UV-vis and fluorescence spectra while other metal ions have nearly no interference on this specific recognition of Fe3+. The 2:1 stoichiometry between RL and Fe3+ was proposed based on a Job’s plot and MS of [2RL+Fe]3+. Besides, the binding constant between RL and Fe3+ was calculated by the Benesie-Hildebrand equation. In addition, the cell imaging of RL with Fe3+ was studied in HeLa cells, the results revealed that the chemosensor RL was highly effective tools for Fe3+ labeling and could detect Fe3+ sensitively at the

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ACCEPTED MANUSCRIPT cellular level, which will be critically important to understand the functions of Fe3+ in related cells and biological organs in the future.

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Acknowledgments This work is sponsored by the Natural Science Foundation of Shanghai (No. 16ZR1408000),

and

the

National

Key

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ACCEPTED MANUSCRIPT Scheme captions: Scheme 1 Synthesis procedure for the chemosensor RL. Scheme 2 Proposed mechanism of the RL response to pH changes.

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Scheme 3 Proposed complexation mechanism of RL with Fe3+.

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Scheme 1 Synthesis procedure for the chemosensor RL.

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Scheme 2 Proposed mechanism of the RL response to pH changes.

Scheme 3 Proposed complexation mechanism of RL with Fe3+.

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ACCEPTED MANUSCRIPT Figure captions: Fig. 1 Time-dependent fluorescence intensity change at 589 nm of RL (10µM) with Fe

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(180 µM) in DMSO/H2O solution, λex=520 nm.

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Fig. 2 Variation of fluorescence intensity at 589 nm of RL solution (10µM) with and without Fe3+ (180 µM) in DMSO/H2O solution (1:1, v/v) as a function of pH, λex=520 nm.

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Fig. 3 UV-vis spectra (a) and fluorescence spectra (b) of RL (10 µM) in DMSO/H2O

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solution with gradual increase of Fe3+ (0-180 µM) , λex=520 nm. Inset: the photos of RL solution before and after the addition of Fe3+ (180 µM) under the visible light (a) and a 365 nm ultraviolet lamp (b).

Fig. 4 UV-vis spectra (a) and fluorescence spectra (b) (λex=520 nm), colorimetric

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performance (c) and fluorescence performance (d) (under a 365 nm ultraviolet lamp) of RL (10 µM) in DMSO/H2O solution in the presence of different metal ions (180 µM for Fe3+ and 900 µM for the other metals ions).From left to

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right(c,d): Free, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Fe3+,

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Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+. Fig. 5 Anti-interference capacity of RL (10 µM) in the presence of various metal ions in DMSO/H2O solution: 180 µM for Fe3+ and 900 µM for the other metal ions. From left to right: Free, Fe3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+. λex=520 nm. Fig. 6 (a) Job’s plot with a total concentration of [RL] and [Fe3+] was 50 µM and (b) Benesi-Hilderbrand plot in DMSO/H2O solution (Fluorescence spectra, λex=520

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overlay of bright field and fluorescence image (Right). Fig. 8 Cell viability values (%) estimated by MTT proliferation test versus different incubation concentrations of RL. HeLa cells were cultured in the presence of RL (10,

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20, 50, 100 µM).

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3+

(180 µM) in DMSO/H2O solution, λex=520 nm.

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with Fe

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Fig. 1 Time-dependent fluorescence intensity change at 589 nm of RL (10µM)

Fig. 2 Variation of fluorescence intensity at 589 nm of RL solution (10µM) with and without Fe3+ (180 µM) in DMSO/H2O solution (1:1, v/v) as a function of pH, λex=520 nm.

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Fig. 3 UV-vis spectra (a) and fluorescence spectra (b) of RL (10 µM) in DMSO/H2O solution with gradual increase of Fe3+ (0-180 µM), λex=520 nm. Inset: the photos of

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RL solution before and after the addition of Fe3+ (180 µM) under the visible light

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(a) and a 365 nm ultraviolet lamp (b).

Fig. 4 UV-vis spectra (a) and fluorescence spectra (b) (λex=520 nm), colorimetric performance (c) and fluorescence performance (d) (under a 365 nm ultraviolet lamp) of RL (10 µM) in DMSO/H2O solution in the presence of different metal ions (180 µM for Fe3+ and 900 µM for the other metals ions).From left to 28

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Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+.

Fig. 5 Anti-interference capacity of RL (10 µM) in the presence of various metal ions in DMSO/H2O solution: 180 µM for Fe3+ and 900 µM for the other metal ions.

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From left to right: Free, Fe3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+,

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Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+. λex=520 nm.

Fig. 6 (a) Job’s plot with a total concentration of [RL] and [Fe3+] was 50 µM and (b) Benesi-Hilderbrand plot in DMSO/H2O solution (Fluorescence spectra, λex=520 nm ).

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Fig. 7 Fluorescence images of HeLa cells treated with RL (10 µM, Top) and RL/ Fe3+ (10 µM/10 µM, Bottom): Bright field image (Left); fluorescence image (Middle); and

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overlay of bright field and fluorescence image (Right).

Fig. 8 Cell viability values (%) estimated by MTT proliferation test versus different incubation concentrations of RL. HeLa cells were cultured in the presence of RL (10, 20, 50, 100 µM).

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Highlights 1. Quick response time less than one minute.

3. Low detection limit as low as 0.28 µM.

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4. Successful imaging application in HeLa cells.

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2. High sensitivity and anti-interference towards Fe3+.